Bentley A Fane

Interests

Research

Virus structure and assembly, virus evolution.

Courses

Scholarly Contributions

Books

• Cherwa, J. E., & Fane, B. A. (2011). Microviridae: Microviruses and Gokushoviruses. eLS.

Chapters

• Fane, B. A., & Roznowski, A. P. (2020). Microviruses. In Microviruses.
• Roznowski, A. P., Doore, S. M., & Fane, B. A. (2017). The Genetics of øX174. In Reference Module in Life Sciences (This is a review article intended for undergraduate/graduate students). doi:10.1016/B978-0-12-809633-8.06897-7
• Fane, B. A. (2011). Microviruses. In Fundamentals of Molecular Virolog(pp 69-76). Hoboken, NJ: John Wiley and Sons.
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  2nd. Edition. Editor(s): Acheson, NH
• Fane, B. A. (2011). The Microviridae. In Virus Taxonomy Ninth Report of the International Committee on Taxonomy of Viruses. London: Elsevier.
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  Editor(s): King, AM | Adams, MJ | Carstens, EB | Lefkowitz, EJ
• Prevelige, P. E., & Fane, B. A. (2011). Viral Nanomachines. In Building the machine, the role of scaffolding proteins in bacteriophage morphogenesis(pp 325-350). New York: Springer.
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  Editor(s): Rao, V | Rossmann, MGR
• Fane, B. (2010). The Genetics of phiX174. In Brenner's Online Encyclopedia of Genetics.
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  Second Edition. Editor(s): Maloy, S | Hughes, K

Journals/Publications

• Love, S. D., Posey, S., Burch, A. D., & Fane, B. A. (2024). Disenfranchised DNA: biochemical analysis of mutant øX174 DNA binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A*
  
  
  
  . Journal of Virology. The article wa further featured as the "Editor's pick".
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  Samuel Love was an undergraduate student in the lab. Sierra Posey was a high school student working with Dr. April Burch at the Berkshire School.
• Mietzsch, M., Kailasan, S., Bennett, A., Chipman, P., Fane, B., Huiskonen, J., Clarke, I., & McKenna, R. (2024). The Structure of Spiroplasma Virus 4: Exploring the Capsid Diversity of the Microviridae. Viruses, 16(7). doi:10.3390/v16071103
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  Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus–host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants.
• Fane, B. A., Lee, H., Baxter, A. J., Bator, C. M., & Hafenstein, S. L. (2022). Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System. Journal of Virology Spotlighted and journal cover. The first Gokushovirus virus structure at atomic resolution., 96(21). doi:10.1128/jvi.00990-22
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  Spotlighted and journal cover
• Ogunbunmi, E. T., Lover, S. D., Rhoades, K. A., Morales, A., Wilch, M. H., Jonas, J., & Fane, B. A. (2022). Low temperature adaptation targets genome packing reactions in an icosahedral single-stranded DNA virus. Journal of Virology.
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  This manuscript include two high school teacher and one high school student author.
• Ogunbunmi, E. T., Roznowski, A. P., & Fane, B. A. (2021). The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid. Journal of virology, 95(18), e0088321.
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  Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA's negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid's outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid's outer surface.
• Roznowski, A. P., Doore, S. M., Kemp, S. Z., & Fane, B. A. (2020). Finally, a role befitting A star: the strongly conserved, unessential microvirus A* (A star) proteins ensure the product fidelity of packaging reactions. Journal of Virology. Spotlighted article and the journal cover..
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  In microviruses, 60 copies of the positively-charged DNA binding protein J guide the single-stranded (ss) DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the φX174, G4, and α3 capsids are nearly identical, their genome length varies widely from 5386 (φX174) to 6067 (α3). As genome size increases, the J protein's length and charge decreases. The φX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23 residue G4 and α3 proteins have respective +6 and +8 charges. While the large φX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, φX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (φXG4J) was generated by replacing the indigenous φX174 J gene with that of G4. The resulting mutant, φXG4J, was not viable on the level of plaque formation without φX174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than wild-type. Complementation-independent φXG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, φXG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene. Double-stranded (ds) DNA viruses typically package their genomes into a preformed capsid. By contrast, ssRNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. ssDNA viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential and/or functionally undefined in-frame versions of A/A*-like proteins.
• Roznowski, A. P., Fisher, J. M., & Fane, B. A. (2020). Mutagenic Analysis of a DNA Translocating Tube's Interior Surface. Viruses, 12(6).
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  Bacteriophage ϕX174 uses a decamer of DNA piloting proteins to penetrate its host. These proteins oligomerize into a cell wall-spanning tube, wide enough for genome passage. While the inner surface of the tube is primarily lined with inward-facing amino acid side chains containing amide and guanidinium groups, there is a 28 Å-long section near the tube's C-terminus that does not exhibit this motif. The majority of the inward-facing residues in this region are conserved across the three ϕX174-like clades, suggesting that they play an important role during genome delivery. To test this hypothesis, and explore the general function of the tube's inner surface, non-glutamine residues within this region were mutated to glutamine, while existing glutamine residues were changed to serine. Four of the resulting mutants had temperature-dependent phenotypes. Virion assembly, host attachment, and virion eclipse, defined as the cell's ability to inactivate the virus, were not affected. Genome delivery, however, was inhibited. The results support a model in which a balance of forces governs genome delivery: potential energy provided by the densely packaged viral genome and/or an osmotic gradient move the genome into the cell, while the tube's inward facing glutamine residues exert a frictional force, or drag, that controls genome release.
• Roznowski, A. P., Young, R. J., Love, S. D., Andromita, A. A., Guzman, V. A., Wilch, M. H., Block, A., McGill, A., Lavelle, M., Romanova, A., Sekiguchi, A., Wang, M., Burch, A. D., & Fane, B. A. (2019). Recessive host range mutants and unsusceptible cells that inactivate virions without genome penetration: ecological and technical implications. Journal of Virology (article was selected for the journal's "Spotlight" section).
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  Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this "host-induced" unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, α3 and ST-1. Despite ∼90% amino acid identity, the natural host of α3 is C; whereas ST-1 is a K12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from co-infected cells did not display the phenotype. Thus, mutant isolation required populations generated in low MOI environments: a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts; culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low MOI environments. However, in laboratory settings, viral propagation includes high MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Lastly, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.
• Blackburn, B. J., Li, S., Roznowski, A. P., Perez, A. R., Villarreal, R. H., Johnson, C. J., Hardy, M., Tuckerman, E. C., Burch, A. D., & Fane, B. A. (2017). Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway. Journal of virology, 91(24).
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  Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.
• Cherwa, J. E., Tyson, J., Bedwell, G. J., Brooke, D., Edwards, A. G., Dokland, T., Prevelige, P. E., & Fane, B. A. (2017). ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro. Journal of Virology (article was selected for the journal's "Spotlight" section), 91(1).
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  During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context.
• Doore, S. M., Schweers, N. J., & Fane, B. A. (2017). Elevating fitness after a horizontal gene exchange in bacteriophage φX174. Virology, 501, 25-34.
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  In an earlier study, protein-based barriers to horizontal gene transfer were investigated by placing the bacteriophage G4 G gene, encoding the major spike protein, into the φX174 genome. The foreign G protein promoted off-pathway assembly reactions, resulting in a lethal phenotype. After three targeted genetic selections, one of two foreign spike proteins was productively integrated into the φX174 system: the complete G4 or a recombinant G4/φX174 protein (94% G4:6% φX174). However, strain fitness was very low. In this study, the chimeras were characterized and experimentally evolved. Inefficient assembly was the primary contributor to low fitness: accordingly, mutations affecting assembly restored fitness. The spike protein preference of the ancestral and evolved strains was determined in competition experiments between the foreign and φX174G proteins. Before adaptation, both G proteins were incorporated into virions; afterwards, the foreign proteins were strongly preferred. Thus, a previously inhibitory protein became the preferred substrate during assembly.
• Levin, E., Lopez-Martinez, G., Fane, B. A., & Davidowitz, G. (2017). Hawkmoths use nectar sugar to reduce oxidative damage from flight. Science, 355, 733-735.
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  Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ13C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar.
• Sun, Y., Roznowski, A. P., Tokuda, J. M., Klose, T., Mauney, A., Pollack, L., Fane, B. A., & Rossmann, M. G. (2017). Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls. Proceedings of the National Academy of Sciences of the United States of America, 114, 13708–13713.
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  Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.
• Christakos, K. J., Chapman, J. A., Fane, B. A., & Campos, S. K. (2016). PhiXing-it, displaying foreign peptides on bacteriophage ΦX174. Virology, 488, 242-248.
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  Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.
• Doore, S. M., & Fane, B. A. (2016). The microviridae: Diversity, assembly, and experimental evolution. Virology (an invited review article), 491, 45-55.
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  The Microviridae, comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries.
• Roznowski, A. P., & Fane, B. A. (2016). Structure-Function Analysis of the ϕX174 DNA-Piloting Protein Using Length-Altering Mutations. Journal of Virology, 90(17), 7956-66.
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  Although the ϕX174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein.
• Doore, S. M., & Fane, B. A. (2015). The Kinetic and Thermodynamic Aftermath of Horizontal Gene Transfer Governs Evolutionary Recovery. Molecular Biology and Evolution, 32(10), 2571-84.
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  Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein-protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera's path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.
• Doore, S. M., Baird, C., The 2012 University of Arizona Virology Undergraduate Lab, ., Roznowski, A. P., & Fane, B. A. (2014). The evolution of genes within genes and the control of DNA replication in Microviruses. Molecular Biology and Evolution, 31(6), 1421-1431.
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  Approximately 20% of the data in this manuscript was generated by students enrolled in the 2012 Summer Virology Lab Course.
• Sun, L., Rossmann, M. G., & Fane, B. A. (2014). High-resolution structure of a virally encoded DNA-translocating conduit and the mechanism of DNA penetration. Journal of Virology, 88(18), 10276-9.
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  The Journal of Virology invited us to submit this article in the GEM platform, which features key developments in virology. Although these articles are peer-reviewed and can report data, they are really more review-like than a manuscript reporting primary data.
• Gordon, E. B., & Fane, B. A. (2013). Effects of an early conformational switch defect during phiX174 morphogenesis are belatedly manifested late in the assembly pathway. Journal of Virology, 87(5).
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  C-terminal, aromatic amino acids in the phiX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B(-) mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B(-) assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.
• Gordon, E. B., & Fane, B. A. (2013). Effects of an early conformational switch defect during φX174 morphogenesis are belatedly manifested late in the assembly pathway. Journal of Virology, 87(5), 2518-2525.
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  PMID: 23255785;PMCID: PMC3571406;Abstract: C-terminal, aromatic amino acids in the φX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B- mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B- assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect. © 2013, American Society for Microbiology.
• Sun, L., Young, L. N., Zhang, X., Budko, S. P., Fokine, A., Zbornik, E., Roznowski, A. P., Moulineux, I. J., Rossmann, M. G., & Fane, B. A. (2014). Icosahedral phiX174 forms a tail for DNA transport.. Nature, 505, 432-435.
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  Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA phiX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the phiX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Angstrom resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Angstron-long alpha-helical barrel. The tube is constructed of ten alpha-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.
• Young, L. N., Hockenberry, A. M., & Fane, B. A. (2014). Mutations in the N-terminus of the phiX174 DNA pilot protein H confer both assembly and host cell attachment defects.. Journal of Virology, 1787-1794.
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  The phiX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.
• Gordon, E. B., Knuff, C. J., & Fane, B. A. (2012). Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation. Journal of Virology, 86(18).
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  Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, X174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.
• Gordon, E. B., Knuff, C. J., & Fane, B. A. (2012). Conformational switch-defective ΦX174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation. Journal of Virology, 86(18), 9911-9918.
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  PMID: 22761377;PMCID: PMC3446603;Abstract: Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, øX174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates. © 2012, American Society for Microbiology.
• Prevelige, P. E., & Fane, B. A. (2012). Building the machines: Scaffolding protein functions during bacteriophage morphogenesis. Advances in Experimental Medicine and Biology, 726, 325-350.
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  PMID: 22297520;Abstract: For a machine to function, it must first be assembled. The morphogenesis of the simplest icosahedral virus would require only 60 copies of a single capsid protein to coalesce. If the capsid protein's structure could be entirely dedicated to this endeavor, the morphogenetic mechanism would be relatively uncomplicated. However, capsid proteins have had to evolve other functions, such as receptor recognition, immune system evasion, and the incorporation of other structure proteins, which can detract from efficient assembly. Moreover, evolution has mandated that viruses obtain additional proteins that allow them to adapt to their hosts or to more effectively compete in their respective niches. Consequently, genomes have increased in size, which has required capsids to do likewise. This, in turn, has lead to more complex icosahedral geometries. These challenges have driven the evolution of scaffolding proteins, which mediate, catalyze, and promote proper virus assembly. The mechanisms by which these proteins perform their functions are discussed in this review. © 2012 Springer Science+Business Media, LLC.
• Cherwa Jr., J. E., & Fane, B. A. (2011). From resistance to stimulation: The evolution of a virus in the presence of a dominant lethal inhibitory scaffolding protein. Journal of Virology, 85(13), 6589-6593.
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  PMID: 21490088;PMCID: PMC3126481;Abstract: By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks ΦX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution. © 2011, American Society for Microbiology.
• Cherwa Jr., J. E., Organtini, L. J., Ashley, R. E., Hafenstein, S. L., & Fane, B. A. (2011). In vitro assembly of the øx174 procapsid from external scaffolding protein oligomers and early pentameric assembly intermediates. Journal of Molecular Biology, 412(3), 387-396.
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  PMID: 21840317;Abstract: Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S* particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S* particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S* particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding-scaffolding protein interactions, as opposed to coat-coat protein interactions between pentamers, constitute the primary lattice-forming contacts. © 2011 Elsevier Ltd. All rights reserved.
• Cherwa, J. E., & Fane, B. A. (2011). From resistance to stimulation: the evolution of a virus in the presence of a dominant lethal inhibitory scaffolding protein. Journal of virology, 85(13).
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  By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks ϕX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.
• Cherwa, J. E., Organtini, L. N., Ashley, R. E., Hafenstein, S. L., & Fane, B. A. (2011). In VITRO ASSEMBLY of the øX174 procapsid from external scaffolding protein oligomers and early pentameric assembly intermediates. Journal of molecular biology, 412(3).
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  Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S⁎ particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S⁎ particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S⁎ particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding-scaffolding protein interactions, as opposed to coat-coat protein interactions between pentamers, constitute the primary lattice-forming contacts.
• Cherwa, J. E., Young, L. N., & Fane, B. A. (2011). Uncoupling the functions of a multifunctional protein: The isolation of a DNA pilot protein mutant that affects particle morphogenesis. Virology, 411(1), 9-14.
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  PMID: 21227478;Abstract: Defective øX174 H protein-mediated DNA piloting indirectly influences the entire viral lifecycle. Faulty piloting can mask the H protein's other functions or inefficient penetration may be used to explain defects in post-piloting phenomena. For example, optimal synthesis of other viral proteins requires de novo H protein biosynthesis. As low protein concentrations affect morphogenesis, protein H's assembly functions remain obscure. An H protein mutant was isolated that allowed morphogenetic effects to be characterized independent of its other functions. The mutant protein aggregates assembly intermediates. Although excess internal scaffolding protein restores capsid assembly, the resulting mutant H protein-containing particles are less infectious. In addition, nonviable phenotypes of am(H) mutants in Su+ hosts, which insert non-wild-type amino acids, do not always correlate with a lack of missense protein function. Phenotypes are highly influenced by host and phage physiology. This phenomenon was unique to am(H) mutants, not observed with amber mutants in other genes. © 2010 Elsevier Inc.
• Cherwa, J. E., Young, L. N., & Fane, B. A. (2011). Uncoupling the functions of a multifunctional protein: the isolation of a DNA pilot protein mutant that affects particle morphogenesis. Virology, 411(1).
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  Defective øX174 H protein-mediated DNA piloting indirectly influences the entire viral lifecycle. Faulty piloting can mask the H protein's other functions or inefficient penetration may be used to explain defects in post-piloting phenomena. For example, optimal synthesis of other viral proteins requires de novo H protein biosynthesis. As low protein concentrations affect morphogenesis, protein H's assembly functions remain obscure. An H protein mutant was isolated that allowed morphogenetic effects to be characterized independent of its other functions. The mutant protein aggregates assembly intermediates. Although excess internal scaffolding protein restores capsid assembly, the resulting mutant H protein-containing particles are less infectious. In addition, nonviable phenotypes of am(H) mutants in Su+ hosts, which insert non-wild-type amino acids, do not always correlate with a lack of missense protein function. Phenotypes are highly influenced by host and phage physiology. This phenomenon was unique to am(H) mutants, not observed with amber mutants in other genes.
• Cherwa Jr., J. E., & Fane, B. A. (2009). Complete virion assembly with scaffolding proteins altered in the ability to perform a critical conformational switch. Journal of Virology, 83(15), 7391-7396.
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  PMID: 19474099;PMCID: PMC2708623;Abstract: In the φX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30° kink to form in α-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Cherwa Jr., J. E., Sanchez-Soria, P., Birkholz, J. A., Dineen, H. A., Grippi, D. C., Kempton, T. L., Kwan, J., Patel, N. N., Toussaint, B. M., Wichman, H. A., & Fane, B. A. (2009). Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type. Journal of Virology, 83(22), 11746-11750.
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  PMID: 19726521;PMCID: PMC2772694;Abstract: Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks ΦX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Fane, B., Cherwa, J. E., & Fane, B. A. (2009). Complete virion assembly with scaffolding proteins altered in the ability to perform a critical conformational switch. Journal of virology, 83(15).
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  In the phiX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30 degrees kink to form in alpha-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.
• Fane, B., Cherwa, J. E., Sanchez-Soria, P., Wichman, H. A., & Fane, B. A. (2009). Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type. Journal of virology, 83(22).
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  Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks phiX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly.
• Fane, B., Ruboyianes, M. V., Chen, M., Dubrava, M. S., Cherwa, J. E., & Fane, B. A. (2009). The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of phiX174 replication. Journal of virology, 83(19).
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  The phiX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (DeltaH). The expression of the DeltaH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory DeltaH proteins, restoring both DNA and protein synthesis to wild-type levels. DeltaH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the DeltaH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled.
• Fane, B., Uchiyama, A., Heiman, P., & Fane, B. A. (2009). N-terminal deletions of the phiX174 external scaffolding protein affect the timing and fidelity of assembly. Virology, 386(2).
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  The first alpha-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5' deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the øX174 genome. Proteins lacking the first seven amino acids were able to rescue a nullD mutant when expressed from a plasmid. However, the lag phase before progeny production was lengthened. The øX174 mutant with the corresponding genomic gene grew very poorly. The molecular basis of the defective phenotype was complex. External scaffolding protein levels were reduced compared to wild-type and most of the viral coat protein in mutant infected cells appears to be siphoned off the assembly pathway. Second-site suppressors of the growth defects were isolated and appear to act via two different mechanisms. One class of suppressors most likely acts by altering mutant external scaffolding protein expression while the second class of suppressors appears to act on the level of protein-protein interactions.
• Ruboyianes, M. V., Chen, M., Dubrava, M. S., Cherwa Jr., J. E., & Fane, B. A. (2009). The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of φX174 replication. Journal of Virology, 83(19), 9952-9956.
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  PMID: 19640994;PMCID: PMC2748053;Abstract: The φX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (ΔH). The expression of the ΔH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory ΔH proteins, restoring both DNA and protein synthesis to wild-type levels. ΔH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the ΔH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Uchiyama, A., Heiman, P., & Fane, B. A. (2009). N-terminal deletions of the øX174 external scaffolding protein affect the timing and fidelity of assembly. Virology, 386(2), 303-309.
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  PMID: 19237183;Abstract: The first α-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5′ deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the øX174 genome. Proteins lacking the first seven amino acids were able to rescue a nullD mutant when expressed from a plasmid. However, the lag phase before progeny production was lengthened. The øX174 mutant with the corresponding genomic gene grew very poorly. The molecular basis of the defective phenotype was complex. External scaffolding protein levels were reduced compared to wild-type and most of the viral coat protein in mutant infected cells appears to be siphoned off the assembly pathway. Second-site suppressors of the growth defects were isolated and appear to act via two different mechanisms. One class of suppressors most likely acts by altering mutant external scaffolding protein expression while the second class of suppressors appears to act on the level of protein-protein interactions. © 2009 Elsevier Inc. All rights reserved.
• Cherwa Jr., J. E., Uchiyama, A., & Fane, B. A. (2008). Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects. Journal of Virology, 82(12), 5774-5780.
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  PMID: 18400861;PMCID: PMC2395140;Abstract: In the φX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D1D 2 and D3D4, in a non-quasi-equivalent structure. To achieve this arrangement, α-helix 3 assumes two different conformations: (i) kinked 30° at glycine residue 61 in subunits D 1 and D3 and (ii) straight in subunits D2 and D4. Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in α-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Fane, B., Cherwa, J. E., Uchiyama, A., & Fane, B. A. (2008). Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects. Journal of virology, 82(12).
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  In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in alpha-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.
• Salim, O., Skilton, R. J., Lambden, P. R., Fane, B. A., & Clarke, I. N. (2008). Behind the chlamydial cloak: The replication cycle of chlamydiaphage Chp2, revealed. Virology, 377(2), 440-445.
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  PMID: 18570973;Abstract: Studying the replication of the chlamydiaphages presents significant challenges. Their host bacteria, chlamydiae, have a unique obligate intracellular developmental cycle. Using qPCR, immunochemistry, and electron microscopy, the life cycle of chlamydiaphage Chp2 was characterised. Chp2 infection has a dramatic inhibitory effect on bacterial cell division. The RB to EB transition is arrested and RBs enlarge without further division. There is a phase of rapid Chp2 genome replication 36 to 48 h post infection that is coincident with the expression of viral proteins and the replication of the host chromosome. The end stage of Chp2 replication is characterised by the appearance of paracrystalline structures followed by bacterial cell lysis. These data indicate that the Chp2 life cycle is closely coordinated with the developmental cycle of its bacterial host. This is a remarkable adaptation by a microvirus to infect and replicate in a bacterial host that has an obligate intracellular developmental cycle. © 2008 Elsevier Inc. All rights reserved.
• Chen, M., Uchiyama, A., & Fane, B. A. (2007). Eliminating the Requirement of an Essential Gene Product in an Already Very Small Virus: Scaffolding Protein B-free øX174, B-free. Journal of Molecular Biology, 373(2), 308-314.
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  PMID: 17825320;Abstract: Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins. © 2007 Elsevier Ltd. All rights reserved.
• Fane, B., Chen, M., Uchiyama, A., & Fane, B. A. (2007). Eliminating the requirement of an essential gene product in an already very small virus: scaffolding protein B-free øX174, B-free. Journal of molecular biology, 373(2).
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  Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.
• Fane, B., Uchiyama, A., Chen, M., & Fane, B. A. (2007). Characterization and function of putative substrate specificity domain in microvirus external scaffolding proteins. Journal of virology, 81(16).
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  Microviruses (canonical members are bacteriophages phiX174, G4, and alpha3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first alpha-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the phiX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that alpha-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction.
• Skilton, R. J., Cutcliffe, L. T., Pickett, M. A., Lambden, P. R., Fane, B. A., & Clarke, I. N. (2007). Intracellular parasitism of chlamydiae: Specific infectivity of chlamydiaphage Chp2 in Chlamydophila abortus. Journal of Bacteriology, 189(13), 4957-4959.
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  PMID: 17468245;PMCID: PMC1913433;Abstract: The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
• Uchiyama, A., Chen, M., & Fane, B. A. (2007). Characterization and function of putative substrate specificity domain in microvirus external scaffolding proteins. Journal of Virology, 81(16), 8587-8592.
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  PMID: 17553892;PMCID: PMC1951351;Abstract: Microviruses (canonical members are bacteriophages φX174, G4, and α3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first α-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the φX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that α-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
• Fane, B. A. (2005). A four-dimensional structure of T4 infection. Nature Structural and Molecular Biology, 12(9), 739-740.
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  PMID: 16142226;Abstract: Biochemical and genetic data defining the assembly pathway and structural biology of the T4 tail apparatus are merging to create a four-dimensional image reconstruction. Human inventions seem to be large-scale replicas of molecular devices honed by evolution. © 2005 Nature Publishing Group.
• Fane, B., Uchiyama, A., & Fane, B. A. (2005). Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging. Journal of virology, 79(11).
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  The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.
• Uchiyama, A., & Fane, B. A. (2005). Identification of an interacting coat-external scaffolding protein domain required for both the initiation of φX174 procapsid morphogenesis and the completion of DNA packaging. Journal of Virology, 79(11), 6751-6756.
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  PMID: 15890913;PMCID: PMC1112155;Abstract: The φX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven α-helices. The protein's core, α-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are φX174, G4, and α3) external scaffolding proteins. On the other hand, the primary sequences of the first α-helices have diverged. The results of previous studies with α3/φX174 chimeric external scaffolding proteins suggest that α-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and αX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. φX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Bernal, R. A., Hafenstein, S., Esmeralda, R., Fane, B. A., & Rossmann, M. G. (2004). The φX174 protein J mediates DNA packaging and viral attachment to host cells. Journal of Molecular Biology, 337(5), 1109-1122.
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  PMID: 15046981;Abstract: Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages φX174, G4, and α3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The φX174 DNA-binding protein, J, is 13 amino acid residues longer than the α3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric φX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric α3 and G4 phages, containing the φX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of α3 packaged with φX174 J protein was determined to 3.5Å resolution and compared with the previously determined structures of φX174 and α3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type α3 virion proteins. The amino-terminal region of the φX174 J protein, which is missing from wild-type α3 virions, is mostly disordered in the α3 chimera. The differences observed between solution properties of wild-type φX174, wild-type α3, and α3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the φX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection. © 2004 Elsevier Ltd. All rights reserved.
• Clarke, I. N., Cutcliffe, L. T., Everson, J. S., Garner, S. A., Lambden, P. R., Pead, P. J., Pickett, M. A., Brentlinger, K. L., & Fane, B. A. (2004). Chlamydiaphage Chp2, a skeleton in the φX174 closet: Scaffolding protein and procapsid identification. Journal of Bacteriology, 186(22), 7571-7574.
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  PMID: 15516569;PMCID: PMC524887;Abstract: Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage φX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.
• Fane, B., Hafenstein, S. L., Chen, M., & Fane, B. A. (2004). Genetic and functional analyses of the øX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains. Virology, 318(1).
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  The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement.
• Fane, B., Novak, C. R., & Fane, B. A. (2004). The functions of the N terminus of the phiX174 internal scaffolding protein, a protein encoded in an overlapping reading frame in a two scaffolding protein system. Journal of molecular biology, 335(1).
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  phiX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5' deleted genes were constructed and the proteins expressed in vivo. The DeltaNH(2) B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.
• Garner, S. A., Everson, J. S., Lambden, P. R., Fane, B. A., & Clarke, I. N. (2004). Isolation, molecular characterisation and genome sequence of a bacteriophage (Chp3) from Chlamydophila pecorum. Virus Genes, 28(2), 207-214.
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  PMID: 14976421;Abstract: Chlamydiae are obligate intracellular pathogens that have a unique developmental cycle. Thirty nine viable isolates representing all nine currently recognised chlamydial species were screened by immunofluorescence with a cross-reacting chlamydiaphage monoclonal antibody. A novel chlamydiaphage (Chp3) was detected in C. pecorum, a chlamydial species not previously known to carry bacteriophages. Chp3 belongs to the Microviridae, members of this virus family are characterised by circular, single-stranded DNA genomes and small T = 1 icosahedral capsids. Double-stranded replicative form Chp3 DNA was purified from elementary bodies and used as a template to determine the complete genome sequence. The genome of Chp3 is 4,554 base pairs and encodes eight open reading frames organised in the same genome structure as other chlamydiaphages. An unrooted phylogenetic tree was constructed based on the major coat proteins of 11 members of the Microviridae and Chp3. This showed that the Microviridae are clearly divided into two discrete sub-families; those that infect the Enterobacteriaceae e.g. ØX174 and the bacteriophages that infect obligate intracellular bacteria or mollicutes including SpV4 (Spiroplasma melliferum), ØMH2K (Bdellovibrio bacteriovorus) and the chlamydiaphages. Comparative analyses demonstrate that the chlamydiaphages can be further subdivided into two groupings, one represented by Chp2/Chp3 and the other by ØCPG1/ØCPAR39.
• Hafenstein, S. L., Chen, M., & Fane, B. A. (2004). Genetic and functional analyses of the øx174 DNA binding protein: The effects of substitutions for amino acid residues that spatially organize the two DNA binding domains. Virology, 318(1), 204-213.
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  PMID: 14972548;Abstract: The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement. © 2003 Elsevier Inc. All rights reserved.
• Morais, M. C., Fisher, M., Kanamaru, S., Przybyla, L., Burgner, J., Fane, B. A., & Rossmann, M. G. (2004). Conformational switching by the scaffolding protein D directs the assembly of bacteriophage φX174. Molecular Cell, 15(6), 991-997.
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  PMID: 15383287;Abstract: The three-dimensional structure of bacteriophage φX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 Å by X-ray crystallography. The crystals belong to space group P41212 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the φX174 procapsid structure. Furthermore, application of the crystallographic 41 symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.
• Novak, C. R., & Fane, B. A. (2004). The functions of the N terminus of the φX174 internal scaffolding protein, a protein encoded in an overlapping reading frame in a two scaffolding protein system. Journal of Molecular Biology, 335(1), 383-390.
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  PMID: 14659765;Abstract: φX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5′ deleted genes were constructed and the proteins expressed in vivo. The ΔNH2 B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins. © 2003 Elsevier Ltd. All rights reserved.
• Bernal, R. A., Hafenstein, S., Olson, N. H., Bowman, V. D., Chipman, P. R., Baker, T. S., Fane, B. A., & Rossmann, M. G. (2003). Structural studies of bacteriophage α3 assembly. Journal of Molecular Biology, 325(1), 11-24.
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  PMID: 12473449;Abstract: Bacteriophage α3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include φX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108 S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The α3 "open" procapsid structural intermediate was determined to 15 Å resolution by cryo-electron microscopy (cryo-EM). Unlike the φX174 "closed" procapsid and the infectious virion, the α3 open procapsid has 30 Å wide pores at the 3-fold vertices and 20 Å wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30 Å wide pores. The structure of the α3 mature virion was solved to 3.5 Å resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the α3 and φX174 virion structures are in the spike and the DNA-binding proteins. The α3 pentameric spikes have a rotation of 3.5° compared to those of φX174. The α3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the φX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in α3 compared to φX174, allowing the building of about 10% of the ribose-phosphate backbone. © 2002 Elsevier Science Ltd. All rights reserved.
• Burch, A. D., & Fane, B. A. (2003). Genetic analyses of putative conformation switching and cross-species inhibitory domains in Microviridae external scaffolding proteins. Virology, 310(1), 64-71.
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  PMID: 12788631;Abstract: Putative conformational switching and inhibitory regions in the Microviridae external scaffolding protein were investigated. Substitutions for glycine 61, hypothesized to promote a postdimerization conformational switch, have dominant lethal phenotypes. In previous studies, chimeric α3/φX174 proteins for structures α-helix 1 and loop 6/α-helix 7 inhibited φX174 morphogenesis when expressed from high copy number plasmids. To determine if inhibition was due to overexpression, chimeric genes were constructed into the φX174 genome. In coinfections with wild-type, protein ratios would be 1:1. The helix 1 chimera has a recessive lethal phenotype; thus, overexpression confers inhibition. In single infections, the mutant cannot form procapsids, suggesting that helix 1 mediates the initial recognition of structural proteins. The lethal chimeric helix 7 protein has a dominant phenotype. Alone, the mutant forms defective procapsids, suggesting a later morphogenetic defect. The results of second-site genetic analyses indicate that the capsid-external scaffolding protein interface is larger than revealed in the crystal structure. © 2003 Elsevier Science (USA). All rights reserved.
• Everson, J. S., Garner, S. A., Lambden, P. R., Fane, B. A., & Clarke, I. N. (2003). Host Range of Chlamydiaphages φCPAR39 and Chp3. Journal of Bacteriology, 185(21), 6490-6492.
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  PMID: 14563888;PMCID: PMC219413;Abstract: The host range of φCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that φCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.
• Fane, B. A., & Prevelige Jr., P. E. (2003). Mechanism of scaffolding-assisted viral assembly. Advances in Protein Chemistry, 64, 259-299.
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  PMID: 13677050;
• Fane, B., Burch, A. D., & Fane, B. A. (2003). Genetic analyses of putative conformation switching and cross-species inhibitory domains in Microviridae external scaffolding proteins. Virology, 310(1).
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  Putative conformational switching and inhibitory regions in the Microviridae external scaffolding protein were investigated. Substitutions for glycine 61, hypothesized to promote a postdimerization conformational switch, have dominant lethal phenotypes. In previous studies, chimeric alpha3/phiX174 proteins for structures alpha-helix 1 and loop 6/alpha-helix 7 inhibited phiX174 morphogenesis when expressed from high copy number plasmids. To determine if inhibition was due to overexpression, chimeric genes were constructed into the phiX174 genome. In coinfections with wild-type, protein ratios would be 1:1. The helix 1 chimera has a recessive lethal phenotype; thus, overexpression confers inhibition. In single infections, the mutant cannot form procapsids, suggesting that helix 1 mediates the initial recognition of structural proteins. The lethal chimeric helix 7 protein has a dominant phenotype. Alone, the mutant forms defective procapsids, suggesting a later morphogenetic defect. The results of second-site genetic analyses indicate that the capsid-external scaffolding protein interface is larger than revealed in the crystal structure.
• Brentlinger, K. L., Hafenstein, S., Novak, C. R., Fane, B. A., Borgon, R., McKenna, R., & Agbandje-McKenna, M. (2002). Microviridae, a family divided: Isolation, characterization, and genome sequence of φMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus. Journal of Bacteriology, 184(4), 1089-1094.
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  PMID: 11807069;PMCID: PMC134817;Abstract: A novel single-stranded DNA phage, φMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage φX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, φMH2K is only distantly related to φX174. Instead, φMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages.
• Everson, J. S., Garner, S. A., Fane, B., Liu, B. -., Lambden, P. R., & Clarke, I. N. (2002). Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus. Journal of Bacteriology, 184(10), 2748-2754.
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  PMID: 11976304;PMCID: PMC135034;Abstract: A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.
• Fane, B., Hafenstein, S., & Fane, B. A. (2002). phi X174 genome-capsid interactions influence the biophysical properties of the virion: evidence for a scaffolding-like function for the genome during the final stages of morphogenesis. Journal of virology, 76(11).
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  During the final stages of phi X174 morphogenesis, there is an 8.5-A radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, phi X174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the phi X174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition.
• Hafenstein, S., & Fane, B. A. (2002). φX174 genome-capsid interactions influence the biophysical properties of the virion: Evidence for a scaffolding-like function for the genome during the final stages of morphogenesis. Journal of Virology, 76(11), 5350-5356.
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  PMID: 11991963;PMCID: PMC137031;Abstract: During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to-virion transition.
• Valentine, C. R., Montgomery, B. A., Miller, S. G., Delongchamp, R. R., Fane, B. A., & Malling, H. V. (2002). Characterization of mutant spectra generated by a forward mutational assay for gene A of ΦX174 from ENU-treated transgenic mouse embryonic cell line PX-2. Environmental and Molecular Mutagenesis, 39(1), 55-68.
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  PMID: 11813297;Abstract: The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for ΦX174 that requires on altered, functionol ΦX174 protein and therefore should have fewer torgets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting o known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in ΦX174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for ΦX174 am3, cs70 (line 54). All six types of base-pair substitution were represented omong both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 μg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R2 = 0.79), with a ninefold increase in mutant frequency at the 400 μg/ml dose. The spontaneous mutant frequency was 1.9 × 10-5 and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 torgets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treotment (P < 0.0001) and 15 showed significant increases. The results suggest that the ΦX174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.
• Burch, A. D., & Fane, B. A. (2000). Efficient complementation by chimeric Microviridae internal scaffolding proteins is a function of the COOH-terminus of the encoded protein. Virology, 270(2), 286-290.
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  PMID: 10792987;Abstract: Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and external species. Both structural and genetic analyses suggest that the COOH-terminus of the internal protein is critical for coat protein recognition and specificity. To test this hypothesis, chimeric internal scaffolding genes between Microviridae members φX174, G4, and α3 were constructed and the proteins expressed in vivo. All of the chimetic proteins were functional in complementation assays. However, the efficient complementation was observed only when the vital coat protein and COOH- terminus of internal scaffolding were of the same origin. Genes with 5' deletions of the φX174 internal scaffolding gene were also constructed and expressed in vivo. Proteins lacking the first 10 amino acids, which self- associate across the twofold axes of symmetry in the atomic structure, efficiently complement φX174 am(B) mutants at temperatures above 24°C. These results suggest that internal scaffolding protein self-associations across the twofold axes of symmetry are required only at lower temperatures. (C) 2000 Academic Press.
• Burch, A. D., & Fane, B. A. (2000). Foreign and chimeric external scaffolding proteins as inhibitors of Microviridae morphogenesis. Journal of Virology, 74(20), 9347-9352.
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  PMID: 11000202;PMCID: PMC112362;Abstract: Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are φX174, G4, and α3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD(r)) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD(r) mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.
• Liu, B. L., Everson, J. S., Fane, B., Giannikopoulou, P., Vretou, E., Lambden, P. R., & Clarke, I. N. (2000). Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. Journal of Virology, 74(8), 3464-3469.
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  PMID: 10729119;PMCID: PMC111853;Abstract: Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the ΦX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.
• Burch, A. D., Josephine, T. a., & Fane, B. A. (1999). Cross-functional analysis of the Microviridae internal scaffolding protein. Journal of Molecular Biology, 286(1), 95-104.
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  PMID: 9931252;Abstract: The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (∅X174, G4 and α3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The ∅X174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21°C. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the ∅X174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.
• Dokland, T., Bernal, R. A., Burch, A., Pletnev, S., Fane, B. A., & Rossmann, M. G. (1999). The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus φX174. Journal of Molecular Biology, 288(4), 595-608.
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  PMID: 10329166;Abstract: An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage φX174. Assembly of the φX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a 'closed' procapsid particle has been determined to 3.5 Å resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 Å resolution electron microscopic reconstruction of the 'open' procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly α-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.
• Rossmann, M. G., Dokland, T., Bemal, R., McKenna, R., Dag, L. L., & Fane, B. A. (1998). Structure of a viral procapsid with molecular scaffolding. FASEB Journal, 12(8), A1332.
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  Abstract: Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Eschcrichia coli bacteriophage 4X174. The XI74 virion contains the proteins F, G, H and I. During assembly, two scaffolding proteins B and D are required for the formation of a 108S, 360-A diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturadon involves packaging of DNA andJ proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel -sandwich motif common to many plant and animal viruses. The structure of a procapsid-like particle at 3.5 A resolution will be described, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and the F protein undergoes conformational changes during capsid maturation.
• Dokland, T., McKenna, R., Llag, L. L., Bowman, B. R., Incardona, N. L., Fane, B. A., & Rossmann, M. G. (1997). Structure of a viral procapsid with molecular scaffolding. Nature, 389(6648), 308-313.
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  PMID: 9305849;Abstract: The assembly of a macromolecular structure proceeds along an ordered morphogenetic pathway, and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Escherichia coil bacteriophage ΦX174 (refs 6, 7). The ΦX174 virion contains the proteins F, G, H and J. During assembly, two scaffolding proteins B and D are required for the formation of a 108S, 360-Å-diameter procapsid from pentameric precursors containing the F, G and H protein. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturation involves packaging of DNA and J proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel β-sandwich motif common to many plant and animal viruses. Here we describe the structure of a procapsid-like particle at 3.5-Å resolution, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and showing that the F protein undergoes conformational changes during capsid maturation.
• Jennings, B., & Fane, B. A. (1997). Genetic analysis of the Φ174 DNA binding protein. Virology, 227(2), 370-377.
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  PMID: 9018136;Abstract: The X174 J protein is 37 amino acids in length and contains 12 basic residues. There are no acidic amino acids in the protein. The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region. To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted. Lysine→leucine and arginine→leucine substitutions were generated within the basic amino acid clusters. At least three substitutions were required to eliminate viability in vivo. Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly. In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced. Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid. The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function.
• McKenna, R., Bowman, B. R., Ilag, L. L., Rossmann, M. G., & Fane, B. A. (1996). Atomic structure of the degraded procapsid particle of the bacteriophage G4: Induced structural changes in the presence of calcium ions and functional implications. Journal of Molecular Biology, 256(4), 736-750.
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  PMID: 8642594;Abstract: Bacteriophage G4 and ∅X174 are members of the Microviridae family. The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein. The atomic structure of the ∅X174 virion had previously been determined by X-ray crystallography. Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization. However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions. The structure of the degraded G4 procapsid has been determined to 3.0 Å resolution. The particles crystallized in the hexagonal space group P6322 with unit cell dimensions a = b = 414.2(5) Å and c = 263.0(3) Å. The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography. Packing considerations indicated there were two particles per unit cell. A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell. Initial phases were calculated to 6 Å resolution, based on the known ∅X174 virion model. Phase information was then extended in steps to 3.0 Å resolution by molecular replacement electron density modification and particle envelope generation. The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of ∅X174. In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence. Redetermining the amino acid sequence confirmed that the density was correct. The r.m.s. deviation between the C(α) backbone of the mature capsid of ∅X174 and the degraded G4 procapsid was 0.36 Å for the F protein and 1.38 Å for the G protein. This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family. Functionally important features between ∅X174 and G4 had greater conservation. Calcium ions (Ca2+) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in ∅X174. Binding of Ca2+ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA.
• Ekechukwu, M. C., & Fane, B. A. (1995). Characterization of the morphogenetic defects conferred by cold-sensitive prohead accessory and scaffolding proteins of φX174. Journal of Bacteriology, 177(3), 829-830.
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  PMID: 7836321;PMCID: PMC176665;Abstract: The morphogenetic defects conferred by the cold-sensitive prohead accessory and scaffolding proteins of φX174 were determined in vivo. The results suggest that the cold-sensitive prohead accessory protein blocks the formation of the 12S assembly intermediate. The cold-sensitive scaffolding protein most likely affects the stability of the prohead.
• Ekechukwu, M. C., Oberste, D. J., & Fane, B. A. (1995). Host and φX 174 mutations affecting the morphogenesis or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate. Genetics, 140(4), 1167-1174.
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  PMID: 7498760;PMCID: PMC1206684;Abstract: The morphogenetic pathway of bacteriophage φX 174 was investigated in rep mutant hosts that specifically block stage III single-stranded DNA synthesis. The defects conferred by the mutant rep protein most likely affect the formation or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate, which consists of a viral prohead and a DNA replicating intermediate (preinitiation complex). φX 174 mutants, ogr (rep), which restore the ability to propagate in the mutant rep hosts, were isolated. The ogr(rep) mutations confer amino acid substitutions in the viral coat protein, a constituent of the prohead, and the viral A protein, a constituent of the preinitiation complex. Four of the six coat protein substitutions are localized on or near the twofold axis of symmetry in the atomic structure of the mature virion.
• Fane, B. A., Shien, S., & Hayashi, M. (1993). Second-site suppressors of a cold-sensitive external scaffolding protein of bacteriophage φX174. Genetics, 134(4), 1003-1011.
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  PMID: 8375644;PMCID: PMC1205568;Abstract: This report describes the isolation and characterization of second-site suppressors of a cold-sensitive (cs) external scaffolding protein, gpD, of bacteriophage φX174. Seven genetically distinct suppressors were isolated. Six of them are located in gene F which encodes the major coat protein of the virus. The seventh is located in gene J which encodes the DNA-binding protein. A subset of the suppressors are trans-acting. These second-site suppressors do not exhibit allele specificity; they are able to suppress defects associated with a csD protein for which they were not selected. The initial characterization of the second-site suppressors and their locations within the major coat protein suggest that the mechanism of suppression may involve both structural and stoichiometric phenomena.
• Dalphin, M. E., Fane, B. A., Skidmore, M. O., & Hayashi, M. (1992). Proteolysis of bacteriophage φX174 prohead accessory protein gpB by Escherichia coli OmpT protease is not essential for phage maturation in vivo. Journal of Bacteriology, 174(7), 2404-2406.
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  PMID: 1532389;PMCID: PMC205867;Abstract: To examine whether cleavage of the φX174 prohead accessory protein, gpB, by the OmpT protease is required for phage development in vivo, a phage mutant lacking the OmpT cleavage site and an Escherichia coli C ΔompT strain were constructed. The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for φX174 development.
• Fane, B. A., Head, S., & Hayashi, M. (1992). Functional relationship between the J proteins of bacteriophages φX174 and G4 during phage morphogenesis. Journal of Bacteriology, 174(8), 2717-2719.
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  PMID: 1532571;PMCID: PMC205913;Abstract: The functions of the small DNA-binding protein, gpJ, of bacteriophages φX174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation. The morphogenetic roles of the two proteins may differ. The φX174 J protein may be required for the formation or stabilization of the φX174 prohead.
• Fane, B. A., & Hayashi, M. (1991). Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage ΦX174. Genetics, 128(4), 663-671.
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  PMID: 1833267;PMCID: PMC1204541;Abstract: This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage ΦX174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type ΦX174 control.
• Fane, B., & King, J. (1991). Intragenic suppressors of folding defects in the P22 tailspike protein. Genetics, 127(2), 263-277.
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  PMID: 1825987;PMCID: PMC1204354;Abstract: Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.
• Fane, B., Villafane, R., Mitraki, A., & King, J. (1991). Identification of global suppressors for temperature-sensitive folding mutations of the P22 tailspike protein. Journal of Biological Chemistry, 266(18), 11640-11648.
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  PMID: 1828803;Abstract: Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84>A, V331>A, and A334>V. The V331>A or A334>V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331>A and A334>V mutations were crossed into well-characterized temperature-sensitive folding (tsf) mutants at various sites in the tailspike protein, they suppressed all of the eight tsf mutants tested. Since the tsf defects destabilize folding intermediates rather than the native conformation, this result implies that the suppressors act in the folding pathway. Strains carrying the isolated suppressor mutations displayed no obvious phenotypic defect and formed native biologically active tailspikes. Thus, these single amino acid substitutions have striking influences on the efficiency of intracellular chain folding, without causing functional defects in the native protein.
• Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J., & King, J. (1991). Global suppression of protein folding defects and inclusion body formation. Science, 253(5015), 54-58.
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  PMID: 1648264;Abstract: Amino acid substitutions at a site in the center of the bacteriophage protein P22 tailspike polypeptide chain suppress temperature-sensitive folding mutations at many sites throughout the chain. Characterization of the intracellular folding and chain assembly process reveals that the suppressors act in the folding pathway, inhibiting the aggregation of an early folding intermediate into the kinetically trapped inclusion body state. The suppressors alone increase the folding efficiency of the otherwise wild-type polypeptide chain without altering the stability or activity of the native state. These amino acid substitutions identify an unexpected aspect of the protein folding grammar-sequences within the chain that carry information inhibiting unproductive off-pathway conformations. Such mutations may serve to increase the recovery of protein products of cloned genes.
• Fane, B., & King, J. (1987). Identification of sites influencing the folding and subunit assembly of the P22 tailspike polypeptide chain using nonsense mutations.. Genetics, 117(2), 157-171.
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  PMID: 2822533;PMCID: PMC1203193;Abstract: Amber mutations have been isolated and mapped to more than 60 sites in gene 9 of P22 encoding the thermostable phage tailspike protein. Gene 9 is the locus of over 30 sites of temperature sensitive folding (tsf) mutations, which affect intermediates in the chain folding and subunit association pathway. The phenotypes of the amber missense proteins produced on tRNA suppressor hosts inserting serine, glutamine, tryosine and leucine have been determined at different temperatures. Thirty-three of the sites are tolerant, producing functional proteins with any of the four amino acids inserted at the sites, independent of temperature. Tolerant sites are concentrated at the N-terminal end of the protein indicating that this region is not critical for conformation or function. Sixteen of the sites yield temperature sensitive missense proteins on at least one nonsense suppressing host. Most of the sites with ts phenotypes map to the central region of the gene which is also the region where most of the tsf mutations map. Mutations at 15 of the sites have a lethal phenotype on at least one tRNA suppressor host. For nine out of ten sites tested with at least one lethal phenotype, the primary defect was in the folding or subunit association of the missense polypeptide chain. This analysis of the tailspike missense proteins distinguishes three classes of amino acid sites in the polypeptide chain; residues whose side chains contribute little to folding, subunit assembly or function; residues critical for maintaining the folding and subunit assembly pathway at the high end of the temperature range of phage growth; and residues critical over the entire temperature range of growth.

Presentations

• Brown, L. T., & Fane, B. A. (2023). Mutational analysis of a coat protein DNA binding pocket in a single-stranded DNA virus.. XXVIII International virus and phage assembly meeting.
• Fane, B. A. (2023). Relaxed parental guidance while packaging a rebellious single-stranded DNA genome. XXVIII Internation virus and phage assembly meeting. Shrigley Hall, Macclesfield UK.
• Ogunbunmi, E. T., & Fane, B. A. (2023). Cis-acting elements covertly regulating single-stranded DNA packaging. XXVIII International virus and phage assembly meeting. Shrigley Hall, Macclesfield UK.
• Fane, B. A. (2022, June). Single-stranded DNA packaging, temperature and Microviridae diversification: parental guidance and self image can curve DNA delinquency.. Virus Structure and Assembly (invited speaker/plenary talk). Southbridge MA: FASEB.
• Fane, B. A. (2022, September). Single-stranded DNA deliquency: causes and solutions (invited/plenary). International Symposium on single-stranded DNA viruses. Sète, Occitanie, France: Agropolis Foundation.
• Ogunbunmi, E. T., & Fane, B. A. (2022, September). The organization and chemical nature of the genome can affect ssDNA packaging and post-packaging phenomena. International Symposium on single-stranded DNA viruses. Sète, Occitanie, France.
• Fane, B. A. (2021, October). SIngle-stranded DNA Delinquency: causes and solutions. Dr. Michael Rossmann Symposium. Purdue University: Purdue University.
• Fane, B. A. (2019, July). The highly conserved yet unessential A* protein operates during packaging to ensure infectivity.. XXVI Biennial Conference on Phage/Virus Assembly.. Brainerd, Minnesota..
• Fane, B. A. (2019, March). Translocating single-stranded DNA in and out of icosahedral symmetry: conduits, energy, and at last, 50 years after the protein was identified, a role befitting Astar. 10th Workshop in Virus Evolution.
• Fane, B. A. (2019, May). Structural, biochemical and genetic analyses of viral host range and DNA translocation: the ephemeral intimacy of an insidious assignation.. Gordon Conference on Cells and Viruses. Barga, Italy.
• Roznowski, A. P., & Fane, B. A. (2019, July). Mutagenic Analysis of a DNA Translocating Tube’s Interior Surface. XXVI Biennial Conference on Phage/Virus Assembly..
• Fane, B. A. (2018, June). Structural changes of tailless microviruses during penetration of bacterial cell walls and their relationship to host cell tropism (plenary talk). FASEB Conference on Virus Structure and Assembly. Steamboat Springs Colorado: FASEB.
• Blackburn, B. J., Li, S., Roznowski, A. P., Perez, A., Villarreal, R., Johnson, C., Hardy, M., Tuckerman, E. C., Burch, A. D., & Fane, B. A. (2017, August). Altering the flux of the assembly intermediates through the øX174 assembly pathway. XXV International Phage and Virus Assembly Meeting. Ellicot City, MD: XXV International Phage and Virus Assembly Meeting.
• Doore, S. M., & Fane, B. A. (2017, March). The Packaging Paradox and the Evolution of Genome Size in Microviruses. Ninth biennial Workshop in Virus Evolution. College Park, PA: Pennsylvania State University.
• Roznowski, A. P., & Fane, B. A. (2017, August). Mutagenic analysis of a DNA translocating tube's interior surface. XXV International Phage and Virus Assembly Meeting. Ellicot City, MD: XXV International Phage and Virus Assembly Meeting.
• Tokuda, J. M., Mauney, A. W., Pollack, L., Budko, S. P., Fokine, A., Klose, T., Sun, L., Sun, Y., Zhang, X., Zbornik, E., Rossmann, M. G., Roznowski, A. P., Young, L. N., Moulineux, I. J., & Fane, B. A. (2017, September). The ephemeral intimacy of an insidious assignation in shockingly graphic atomic detail. A Celebration of Structural Biology Honoring Professor Michael G. Rossmann. Purdue University: Purdue University.
• Cherwa, J. E., Bedwell, G., Brooke, D., Tyson, J. D., Dokland, T., Prevelige, P. E., & Fane, B. A. (2016, July). Biochemical Analysis of a Dominant Lethal Scaffolding Protein and Evolutionary Mechanisms of Resistance. FASEB Virus Structure and Assembly Meeting. Steamboat Springs Colorado: FASEB.
• Fane, B. A. (2015, June). Presentations by graduate students aaron Roznowski and post doc Sarah Doore, who won award for her talk.. XXIV International Phage and Virus Assembly Meeting. Les Diabrelets, Switzerland: XXIV International Phage and Virus Assembly Meeting.
• Fane, B. A. (2015, June). Structure-function of a virally encoded DNA translocating conduit.. Plenary Talk at the American Society for Microbiology (ASM) General Meeting. New Orleans: American Society for Microbiology.
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  Plenary Talk
• Fane, B. A. (2015, March). The ephemeral intimacy of an insidious assignation, in shockingly graphic atomic detail.. Departmental Seminar Speaker, Biology, Washington State University. Pullman, Washington: Washingston State University.
• Fane, B. A. (2014, January). Atomic Structure and Function of a novel viral DNA Delivering Device. Seminar, University of Missouri-Kansas City, School of Biological Sciences Seminar Series.
• Doore, S. M., & Fane, B. A. (2013, Fall). Particle Morphogenesis: the major barrier to horizontal gene exchange in microviruses. XXIII Virus Assembly Meeting. Lake Arrowhead, CA: XXIII Virus Assembly Meeting.
• Doore, S. M., & Fane, B. A. (2013, Fall). Regulation the switch between dsDNA and ssDNA biosynthesis.. XXIII Virus Assembly Meeting. Lake Arrowhead, CA: XXIII Virus Assembly Meeting.
• Doore, S. M., & Fane, B. A. (2013, Spring). Temporally temperamental protein-protein interactions after a horizontal gene tranfer. International Workshop on Virus Evolution. Pennsylvania State University: International Workshop on Virus Evolution.
• Fane, B. A. (2013, Fall). Travel Accessories For the Strictly Icosahedral: Atomic Structure and Function of DNA translocating conduit.. Invited Seminar at Michigan State University. Michigan State University: Department of Biochemistry.
• Fane, B. A. (2013, Spring). Atomic structure of DNA translocating conduit. International Workshop on Virus Evolution. Pennsylvania State University: International Workshop on Virus Evolution.
• Roznowski, A. P., & Fane, B. A. (2013, Fall). Structure-function analysis of the phiX174 DNA pilot protein, prefabricated on-site tail assembly. XXIII Virus Assembly Meeting. Lake Arrowhead, CA: XXIII Virus Assembly Meeting.
• Fane, B. A. (2012, June). FASEB Conference on Virus Structure and Assembly. Saxton River, Vermont.
• Fane, B. A. (2011, March). After desolate decades, procapsids produced a plenty in vitroInstitute for Bioinformatics and Evolutionary Studies.
• Fane, B. A. (2011, October). The evolution of resistance to an inhibitor of virus assembly: a poem of poison, passion and pick-ups. Tucson, AZ: Department of Immunobiology, University of Arizona.

Poster Presentations

• Brown, L. T., & Fane, B. A. (2024). Mutational analysis of a coat protein DNA bidning pocket in a single-stranded DNA virus.. FASEB conference on Virus Structure and Assembly. Southbridge MA: FASEB.
• Love, S. D., Ogunbunmi, E. T., & Fane, B. A. (2024). In the dark, dank doom of DNA packaging, A star bedazzles: The A* protein and its DNA target sites orchestrate single-stranded DNA packaging.
  

   

  . FASEB Conference on Virus Structure and Assembly. Southbridge MA: FASEB.
• Ogunbunmi, E. T., & Fane, B. A. (2022, June). The organization and chemical nature of the genome can affect single-stranded DNA packaging and post-packaging phenomena. Virus Structure and Assembly. Southbridge MA: FASEB.
• Fane, B. A., & Roznowski, A. P. (2018, June). Mutagenic Analysis of a DNA Translocating Tube's Interior Surface. FASEB Conference on Virus Structure and Assembly. Steamboat Springs Colorado: FASEB.
• Doore, S. M., & Fane, B. A. (2016, July). Procapsid Permanence, DNA dimensions, and genome grappling during microvirus packaging. FASEB Virus Structure and Assembly Meeting. Steamboat Springs Colorado: FASEB.
• Roznowski, A. P., & Fane, B. A. (2016, July). Structure-Function Analysis of the phiX174 DNA Piloting Protein using Length Altering Mutations. FASEB Virus Structure and Assembly Meeting. Steamboat Springs Colorado: FASEB.
• Fane, B. A. (2014, June). Poster presentations by two graduate students, Sarah Doore and Aaron Roznowski.. FASEB International Meeting on Virus Structure and Assembly. Saxton's River Vermont: FASEB.
  More info

  Sarah Doores abstract was also selected for a graduate student short talk.
• Fane, B. A., & Doore, S. (2012, June). Temporally temperamental protein-protein interactions as a barrier to productive gene exchang. Not Provided in APROL. Saxton.

Reviews

• Fane, B. A., & Roznowski, A. P. (2019. Icosahedral Phages - ssDNA(pp doi.org/10.1016/B978-0-12-809633-8.20944-8). https://www.sciencedirect.com/science/article/pii/B9780128096338209448?via%3Dihub.