Autophagy is an important cellular process by which ATG5 initiates the formation of double membrane vesicles (DMVs). Upon infection, DMVs have been shown to harbor the replicase complex of positive-strand RNA viruses such as MHV, poliovirus, and equine arteritis virus. Recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis C virus (HCV) infection. Here, we identified ATG5 as an interacting protein for the HCV NS5B. ATG5/NS5B interaction was confirmed by co-IP and metabolic labeling studies. Furthermore, ATG5 protein colocalizes with NS4B, a constituent of the membranous web. Importantly, immunofluorescence staining demonstrated a strong colocalization of ATG5 and NS5B within perinuclear regions of infected cells at 2 days postinfection. However, colocalization was completely lacking at 5 DPI, suggesting that HCV utilizes ATG5 as a proviral factor during the onset of viral infection. Finally, inhibition of autophagy through ATG5 silencing blocks HCV replication.
During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 & 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 & Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.
During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.
Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra-and intermolecular interactions required for NS4B structure/ function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus responsible for 170 million cases of chronic infections worldwide. HCV is the only member of the genus Hepacivirus in the family Flaviviridae (51, 63), which includes other human pathogens, such as West Nile virus and dengue virus. Translation of the virus genome yields at least three structural proteins (core, E1, and E2), the highly hydrophobic p7 peptide, and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS proteins, including NS3 to NS5B, are sufficient to promote HCV replication in vitro (10,43). However, with the advent of the HCV cell culture system, many of the NS proteins (NS2, NS3, and NS5A) have been reported to play an active role in HCV assembly (5,46,50,69,73), further supporting the idea that the NS proteins in general have multiple functions in the HCV life cycle.NS3 is illustrative of multifunctionality. Its N-terminal serine protease activity is responsible for processing the NS proteins into their mature forms, whereas the C-terminal helicase activity may be required for the unwinding of HCV RNA (35,66). Similarly, NS4A is a cofactor of NS3 serine protease; it also assists NS3 in binding to host membranes (72) and facilitates the association of NS3 with the HCV replication complex (RC). NS5A may have multiple functions, including inhibition of the interferon response to virus infection and HCV RNA ...
. 85:6963-6976, 2011) have also reported NS4B's function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B's function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state. Hepatitis C virus (HCV) infects 2 to 3% of the world population, with ca. 160 to 170 million individuals chronically infected and more than 350,000 deaths annually due to complications from cirrhosis and hepatocellular carcinoma (1, 2). As a result of the error-prone nature of its polymerase (3), HCV is classified into at least 6 genotypes and more than 50 subtypes (4). HCV is an enveloped, positive-sense RNA virus with a 9.6-kb genome flanked by 5= and 3= noncoding regions (NCR) and a long open reading frame encoding one polyprotein ϳ3,011 amino acids (aa) in length. Processing of the polyprotein by host and viral proteases occurs co-or posttranslationally, giving rise to three structural proteins (the capsid protein core and the envelope glycoproteins E1 and E2), the viroporin protein p7, and six nonstructural (NS) proteins (NS2, -3, -4A, -4B, -5A, and -5B) (5). The p7 and NS2 proteins are involved in HCV assembly (6-8), while NS3 to NS5B are sufficient to promote virus genome replication in vitro (9, 10). Recently, many of the replicase proteins (NS3, NS4B, and NS5A) were also found to play an active role in HCV production (11-15), consistent with the interpretation that the NS proteins have multiple functions in the HCV life cycle.Recent studies suggest that NS5A physically links the HCV replication complex to the site of HCV assembly on lipid droplets (LDs) or the endoplasmic reticulum (ER) (6,16). This is possible in part because NS5A is a phosphoprotein that exists in two states, based on its migration distance after SDS-PAGE. Basal phosphorylation (NS5A p56) favors HCV genome re...
The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higherthan-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.Simian virus 40 (SV40) has long been used as the model system to elucidate the mechanism of eukaryotic DNA replication initiation (7,20,58). Compared to the multiple origins of replication in eukaryotic cells, a single well-defined origin is utilized in SV40 DNA replication. The 64-bp core origin DNA is composed of three functional regions: a 23-bp perfect palindrome (site II) consisting of four GAGGC pentanucleotides, an imperfect palindrome (EP region), and a 17-bp AT-rich domain (15).The large tumor antigen (LTag) is a multifunctional protein possessing vital roles in coordinating viral DNA replication and transformation (7,21,58,67). It contains 708 amino acids that fold into several domains (3, 21, 58), including an Nterminal J domain, origin DNA binding domain (OBD), a helicase domain, and a C-terminal domain where host range functions are located (58). So far, the structures of the first three domains have been solved separately using X-ray crystallography and nuclear magnetic resonance techniques. The J domain, representing the first 82 amino acids, shows sequence similarities with DnaJ from Escherichia coli. It interacts with chaperone proteins and is involved in virus replication and transformation (8, 33). The OBD spans residues 131 to 260 and plays an important role in sequence-specific initiation of DNA replication. The recently solved crystal structure of T antigen's OBD reveals that the OBD monomers can form a left-handed spiral hexame...
Context Despite the increase of importance placed on research, both by residency program directors and the medical field at large, osteopathic medical students (OMS) have significantly fewer research experiences than United States (U.S.) allopathic medical students and non-U.S. international medical graduates. However, few studies have addressed this long-standing discrepancy, and none directly have focused on osteopathic medical students to assess their unique needs. The literature would benefit from identifying the barriers osteopathic medical students encounter when participating in research and understanding the currently available resources. Objectives To assess the barriers that OMS face when seeking research opportunities, identify resources currently available to osteopathic medical students at their respective schools, and investigate factors that contribute to an osteopathic medical student’s desire to pursue research opportunities. Additionally, to investigate osteopathic medical students’ confidence in research methodology. Methods A survey was created by the investigators and administered to participants over a three-month period via a GoogleForm. Research participants were surveyed for demographic information, as well as their involvement in research projects in the past, mentor availability, institutional resources, motivation to participate in research, individual barriers to participation, and confidence in their ability to do independent research. Responses were de-identified and analyzed using Microsoft Excel functions to count data and calculate percentages, as well as Pearson’s chi square analysis. Results After relevant exclusion, 668 responses were included. Of the students surveyed, 85.9% (574) indicated they currently and/or in the past were involved in research. More than half of the respondents that are not currently involved in research are interested in pursuing it (86.9%; 344). The primary barriers students reported facing include lack of time (57.8%; 386), feeling overwhelmed and unsure how to start (53.4%; 357), and lack of access to research (53%; 354). 34.7% (232) of students stated they either did not have resources from their school or were unsure whether these resources were available. The two most cited motivations to pursue research included boosting their residency application and/or interest in the area of study. Male gender and current research were associated with reported confidence in research ( [4, n=662]=10.6, p<0.05). Conclusions Findings from this study provide a synopsis of the barriers to research opportunities among osteopathic medical students. Notably, ⅓ of OMSs reported an absence or unawareness of available research resources at their osteopathic medical schools.
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