Browsing by Subject "Virus Replication"
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Item Characterization of the Antiviral Effector IFI6(2018-11-26) Richardson, Ryan Blake; Yan, Nan; Schoggins, John W.; Levine, Beth; Pfeiffer, Julie K.The innate immune response is a critical line of host defense against invading pathogens. The production of interferon (IFN) and the subsequent expression of interferon stimulated genes (ISGs) are major contributors to the innate immune response, which establish an antiviral state in the cell. Flaviviruses such as dengue virus, Zika virus, and West Nile virus rely intimately on host pathways for completing a replication cycle, and have developed strategies to overcome the inhibitory effect of the innate immune response. To identify host factors required during an IFN response to flavivirus infection, a genome-wide CRISPR screen was carried out. Two of the top hits from the screen were IFI6, a previously identified ISG long predicted to be antiviral, and BiP, a luminal chaperone in the endoplasmic reticulum (ER). I questioned whether IFI6 was important for the antiviral response to flaviviruses and sought to investigate its role during infection. I confirmed the results from the CRISPR screen and showed that cells lacking IFI6 were insensitive to IFN, suggesting a key role in the innate immune response to flaviviruses. This was complemented by overexpression studies which showed IFI6 is potently inhibitory to flavivirus infection. I further demonstrated that BiP is required for an intact IFN response and importantly mediates expression of IFI6, which it binds in a chaperone-dependent manner. I also showed that IFI6 is localized to the ER and is an integral membrane protein. Importantly, IFI6 acts during the flavivirus life cycle to inhibit replication and formation of replication complexes, which are formed by rearrangement of ER membranes. IFI6 specifically inhibits flaviviruses, since other viruses that replicate at the ER such as hepatitis C virus (HCV) are not affected by IFI6. I hypothesize the key to this specificity lies in the orientation of the replication complexes - HCV complexes extend outwards into the cytoplasm while flaviviruses bud inwards into the lumen. Taken together, these data support a model where IFI6 is sensitive to membrane alterations specifically induced by flaviviruses but not other viruses, which provides the innate immune response with a potent and specific ISG to block viral infection.Item Identification and Characterization of a NEF-Associated Kinase(2004-05-04) Arora, Vivek Kumar; Bennett, MichaelThe nef gene encoded by primate lentiviruses is a major determinant of virulence in vivo. It is expressed early in the viral life cycle; its importance likely stems from its ability to prime the host environment for efficient viral replication. Reasonable models by which cellular phenotypes associated with Nef expression could enhance viral replication in vivo have been proposed. The molecular mechanisms by which Nef executes its functions, however, are poorly understood. The work presented here investigates the regulation of cellular proteins by Nef. I first identified a previously described Nef-associated kinase as the cellular kinase p21 activated kinase 2 (Pak2). This was done using proteolytic digestion of the Nef-associated kinase in multiple systems as well as by demonstrating the presence of active, ectopically expressed Pak2 associated with Nef in a cellular expression system. I further demonstrated that Nef induces Pak2 activation in vivo using multiple systems. First, Nef dependent activation of ectopically expressed tagged Pak2 was demonstrated in vivo. Second, an in gel renaturable kinase activity assay showed in cell extracts a Nef dependent kinase activity I subsequently demonstrated to be Pak2 by proteolytic digestion. Third, I showed that in vivo Nef expression induces the phosphorylation of Merlin at S518, a known and specific Pak2 substrate. The mechanism by which Nef leads to Pak2 activation was also addressed. Rho family GTPases are well-described endogenous activators of Pak2. Inhibition of Rho family GTPase activity in vivo also blocked Nef mediated activation of Pak2 as did mutation of the Rho GTPase binding site in Pak2. Thus, Nef induced Pak2 activation is dependent on endogenous Rho family GTPases. No Nef dependent effect on Rho GTPase activity levels, however, was detected. Instead, biochemical separation and cellular localization suggested that Nef mediates Pak2 activation by recruiting Pak2 to membranes where it encounters high concentrations of constituitively active Rho family GTPases. In summary, this work conclusively demonstrated that Pak2 associates with Nef and is activated by Nef via a endogenous Rho GTPase. Lastly, the potential roles of these molecular events in mediating Nef's pathogenic effects are discussed.Item Unexpected Factors That Influence Coxsackievirus B3 Replication in Mouse Intestine(2017-04-14) Wang, Yao; Kahn, Jeffrey; Pfeiffer, Julie K.; Hooper, Lora V.; Ye, JinCoxsackievirus is a human pathogen that frequently infect humans. Although many infections are asymptomatic, there can be severe outcomes, including heart inflammation and pancreas inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. I used a cell culture-adapted strain of coxsackievirus B3 (CVB3), CVB3-Nancy, to examine viral replication and pathogenesis in orally inoculated mice. Using HeLa cell plaque assays with agar overlays, I noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. I identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. I determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. This work reinforce the idea that culture-adapted laboratory virus strains can have reduced fitness in vivo. N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies. I also explored other factors that influence CVB3 infection in mouse intestine. First, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. We sought to examine if the phenomenon can be seen in mice and to further understand the mechanisms. Here we orally infected mice with CVB3 and confirmed that CVB3 replication in the intestine is sex-dependent. CVB3 replicated efficiently in male mice intestine, but not female mice. Overall these data suggest that sex and the immune response play an important role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease. Previously, our lab has shown that intestinal microbiota promote replication and pathogenesis of several viruses, including poliovirus (PV), reovirus and CVB3. With that finding, we wanted to examine how microbiota enhance CVB3 infection. Bacteria in the colon produce millimolar quantities of butyrate and other short-chain fatty acids (SCFAs) through fermentation of dietary fiber. SCFAs are among the most abundant molecules in the distal gastrointestinal tract. To determine whether bacterial-derived SCFAs such as butyrate impact CVB3 replication in the intestine, I antibiotic treated mice and then supplied them with tributyrin, a form of butyrate that is absorbed in the distal gastrointestinal tract. I found that CVB3 replication and pathogenesis was restored in antibiotic-treated mice that received tributyrin. These results suggest that butyrate is sufficient to promote CVB3 replication. My preliminary data demonstrate that oral delivery of a histone deacetylase (HDAC) inhibitor, Vorinostat, is sufficient to restore CVB3 replication in antibiotic-treated mice, suggesting that the HDAC activity of butyrate may promote CVB3 infection. Taken all together, I identified several unexpected factors that may influence CVB3 replication in mouse intestine although much remains open for exploration.Item Viral and Host Genetic Determinants of Hepatitis C Virus Persistence and Interferon Resistance(2006-05-16) Sumpter, Rhea Myers, Jr.; Gale, Michael, Jr.Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is an important cause of cirrhosis and hepatocellular carcinoma. HCV replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. The development of the HCV RNA replicon system has allowed the study of persistent HCV RNA replication in tissue culture. We evaluated HCV/IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV genotype 1b subgenomic RNA replicon that differed only at two sites within the NS5A coding region. A replicon with a lysine (K) insertion at HCV codon 2040 (K2040) replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an leucine (L) to serine (S) point mutation at HCV codon 2198 (L2198S) replicated poorly and triggered a cellular response characterized by IFN-! production and low-level interferon-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high passage (HP) variant with 6 additional point mutations throughout the HCV protein-coding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-" treatment and was associated with viral-directed reduction in host cell expression and action of ISG56, an antagonist of HCV RNA translation. We also demonstrated that HCV subgenomic RNA replicons can be used to model the early events of HCV infection. We found that HCV RNA replicons rapidly induce the cellular antiviral response upon their transfection into host Huh7 cells and we determined that intracellular HCV double stranded RNA (dsRNA) is a potent agonist of host dsRNAactivated pathways. A Huh7 derived cell line that is highly permissive for transduction by HCV replicons is specifically defective in the activation of interferon regulatory factor (IRF)-3 by virus infection or HCV dsRNA transfection. We found that a mutation in the caspase recruitment domain (CARD) of the DExH/D-box helicase protein RIG-I, a component of the TLR3-independent intracellular dsRNA-responsive IRF-3 activation pathway, was responsible for this phenotype. Restoration of RIG-I-mediated IRF-3 activation through genetic complementation resulted in decreased permissiveness to HCV RNA replication. These results establish the RIG-I!IRF-3 pathway as a critical determinant of HCV persistence.