Kurt E Gustin

Interests

Research

Picornaviruses, RNA virus biology, host-pathogen interactions, RNA biology, Innate immune response, stress response, nuclear transport and nuclear pore complex

Courses

Scholarly Contributions

Chapters

• Gustin, K. E., Dougherty, J. D., Park, N., & Lloyd, R. E. (2014). Interference with Cellular Gene Expression. In Picornaviruses(pp 163-180). ASM Press. doi:10.1128/9781555816698.ch10
• Dougherty, J. D., Park, N., Gustin, K. E., & Lloyd, R. E. (2010). Picornavirus Interference with Cellular Gene Expression. In In Picornaviruses: Molecular Biology, Evolution and Pathogenesis. ASM Press.
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  ISBN: 978-1-55581-603-2
• Dougherty, J. D., Park, N., Gustin, K. E., & Lloyd, R. E. (2010). Picornavirus Interference with Cellular Gene Expression. In Picornaviruses: Molecular Biology, Evolution and Pathogenesis. American Society of Microbiology. doi:10.1128/9781555816698.ch10
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  This chapter focuses on new advances in understanding viral inhibition of host gene expression at four levels: transcription, nucleocytoplasmic trafficking, translation initiation, and manipulation of mRNA granules that store or process mRNA. Blockage of host gene expression serves multiple functions of liberating ribonucleotides, charged amino- acyl tRNAs, and ribosomal machinery for viral use and also restricting expression of innate immune response polypeptides that could counter viral replication. Further, blockage of host gene expression can hamper premature cell apoptosis and promote cell lysis after viral assembly. Recently, viral interference with RNA metabolism has been shown to extend to spliceosome assembly. Interestingly, in contrast to enteroviruses, cardioviruses appear to inhibit mRNA export, and this difference may be due to the different mechanisms utilized by these viruses to inhibit nuclear transport. In addition to the cleavages of eIF4G and PABP, which have major functional consequences, picornavirus infection leads to the proteolytic processing of other accessory translation factors that likely contribute to host cell translation shutoff. Mechanistically, various cellular stresses, such as oxidative stress, heat shock, or nutrient deprivation, induce SG formation by driving phosphorylation of eIF2α, which causes generalized translational arrest, and accumulation of mRNPs with stalled 40S ribosome subunits in stress granules (SGs). In the future, a more complete understanding of the mechanisms by which these fascinating viruses manipulate host gene expression and linkage to specific pathologies could lead to the rational design of novel antiviral drugs and therapies to combat these viruses and limit or interrupt disease progression.
• Choi, D., McIlroy, D. N., Nagler, J., Aston, E., Hrdlicka, P., Gustin, K. E., Hill, R., Stenkamp, D., & Branen, J. (2009). 1-Dimensional Silica Structures and their Applications to the Biological Sciences. In Nanostructured Oxides. In Volume 2 of Nanomaterials for Life Sciences. Wiley-VCH.
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  ISBN: 978-3-527-32152-0
• Beaux, M. F., McIlroy, D. N., & Gustin, K. E. (2008). Utilization of Solid Nanomaterial for Drug Delivery. In Expert Opinion on Drug Delivery(pp 725-735).

Journals/Publications

• Ke, H., Han, M., Kim, J., Gustin, K. E., & Yoo, D. (2019). Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion. Journal of virology, 93(14).
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  Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1β) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1β, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1β. The nsp1β L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1β-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-α/β), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1β, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1β protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1β binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1β binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.
• Gustin, K. E., Schweers, N. J., & Park, N. (2015). Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A ^pro. Journal of Virology, 89(21), 11069-11079. doi:10.1128/jvi.00956-15
• Kotla, S., & Gustin, K. E. (2015). Proteolysis of MDA5 and IPS-1 is not required for inhibition of the type I IFN response by poliovirus. Virology journal, 12, 158.
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  The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined.
• Park, N., Schweers, N. J., & Gustin, K. E. (2015). Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A(pro). Journal of virology, 89(21), 11069-79.
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  Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2A(pro)). Incubation of bacterially expressed or in vitro-translated Nup98 with 2A(pro) results in proteolytic cleavage at multiple sites in vitro, indicating that 2A(pro) cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2A(pro) proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected.
• Pathak, A., Adams, R. H., Shah, N. C., & Gustin, K. E. (2013). Persistent human rhinovirus type C infection of the lower respiratory tract in a pediatric cord blood transplant recipient. Bone Marrow Transplantation, 747-748.
• Gustin, K., Park, N., Skern, T., & Gustin, K. E. (2010). Specific cleavage of the nuclear pore complex protein Nup62 by a viral protease. The Journal of biological chemistry, 285(37).
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  Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A(pro)) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A(pro) is a major contributor to Nup62 processing. The ability of purified 2A(pro) to cleave bacterially expressed and purified Nup62 demonstrated that 2A(pro) directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A(pro) in vitro. This analysis revealed that 2A(pro) cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.
• Gustin, K., Piotrowska, J., Hansen, S. J., Park, N., Jamka, K., Sarnow, P., & Gustin, K. E. (2010). Stable formation of compositionally unique stress granules in virus-infected cells. Journal of virology, 84(7).
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  Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.
• Park, N., Skern, T., & Gustin, K. E. (2010). Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease. Journal of Biological Chemistry, 28796-28805.
• Piotrowska, J., Hansen, S. J., Park, N., Jamka, K., Sarnow, P., & Gustin, K. E. (2010). Stable Formation of Compositionally Unique Stress Granules in Virus-infected Cells. Journal of Virology, 3654-3665.
• Stenkamp, D. L., Nagler, J. J., Mcilroy, D. N., Hrdlicka, P. J., Hill, R., Gustin, K. E., Choi, D. S., Branen, J., & Aston, E. (2010). One-Dimensional Silica Structures and Their Applications to the Biological Sciences. Nanotechnologies for the Life Sciences. doi:10.1002/9783527610419.ntls0139
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  The sections in this article are Introduction Synthesis of Silica Nanowires and Nanosprings Catalyst Preparation and Application Methods for VLS Synthesis of Nanowires Flow Reaction Formation of Nanowires Laser Ablation of Nanowires Chemical Vapor Deposition and Plasma-Enhanced Chemical Vapor Deposition of Nanowires Functionalization of Silica 1-D Silica Nanomaterials Toxicology Studies on 1-D Silica Nanomaterials Intracellular Targeted Delivery A Typical Cellular Targeting Strategy Using 1-D NS-Based Nanostructures In Vitro Toxicity of 1-D Nanostructures In Vivo Toxicity of 1-D Nanostructures Biological Applications of 1-D Silica Nanomaterials Biodetection Keywords: silica nanowires; functionalization; biomolecule; protein; toxicology; biosensor
• Kotla, S., Major, S. C., & Gustin, K. E. (2009). Rapid detection and quantitation of poliovirus and rhinovirus sequences in viral stocks and infected cells. Journal of virological methods, 157(1).
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  Laboratories working with closely related viruses need simple and cost-effective ways to rapidly validate viral stocks, detect contamination and measure the abundance of viral RNA species. Using RT-PCR and specific primers an approach for the specific detection of rhinovirus type 14 (RV14) or poliovirus type 1 (PV1) is presented. It is demonstrated that viral sequences can be amplified directly from viral stocks or from infected cells. In addition, the utility of this protocol for the detection of low levels of contaminating PV1 in RV14 stocks is shown. Further, using quantitative real-time PCR It is shown that this approach can be used for the quantitative analysis of viral RNA and replication kinetics in infected cells. This method should be useful for laboratories working with PV and RV14 and could be adapted easily for use by laboratories working with other rhinovirus and enterovirus serotypes.
• Ricour, C., Delhaye, S., Hato, S. V., Olenyik, T. D., Michel, B., van Kuppeveld, F. J., Gustin, K. E., & Michiels, T. (2009). Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein. The Journal of general virology, 90(Pt 1).
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  Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice.
• Ricour, C., Delhaye, S., Stanleyson, H. V., Olenyik, T. D., Michel, B., van Kuppeveld, F. J., Gustin, K. E., & Micheils, T. (2009). Inhibition of mRNA export and IRF-3 dimerization by Theiler's virus 1 leader protein.. Journal of General Virology, 177-186.
• Adili, A., Crowe, S., Beaux, M. F., McIlroy, D. N., & Gustin, K. E. (2008). Differential cytotoxicity exhibited by silica nanowires and nanoparticles. Nanotoxicology, 1-8.
• Beaux, M. F., McIlroy, D. N., & Gustin, K. E. (2008). Utilization of solid nanomaterials for drug delivery. Expert opinion on drug delivery, 5(7).
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  Background: Solid nanostructures are versatile platforms for constructing hybrid drug delivery systems that have tremendous potential for improving disease prevention and treatment. The rationale and application of solid nanostructures in the context of drug delivery are explored in this article. Objective: The purpose of this paper is to provide a concise review of the major attributes of solid nanostructures as they relate to drug delivery and to describe the outstanding issues that need to be addressed in order to develop these materials into clinically useful reagents. Methods: The scope of this opinion has been restricted to solid nanostructures, where solid nanostructures are defined as those that are not biodegradable. The opinion has been further limited to the three primary types of nanostructures: nanoparticles, nanowires and nanotubes. Results/conclusion: There is a need for cross-disciplinary training and standardized protocols for developing and evaluating the efficacy of solid nanomaterials.
• Kotla, S., Peng, T., Bumgarner, R. E., & Gustin, K. E. (2008). Attenuation of the type I interferon response in cells infected with human rhinovirus. Virology, 374(2).
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  The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, IRF-3 and ATF-2 which in turn activate transcription from the IFN-beta promoter. Here we have examined the type I interferon response in rhinovirus type 14-infected A549 cells, with particular emphasis on the status of the transcription factor IRF-3. Our results indicate that although rhinovirus type 14 (RV14) infection induces the activation of NF-kappaB and ATF-2, only very low levels of IFN-beta mRNA are detected. Analysis of ISG54 mRNA levels revealed very little induction of this IRF-3 responsive transcript and suggested that IRF-3 activation might be impaired. Examination of IRF-3 in RV14-infected cells demonstrated only low levels of phosphorylation, a lack of homodimer formation and an absence of nuclear accumulation indicating that this transcription factor is not activated. Inhibition of viral protein synthesis following infection resulted in an increase in IFN-beta mRNA levels indicating that viral gene products prevent induction of this pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.
• Park, N., Katikaneni, P., Skern, T., & Gustin, K. E. (2008). Differential targeting of nuclear pore complex proteins in poliovirus-infected cells. Journal of virology, 82(4).
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  Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2A(pro) to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.
• Ferens, W. A., Halver, M., Gustin, K. E., Ott, T., & Hovde, C. J. (2007). Differential sensitivity of viruses to the antiviral activity of Shiga toxin 1 A subunit. Virus research, 125(1).
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  The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10 microg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48 h post challenge. Cells treated with 0.1 microg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01 microg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner.
• Ferens, W. A., Halver, M., Gustin, K. E., Ott, T., & Hoyde, C. J. (2007). Differential sensitivity of viruses to the antiviral activity of Shiga toxin 1 A subunit. Virus Research, 104-108.
• Peng, T., Kotla, S., Bumgarner, R. E., & Gustin, K. E. (2007). Human rhinovirus attenuates the type I interferon response by disrupting activation of interferon regulatory factor 3. Journal of virology, 81(11).
• Peng, T., Kotla, S., Bumgarner, R. E., & Gustin, K. E. (2006). Human rhinovirus attenuates the type I interferon response by disrupting activation of interferon regulatory factor 3. Journal of virology, 80(10).
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  The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, interferon regulatory factor 3 (IRF-3), and ATF-2, which in turn activate transcription from the IFN-beta promoter. Synthesis and subsequent secretion of IFN-beta activate the Jak/STAT signaling pathway, resulting in the transcriptional induction of the full spectrum of antiviral gene products. We utilized high-density microarrays to examine the transcriptional response to rhinovirus type 14 (RV14) infection in HeLa cells, with particular emphasis on the type I interferon response and production of IFN-beta. We found that, although RV14 infection results in altered levels of a wide variety of host mRNAs, induction of IFN-beta mRNA or activation of the Jak/STAT pathway is not seen. Prior work has shown, and our results have confirmed, that NF-kappaB and ATF-2 are activated following infection. Since many viruses are known to target IRF-3 to inhibit the induction of IFN-beta mRNA, we analyzed the status of IRF-3 in infected cells. IRF-3 was translocated to the nucleus and phosphorylated in RV14-infected cells. Despite this apparent activation, very little homodimerization of IRF-3 was evident following infection. Similar results in A549 lung alveolar epithelial cells demonstrated the biological relevance of these findings to RV14 pathogenesis. In addition, prior infection of cells with RV14 prevented the induction of IFN-beta mRNA following treatment with double-stranded RNA, indicating that RV14 encodes an activity that specifically inhibits this innate host defense pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.
• Perez-Romero, P., Gustin, K. E., & Imperiale, M. J. (2006). Dependence of the encapsidation function of the adenovirus L1 52/55-kilodalton protein on its ability to bind the packaging sequence. Journal of virology, 80(4).
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  The adenovirus IVa2 and L1 52/55-kDa proteins are involved in the assembly of new virus particles. Both proteins bind to the packaging sequence of the viral chromosome, and the lack of expression of either protein results in no virus progeny: the absence of the L1 52/55-kDa protein leads to formation of only empty capsids, and the absence of the IVa2 protein results in no capsid assembly. Furthermore, the IVa2 and L1 52/55-kDa proteins interact with each other during adenovirus infection. However, what is not yet clear is when and how this interaction occurs during the course of the viral infection. We defined the domains of the L1 52/55-kDa protein required for interaction with the IVa2 protein, DNA binding, and virus replication by constructing L1 52/55-kDa protein truncations. We found that the N-terminal 173 amino acids of the L1 52/55-kDa protein are essential for interaction with the IVa2 protein. However, for both DNA binding and complementation of the pm8001 mutant virus, which does not express the L1 52/55-kDa protein, the amino-terminal 331 amino acids of the L1 52/55-kDa protein are necessary. These results suggest that the production of infectious virus particles depends on the ability of the L1 52/55-kDa protein to bind to DNA.
• Graham, K. L., Gustin, K. E., Rivera, C., Kuyumcu-Martinez, N. M., Choe, S. S., Lloyd, R. E., Sarnow, P., & Utz, P. J. (2004). Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection. Journal of virology, 78(12).
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  DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.
• Graham, K. L., Gustin, K. E., Rivera, C., Kuyumcu-Martinez, N. M., Choe, S. S., Lloyd, R. E., Sarnow, P., & Utz, P. J. (2004). Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection.. J. Virology, 6313-6321.
• Gustin, K. E. (2003). Inhibition of nucleo-cytoplasmic trafficking by RNA viruses: targeting the nuclear pore complex. Virus research, 95(1-2).
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  Analysis of virus-host interactions has revealed a variety of ways in which viruses utilize and/or alter host functions in an effort to facilitate efficient replication. Recent work has suggested that certain RNA viruses that replicate in the cytoplasm disrupt the normal trafficking of cellular RNAs and proteins within the host cell. This review will examine the recent evidence showing that poliovirus and vesicular stomatitis virus (VSV) can inhibit nucleo-cytoplasmic transport within cells. Interestingly, the data indicate that inhibition by both viruses involves targeting components of the nuclear pore complex (NPC). Following this, several possible explanations for why viruses might disrupt nucleo-cytoplasmic transport are discussed. Finally, the possibility that disruption of nucleo-cytoplasmic trafficking may be a more common feature of RNA virus-host interactions than previously thought is examined.
• Gustin, K. E., & Sarnow, P. (2002). Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus. Journal of virology, 76(17).
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  Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.
• Gustin, K. E., & Sarnow, P. (2001). Effects of Poliovirus Infection on Nucleo-cytoplasmic Trafficking and Nuclear Pore Complex Composition. EMBO J, 240-249.
• Gustin, K. E., & Burk, R. D. (2000). PCR-Directed Linker Scanning Mutagenesis. Methods in Molecular Biology, 85-90.
• Imperiale, M. J., & Gustin, K. E. (1998). Encapsidation of viral DNA requires the adenovirus L1 52/55-kilodalton protein.. Journal of virology, 72(10), 7860-70. doi:10.1128/jvi.72.10.7860-7870.1998
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  Previous work demonstrated that the adenovirus L1 52/55-kDa protein is required for assembly of viral particles, although its exact role in the assembly process is unclear. The 52/55-kDa protein's early expression, however, suggests that it might have other roles at earlier times during infection. To uncover any role the 52/55-kDa protein might have at early times and to better characterize its role in assembly, a mutant adenovirus incapable of expressing the 52/55-kDa protein was constructed (H5pm8001). Analysis of the onset and extent of DNA replication and late protein synthesis revealed that H5pm8001-infected 293 cells entered the late stage of infection at the same time as did adenovirus type 5 (Ad5)-infected cells. Interestingly, H5pm8001-infected cells displayed slightly lower levels of replicated viral DNA and late proteins, suggesting that although not required, the 52/55-kDa protein does augment these activities during infection. Analysis of transcripts produced from the major late and IVa2 promoters indicated a slight reduction in H5pm8001-infected compared to Ad5-infected cells at 18 h postinfection that was not apparent at later times. Analysis of particles formed in H5pm8001 cells revealed that empty capsids could form, suggesting that the 52/55-kDa protein does not function as a scaffolding protein. Subsequent characterization of these particles demonstrated that they lacked any associated viral DNA. These findings indicate that the 52/55 kDa-protein is required to mediate stable association between the viral DNA and empty capsid and suggest that it functions in the DNA encapsidation process.
• Lutz, P. G., Lutz, P., Imperiale, M. J., & Gustin, K. E. (1996). Interaction of the adenovirus L1 52/55-kilodalton protein with the IVa2 gene product during infection.. Journal of virology, 70(9), 6463-7. doi:10.1128/jvi.70.9.6463-6467.1996
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  The adenovirus L1 52/55-kDa protein is expressed both in the early and late stages of infection, raising the possibility that it has multiple roles in the viral life cycle. To obtain possible insights into these roles, the yeast two-hybrid system was used to examine the interactions of the 52/55-kDa protein with viral and cellular factors. cDNA expression libraries from human 293 cells at both early and late stages of adenovirus type 5 infection were constructed and screened, with the 52/55-kDa protein being used as bait. Characterization of positive clones revealed that the adenovirus IVa2 gene product interacted specifically with the 52/55-kDa protein. In addition, the IVa2 protein was shown to interact with a bacterial glutathione S-transferase-52/55-kDa fusion protein in vitro, further supporting the finding with the yeast two-hybrid system. Finally, coimmunoprecipitation studies confirmed that the 52/55-kDa protein and IVa2 polypeptide interact specifically during the course of adenovirus infection. A potential role for the IVa2-52/55-kDa protein interaction in the regulation of transcription from the major late promoter and in viral assembly is discussed.

Presentations

• Traugutt, E., Sattler, R., & Gustin, K. E. (2022, April). Genetic Mutations Associated with the Development of Amyotrophic Lateral Sclerosis Enhance Replication of a Neurotropic Virus. 7th Annual ABRC-Flinn Research Conference. Phoenix, AZ: ABRC- Flinn.
• Altemus, J., & Gustin, K. E. (2019, August). Role of Nup98 proteolysis in evasion of the antiviral response by an RNA virus. 3rd Annual Arizona RNA Salon Symposium. University of Arizona, Tucson AZ.
• Gustin, K. E. (2019, January). Transcriptional Memory Enhances the Innate Immune Response to Viral Infection.. 2nd Annual ASU-UA Virology Symposium. University of Arizona, Tucson AZ.
• Gustin, K. E. (2019, January). Transcriptional Memory Enhances the Innate Immune Response to Viral Infection.. Arizona RNA Salon. COM-Phoenix.
• Molloy, J., Le, S., Sullivan, B. G., & Gustin, K. E. (2019, June). Epigenetic Transcriptional Memory Attenuates Virus Replicatio. Arizona Symposium on Virology, Immunology, Microbiomes and Infectious Disease. COM-Phoenix.
• Gustin, K. E., Schweers, N., Sullivan, B., & Aintablian, H. (2018, March). Transcriptional Regulation of the Type I Interferon Response by a Nuclear Pore Protein.. COM-Phoenix Scholarly Project Student Research Symposium. COM-Phoenix: COM-Phoenix.
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  Mr. Aintablian's abstract was selected (out of ~80) for an oral presentation.
• Gustin, K. E., Sullivan, B. G., & Le, S. (2018, May). Transcriptional Memory Potentiates the Anti-viral Response. 2nd Annual Arizona RNA Salon Symposium. COM-Phoenix: The RNA Socieity, Arizona Biomedical Research Centre.
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  Ms. Le, a technician in my lab presented a short talk at the symposium.
• Gustin, K. E. (2017, December). Role for Nuclear Events in Stress Granule Formation Following Virus Infection. Arizona RNA Salon Regular Meeting. University of Arizona College of Medicine, Phoenix AZ: Arizona RNA Salon.
• Kotla, S., Bradley, C., Schweers, N., Aintablian, H., & Gustin, K. E. (2015, June). Role for a Nuclear Pore Protein in the Host Antiviral Response. Department of Basic Medical Sciences Research Symposium. Phoenix, AZ.
• Kotla, S., Park, N., Bradley, C., & Gustin, K. E. (2015, March). Modulation of the Host Antiviral Response by an RNA Virus. Immunobiology Symposium. Tucson: University of Arizona.
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  Oral Presentation
• Hansen, S. J., & Gustin, K. E. (2013, March). Mechanisms Responsible for Anti-viral Granule Formation in the Cytoplasm of Poliovirus-infected Cells. Immunobiology Symposium. University of Arizona, Tuscon Arizona.
• Bradley, C. A., Hansen, S. J., Hwang, H., Polpitiya, A., Liu, J., Petritis, K., & Gustin, K. E. (2012, July). Proteomic analysis reveals the consequences of poliovirus infection on nuclear pore complex composition. American Society of Virology. Madison, Wisconsin.
• Bradley, C. A., Hansen, S. J., Hwang, H., Polpitiya, A., Liu, J., Petritis, K., & Gustin, K. E. (2012, June). Comparative Proteomics Reveals Alterations to the Nuclear Pore Complex Proteome Following Poliovirus Infection. European Study Group on the Molecular Biology of Picornaviruses. St. Raphael, France.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2008, July). Inhibition of IRF-3 Activation in Rhinovirus and Poliovirus-infected Cells. American Society of Virology. Ithaca, New.
• Park, N., Skern, T., & Gustin, K. E. (2008, July). Proteolysis of Nuclear Pore Complex Proteins by the Picornavirus 2A Protease. American Society of Virology. Ithaca, New York.
• Park, N., Skern, T., & Gustin, K. E. (2008, May). Proteolysis of Nuclear Pore Complex Proteins by the Picornavirus 2A Protease. European Study Group on the Molecular Biology of Picornaviruses. Stiges, Spain.
• Piotrowska, J., Park, N., Jamka, K., & Gustin, K. E. (2008, July). Stress Granule Formation in Poliovirus-infected Cells. American Society of Virology. Ithaca, New York.
• Gustin, K. E. (2007, March). Disruption of Nucleo-cytoplasmic Trafficking by an RNA Virus. American Society of Microbiology Northwest Branch. Seattle, Washington.
• Park, N., Skern, T., & Gustin, K. E. (2007, July). Role of 2A protease in picornavirus-induced alterations to the nuclear pore complex. American Society of Virology. Corvallis Oregon.
• Gustin, K. E. (2006, April). Host-pathogen Interactions in Picornavirus-infected Cells. Western INBRE States Infectious Disease Symposium.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2006, July). Inhibition of IRF-3 Dimerization by Rhinovirus Attenuates the Type I Interferon Response. American Society of Virology. Madison, Wisconsin.
• Park, K., Katikaneni, P., Skern, T., & Gustin, K. E. (2006, July). Disruption of the Nuclear Pore Complex by Picornaviruses.. American Society of Virology. Madison Wisconsin.
• Gustin, K. E. (2005, Spring). Alteration of the Nuclear Pore Complex by Picornaviruses.. Vertebrate Nuclear Pore Group.
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  Oral Presentation
• Gustin, K. E. (2005, Spring). Evasion of Innate Immune Responses by Human Rhinovirus. INBRE Research Conference.
• Gustin, K. E., Skern, T., Park, N., & Halver, M. (2005, Spring). Rhinovirus-induced Alterations to the Nuclear Pore Complex. European Study Group on the Molecular Biology of Picornaviruses.. Lunteren, The Netherlands.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2005, Spring). Rhinovirus Inhibits Host Antiviral Responses Downstream of Activation of NF-kB, IRF-3 and ATF-2. Northwest Microarray Meeting.
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  Oral Presentation

Poster Presentations

• Vargua, S., Sattler, R., & Gustin, K. E. (2023, April). Amyotrophic Lateral Sclerosis Mutations Enhance Replication of a Neurotropic Virus. 8th Annual ABRC-Flinn Research Conference. Phoenix AZ: Flinn and ABRC.
• Gustin, K. E. (2020, May). Potentiation of Transcriptional Memory by a Nuclear Pore Protein.. 25th Annual Meeting of the RNA Society. Virtual: RNA Society.
• Gustin, K. E. (2020, September). HRV-C’s Impact on Pediatric Cystic Fibrosis Patients: A Prospective Cohort Study.. North American Cystic Fibrosis Conference. Phoenix, AZ: The Cystic Fibrosis Foundation.
• Gustin, K. E., Sullivan, B. G., & Le, S. (2019, February). Transcriptional Memory at the IFN-beta Promoter Enhances the Type I Interferon Response. Keystone Meeting on Transcription and RNA Regulation in Inflammation and Immunity. Lake Tahoe, CA: Keystone Symposium.
• Aintablian, H., Schweers, N., & Gustin, K. E. (2017, March). Transcriptional Regulation of the Type I Interferon Response by a Nuclear Pore Protein. Scholarly Project Student Research Symposium. University of Arizona College of Medicine, Phoenix AZ.
• Almader, A., & Gustin, K. E. (2017, March). Impact of HRV-C on Pediatric Cystic Fibrosis Patients. Scholarly Project Student Research Symposium. University of Arizona College of Medicine, Phoenix AZ.
• Gustin, K. E., Bradley, C. A., Park, N., & Schweers, N. (2015, July). Role of Nup98 in the Cellular Response to Poliovirus Infection. American Society of Virology. London, Ontario Canada.
• Hansen, S. J., & Gustin, K. E. (2012, June). Role of Host-cell Transcription and Nuclear Export in Poliovirus-Induced Stress Granule Formation. European Study Group on the Molecular Biology of Picornaviruses.. St. Raphael, France.
• Liu, J., Bradley, C. A., Polpitiya, A., Hansen, S. J., Tegler, T., Hwang, H., Gustin, K. E., & Petritis, K. (2012, May). Analysis of the Nuclear Pore Complex upon Poliovirus Infection Using Mass Spectrometry. American Society for Mass Spectrometry Annual Conference on Mass Spectrometry and Allied Topics.
• Bradley, C. A., Hansen, S. J., Hwang, H., Polpitiya, A., Liu, J., Petritis, K., & Gustin, K. E. (2011, November). A Proteomic Analysis of the nuclear Pore Complex Following Poliovirus Infection. Translational Genomics/Van Andel Research Institute Scientific Retreat.
• Hansen, S. J., & Gustin, K. E. (2011, July). Nuclear Events Required For Poliovirus-induced Stress Granule Formation. American Society of Virology. Minneapolis Minnesota.
• Hansen, S., & Gustin, K. E. (2010, July). Nucleo-cytoplasmic transport in poliovirus-infected cell; analysis of the nuclear pore complex and the Crm1 export pathway. American Society of Virology. Bozeman, Montana.
• Flerchinger, E., Piotrowska, A., & Gustin, K. E. (2008, August). Viral Induction of Stress Granule Formation. INBRE Research Conference. Boise, Idaho.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2008, February). Human Rhinovirus Attenuates the Type I Interferon Response by Inhibiting Homodimerization of IRF-3.. Keystone Meeting on Innate Immunity. Keystone, CO.
• Kotla, S., & Gustin, K. E. (2007, August). Analysis of the host innate immune response in rhinovirus-infected cells. INBRE Research Conference. Moscow, Idaho.
• Major, S. C., Kotla, S., & Gustin, K. E. (2007, August). Developing a Quantitative Real Time PCR Assay to Measure RNA Levels in Human Rhinovirus-Infected Cells. EPSCOR REU2 Research Symposium. Moscow, Idaho.
• McGuckin, K. A., & Gustin, K. E. (2007, August). Determining the Role of Rhinovirus 2A Protease in Inhibition of Nuclear Import. INBRE Research Conference. Moscow, Idaho.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2006, February). Human Rhinovirus Attenuates the Type I Interferon Response by Disrupting Activation of IRF-3. Keystone Meeting on Cell Biology of Virus Entry, Replication and Pathogenesis. Santa Fe, New Mexico.
• Kotla, S., Peng, T., Bumgarner, R., & Gustin, K. E. (2006, March). Human Rhinovirus Attenuates the Type I Interferon Response by Inhibiting Homodimerization of IRF-3. Keystone Meeting on Viral Immunity: From Basic Mechanisms to Vaccines.. Steamboat Springs, Colorado.
• Park, K., Katikaneni, P., & Gustin, K. E. (2006, July). Disruption of the Nuclear Pore Complex by Picornaviruses. National IDeA Symposium of Biomedical Research Excellence. Washington, D.C..
• Park, N., Gustin, K. E., & Katikaneni, P. (2006, December). Disruption of the Nuclear Pore Complex by Picornaviruses. American Society of Cell Biology.
• Baca, T. D., & Gustin, K. E. (2005, August). Alterations in Nuclear Transport by Infection with Theiler's Murine Encephalomyelitis Virus.. INBRE Research Conference..
• Kotla, S., & Gustin, K. E. (2005, June). Rhinovirus Inhibits Host Antiviral Responses Downstream of Activation of NF-kB, IRF-3 and ATF-2. American Society of Virology.
• McGuckin, K. A., & Gustin, K. E. (2005, August). Identification of Rhinovirus Proteins Responsible for Disrupting Nucleo-cytoplasmic Transport.. INBRE Research Conference.

Reviews

• Gustin, K. E., & Sarnow, P. (2006. Positive-strand RNA Viruses and the Nucleus in Viruses and the Nucleus(pp 161-184).
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  Edited by Julian Hiscox

Others

• Pathak, A. K., Adams, R. H., Shah, N. C., & Gustin, K. E. (2013, May). Persistent human rhinovirus type C infection of the lower respiratory tract in a pediatric cord blood transplant recipient. Bone marrow transplantation.