Samuel K Campos
- Assistant Professor, Immunobiology
- Assistant Professor
- Assistant Professor, Cancer Biology - GIDP
My research focuses on the molecular mechanisms of Human Papillomavirus- host cell interactions, including entry, subcellular trafficking & transport, and evasion/antagonism of innate immune sensory pathways during infection.
- Ph.D. Biochemistry & Cell Biology
- Rice University, Houston, Texas, United States
- Metabolic biotinylation of the adenoviral capsid: Avidin-based applications and studies of ligand-targeted gene delivery
- B.S. Biology
- Virginia Polytechnic Institute & State University, Blacksburg, Virginia, United States
- Assistant Professor, Immunobiology, University of Arizona (2011 - Ongoing)
- Assistant Professor, Molecular & Cellular Biology, University of Arizona, Tucson, Arizona (2011 - Ongoing)
- Research Assistant Professor, BIO5 Institute, University of Arizona, Tucson, Arizona (2008 - 2011)
- Postdoctoral Fellow, Molecular Genetics & Microbiology, University of New Mexico, Albuquerque, New Mexico (2005 - 2008)
Virology, Papillomavirus, Cell Biology, Molecular Biology, Biochemistry, Protein Structure, Structural Biology, Protein-Membrane Interactions
CBIO GIDP Seminar SeriesCBIO 596H (Fall 2016)
Cancer BiologyCBIO 552 (Fall 2016)
DissertationIMB 920 (Fall 2016)
Honors Independent StudyPSIO 499H (Fall 2016)
Introduction to ResearchMCB 795A (Fall 2016)
ResearchCBIO 900 (Fall 2016)
Research ConferenceCBIO 695A (Fall 2016)
ThesisCMM 910 (Summer I 2016)
DissertationIMB 920 (Spring 2016)
Introduction to ResearchMCB 795A (Spring 2016)
Prins+Molec MechanismsIMB 565 (Spring 2016)
Cancer BiologyCBIO 552 (Fall 2015)
DissertationIMB 920 (Fall 2015)
ResearchIMB 900 (Fall 2015)
Directed RsrchMCB 392 (Spring 2015)
DissertationIMB 920 (Spring 2015)
Honors Independent StudyMCB 499H (Spring 2015)
Introduction to ResearchMCB 795A (Spring 2015)
Prins+Molec MechanismsIMB 565 (Spring 2015)
ResearchIMB 900 (Spring 2015)
Cancer BiologyCBIO 552 (Fall 2014)
Directed ResearchBIOC 392 (Fall 2014)
DissertationIMB 920 (Fall 2014)
Honors Independent StudyMCB 399H (Fall 2014)
ResearchIMB 900 (Fall 2014)
Directed RsrchMCB 492 (Spring 2014)
Honors Independent StudyECOL 399H (Spring 2014)
ResearchIMB 900 (Spring 2014)
Directed RsrchMCB 492 (Fall 2013)
ResearchIMB 900 (Fall 2013)
- Bronnimann, M. P., Calton, C. M., Chiquette, S. F., Li, S., Lu, M., Chapman, J. A., Bratton, K. N., Schlegel, A. M., & Campos, S. K. (2016). Furin Cleavage of L2 During Papillomavirus Infection: Minimal Dependence on Cyclophilins. Journal of virology.More infoDespite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during Human Papillomavirus Type 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N-terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N-terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurs on both the HaCaT cell surface and ECM. Contrary to current models, experiments on the involvement of cyclophilins revealed little if any role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12 as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues reveal that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.
- Christakos, K. J., Chapman, J. A., Fane, B. A., & Campos, S. K. (2015). PhiXing-it, displaying foreign peptides on bacteriophage ΦX174. Virology, 488, 242-248.More infoAlthough 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.
- Campos, S., Campos, S., Bronnimann, M. P., Bronnimann, M. P., Chapman, J. A., Chapman, J. A., Park, C. K., Park, C. K., Campos, S. K., & Campos, S. K. (2013). A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection. Journal of virology, 87(1).More infoDuring cellular invasion, human papillomavirus type 16 (HPV16) must transfer its viral genome (vDNA) across the endosomal membrane prior to its accumulation at nuclear PML bodies for the establishment of infection. After cellular uptake, the capsid likely undergoes pH-dependent disassembly within the endo-/lysosomal compartment, thereby exposing hidden domains in L2 that facilitate membrane penetration of L2/vDNA complexes. In an effort to identify regions of L2 that might physically interact with membranes, we have subjected the L2 sequence to multiple transmembrane (TM) domain prediction algorithms. Here, we describe a conserved TM domain within L2 (residues 45 to 67) and investigate its role in HPV16 infection. In vitro, the predicted TM domain adopts an alpha-helical structure in lipid environments and can function as a real TM domain, although not as efficiently as the bona fide TM domain of PDGFR. An L2 double point mutant renders the TM domain nonfunctional and blocks HPV16 infection by preventing endosomal translocation of vDNA. The TM domain contains three highly conserved GxxxG motifs. These motifs can facilitate homotypic and heterotypic interactions between TM helices, activities that may be important for vDNA translocation. Disruption of some of these GxxxG motifs resulted in noninfectious viruses, indicating a critical role in infection. Using a ToxR-based homo-oligomerization assay, we show a propensity for this TM domain to self-associate in a GxxxG-dependent manner. These data suggest an important role for the self-associating L2 TM domain and the conserved GxxxG motifs in the transfer of vDNA across the endo-/lysosomal membrane.
- Campos, S., Campos, S., Calton, C. M., Calton, C. M., Schlegel, A. M., Schlegel, A. M., Chapman, J. A., Chapman, J. A., Campos, S. K., & Campos, S. K. (2013). Human papillomavirus type 16 does not require cathepsin L or B for infection. The Journal of general virology, 94(Pt 8).More infoCathepsin L (CatL) and cathepsin B (CatB) are lysosomal proteases that many viruses utilize for capsid disassembly. We tested whether CatL and CatB are required for infection by human papillomavirus type 16 (HPV16). CatL- and CatB-deficient mouse embryonic fibroblasts had higher levels of infection when compared with wild-type cells. Similar results were obtained in HaCaT keratinocytes treated with CatL- or CatB-specific small interfering RNA. Thus, CatL and CatB are not required for HPV16 infection but instead appear to restrict infection.
- Bergant Marušič, M., Ozbun, M. A., Campos, S. K., Myers, M. P., & Banks, L. (2012). Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17. Traffic (Copenhagen, Denmark), 13(3), 455-67.More infoThe human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection, but the molecular mechanisms underlying its mode of action remain obscure. Using a proteomic approach, we have identified the adaptor protein, sorting nexin 17 (SNX17) as a strong interacting partner of HPV L2. This interaction occurs through a highly conserved SNX17 consensus binding motif, which is present in the majority of HPV L2 proteins analysed. Using mutants of L2 defective for SNX17 interaction, or siRNA ablation of SNX17 expression, we demonstrate that the interaction between L2 and SNX17 is essential for viral infection. Furthermore, loss of the L2-SNX17 interaction results in enhanced turnover of the L2 protein and decreased stability of the viral capsids, and concomitantly, there is a dramatic decrease in the efficiency with which viral genomes transit to the nucleus. Indeed, using a range of endosomal and lysosomal markers, we show that capsids defective in their capacity to bind SNX17 transit much more rapidly to the lysosomal compartment. These results demonstrate that the L2-SNX17 interaction is essential for viral infection and facilitates the escape of the L2-DNA complex from the late endosomal/lysosomal compartments.
- Campos, S. K., Chapman, J. A., Deymier, M. J., Bronnimann, M. P., & Ozbun, M. A. (2012). Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration. Journal of virology, 86(8), 4169-81.More infoCell invasion by human papillomavirus type 16 (HPV16) is a complex process relying on multiple host cell factors. Here we describe an investigation into the role of cellular protein disulfide isomerases (PDIs) by studying the effects of the commonly used PDI inhibitor bacitracin on HPV16 infection. Bacitracin caused an unusual time-dependent opposing effect on viral infection. Enhanced cellular binding and entry were observed at early times of infection, while inhibition was observed at later times postentry. Bacitracin was rapidly taken up by host cells and colocalized with HPV16 at late times of infection. Bacitracin had no deleterious effect on HPV16 entry, capsid disassembly, exposure of L1/L2 epitopes, or lysosomal trafficking but caused a stark inhibition of L2/viral DNA (vDNA) endosomal penetration and accumulation at nuclear PML bodies. γ-Secretase has recently been implicated in the endosomal penetration of L2/vDNA, but bacitracin had no effect on γ-secretase activity, indicating that blockage of this step occurs through a γ-secretase-independent mechanism. Transient treatment with the reductant β-mercaptoethanol (β-ME) was able to partially rescue the virus from bacitracin, suggesting the involvement of a cellular reductase activity in HPV16 infection. Small interfering RNA (siRNA) knockdown of cellular PDI and the related PDI family members ERp57 and ERp72 reveals a potential role for PDI and ERp72 in HPV infection.
- Frietze, K. M., Campos, S. K., & Kajon, A. E. (2012). No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection. BMC research notes, 5, 429.More infoSubspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.
- Frietze, K. M., Campos, S. K., & Kajon, A. E. (2010). Open reading frame E3-10.9K of subspecies B1 human adenoviruses encodes a family of late orthologous proteins that vary in their predicted structural features and subcellular localization. Journal of virology, 84(21), 11310-22.More infoSubspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism.
- Campos, S. K., & Ozbun, M. A. (2009). Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes. PloS one, 4(2), e4463.More infoMinor capsid protein L2 performs an indispensable but uncharacterized role in human papillomavirus infections. A neutralizing B cell epitope has recently been mapped to the N-terminus of HPV16 L2, residues 17-36, and exposure of this region of L2 has been implicated in translocation of incoming virions from the endo/lysosomal compartment to the cellular cytoplasm. Here we examine the redox state of Cys22 and Cys28 two highly conserved cysteines located within this epitope. We also investigate the infectivity of virions containing L2 single and double cysteine point mutants.
- Smith, J. L., Campos, S. K., Wandinger-Ness, A., & Ozbun, M. A. (2008). Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating. Journal of virology, 82(19), 9505-12.More infoHigh-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.
- Campos, S. K., & Barry, M. A. (2007). Current advances and future challenges in Adenoviral vector biology and targeting. Current gene therapy, 7(3), 189-204.More infoGene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting.
- Smith, J. L., Campos, S. K., & Ozbun, M. A. (2007). Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes. Journal of virology, 81(18), 9922-31.More infoPapillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.
- Wu, Y., Campos, S. K., Lopez, G. P., Ozbun, M. A., Sklar, L. A., & Buranda, T. (2007). The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy. Analytical biochemistry, 364(2), 180-92.More infoThe use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.
- Campos, S. K., & Barry, M. A. (2006). Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting. Virology, 349(2), 453-62.More infoThe direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.
- Marsh, M. P., Campos, S. K., Baker, M. L., Chen, C. Y., Chiu, W., & Barry, M. A. (2006). Cryoelectron microscopy of protein IX-modified adenoviruses suggests a new position for the C terminus of protein IX. Journal of virology, 80(23), 11881-6.More infoRecombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.
- Campos, S. K., & Barry, M. A. (2004). Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination. Human gene therapy, 15(11), 1125-30.More infoThere are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.
- Campos, S. K., Parrott, M. B., & Barry, M. A. (2004). Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector. Molecular therapy : the journal of the American Society of Gene Therapy, 9(6), 942-54.More infoWhile genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.
- Barry, M. A., Campos, S. K., Ghosh, D., Adams, K. E., Mok, H., Mercier, G. T., & Parrott, M. B. (2003). Biotinylated gene therapy vectors. Expert opinion on biological therapy, 3(6), 925-40.More infoThe avidin-biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.
- Parrott, M. B., Adams, K. E., Mercier, G. T., Mok, H., Campos, S. K., & Barry, M. A. (2003). Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification. Molecular therapy : the journal of the American Society of Gene Therapy, 8(4), 688-700.More infoDevelopment of cell-targeting vectors is an important focus for gene therapy. While some ligands can be genetically inserted into virus capsid proteins for cell targeting, for many ligands, this approach can disrupt either ligand function or vector function. To address this problem for adenovirus type 5 vectors, the fiber capsid protein was genetically fused to a biotin acceptor peptide (BAP). Adenovirus particles bearing this BAP were metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells to produce covalently biotinylated virions. The resulting biotinylated vector could be retargeted to new receptors by conjugation to biotinylated antibodies using tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). Finally, this vector was used as a ligand screening platform for dendritic cells in which a variety of structurally diverse protein, carbohydrate, and nucleic acid ligands were easily added to the vector using the biotin-avidin interaction. This work demonstrates the utility of metabolically biotinylated viruses for ligand screening, vector targeting, and virus purification applications.
- Campos, S. K. (2016, July 20). Spanning and Penetration of the Limiting Membrane by the L2 Capsid Protein. DNA Tumor Virus Meeting. Montreal, Quebec.
- Bronnimann, M. P., Williamson, T., Berumen, M., Calton, C. M., & Campos, S. K. (2015, July). Furin Cleavage Facilitates the Cytoplasmic Translocation and Oligomerization of the Human Papillomavirus Minor Capsid Protein L2. ICGEB DNA Tumor Virus Meeting. Trieste, Italy: International Centre for Genetic Engineering and Biotechnology.More infoPoster Presentation
- Calton, C. M., Bronnimann, M. P., Chapman, J. A., Berumen, M., Williamson, T., Manson, A., & Campos, S. K. (2015, July). A Role for Mitosis in the Membrane Translocation of the HPV16 L2/Genome Complex. ICGEB DNA Tumor Virus Meeting. Trieste, Italy: International Centre for Genetic Engineering and Biotechnology.More infoOral Presentatiobn
- Campos, S. K., & Bronnimann, M. P. (2015, October). Furin Cleavage has Conserved Roles for Human Papillomavirus Type 16 and Bacterial Toxin Entry Pathways. University of Texas at El Paso, Chemistry & Biochemistry Departmental Seminar. El Paso, TX: University of Texas at El Paso.
- Campos, S. K. (2014). New structural model of adenoviral cement proteins is not yet concrete. Proceedings of the National Academy of Sciences of the United States of America.