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Background: Many people who inject drugs (PWID) use syringes with detachable needles, which have high dead space (HDS). Contaminated HDS blood may substantially contribute to the transmission of HIV, hepatitis C (HCV), and other blood-borne viruses within this population. Newly designed low dead space (LDS) syringe-needle combinations seek to reduce blood-borne virus transmission among PWID. We evaluated the infectivity of HCV-contaminated residual volumes recovered from two LDS syringe-needle combinations. Methods: We tested two different design approaches to reducing the dead space. One added a piston to the plunger; the other reduced the dead space within the needle. The two approaches cannot be combined. Recovery of genotype-2a reporter HCV from LDS syringe-needle combinations was compared to recovery from insulin syringes with fixed needles and standard HDS syringe-needle combinations. Recovery of HCV from syringes was determined immediately following their contamination with HCV-spiked plasma, after storage at 22°C for up to 1 week, or after rinsing with water. Results: Insulin syringes with fixed needles had the lowest proportion of HCV-positive syringes before and after storage. HCV recovery after immediate use ranged from 47%±4% HCV-positive 1 mL insulin syringes with 27-gauge ½ inch needles to 98%±1% HCV-positive HDS 2 mL syringes with 23-gauge 1¼ inch detachable needles. LDS combinations yielded recoveries ranging from 65%±5% to 93%±3%. Recovery was lower in combinations containing LDS needles than LDS syringes. After 3 days of storage, as much as 6-fold differences in virus recovery was observed, with HCV recovery being lower in combinations containing LDS needles. Most combinations with detachable needles required multiple rinses to reduce HCV infectivity to undetectable levels whereas a single rinse of insulin syringes was sufficient. Conclusions: Our study, the first to assess the infectivity of HCV in residual volumes of LDS syringes and needles available to PWID, demonstrates that LDS syringe-needle combination still has the greater potential for HCV transmission than insulin syringes with fixed needles. Improved LDS designs may be able to further reduce HCV recovery, but based on the designed tested, LDS needles and syringes remain intermediate between fixed-needle syringes and HDS combinations in reducing exposure to HCV. [ABSTRACT FROM AUTHOR]

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly. [ABSTRACT FROM AUTHOR]

Background. Healthcare workers may come into contact with fomites that contain infectious hepatitis C virus (HCV) during preparation of plasma or following placement or removal of venous lines. Similarly, injection drugs users may come into contact with fomites. Hypothesizing that prolonged viability of HCV in fomites may contribute significantly to incidence, we determined the longevity of virus infectivity and the effectiveness of antiseptics.Methods. We determined the volume of drops misplaced during transfer of serum or plasma. Aliquots equivalent to the maximum drop volume of plasma spiked with the 2a HCV reporter virus were loaded into 24-well plates. Plates were stored uncovered at 3 temperatures: 4°C, 22°C, and 37°C for up to 6 weeks before viral infectivity was determined in a microculture assay.Results. The mean volume of an accidental drop was 29 µL (min–max of 20–33 µL). At storage temperatures 4°C and 22°C, we recovered viable HCV from the low-titer spots for up to 6 weeks of storage. The rank order of HCV virucidal activity of commonly used antiseptics was bleach (1:10) > cavicide (1:10) > ethanol (70%).Conclusions. The hypothesis of potential transmission from fomites was supported by the experimental results. The anti-HCV activity of commercial antiseptics varied. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) NS2 protein is essential for particle assembly, but its function in this process is unknown. We previously identified critical genetic interactions between NS2 and the viral E1-E2 glycoprotein and NS3-NS4A enzyme complexes. Based on these data, we hypothesized that interactions between these viral proteins are essential for HCV particle assembly. To identify interaction partners of NS2, we developed methods to site-specifically biotinylate NS2 in vivo and affinity capture NS2-containing protein complexes from virus-producing cells with streptavidin magnetic beads. By using these methods, we confirmed that NS2 physically interacts with E1, E2, and NS3 but did not stably interact with viral core or NS5A proteins. We further characterized these protein complexes by blue native polyacrylamide gel electrophoresis and identified ≈520-kDa and ≈680-kDa complexes containing E2, NS2, and NS3. The formation of NS2 protein complexes was dependent on coexpression of the viral p7 protein and enhanced by cotranslation of viral proteins as a polyprotein. Further characterization indicated that the glycoprotein complex interacts with NS2 via E2, and the pattern of N-linked glycosylation on E1 and E2 suggested that these interactions occur in the early secretory pathway. Importantly, several mutations that inhibited virus assembly were shown to inhibit NS2 protein complex formation, and NS2 was essential for mediating the interaction between E2 and NS3. These studies demonstrate that NS2 plays a central organizing role in HCV particle assembly by bringing together viral structural and nonstructural proteins. [ABSTRACT FROM AUTHOR]

Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication. [ABSTRACT FROM AUTHOR]

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2′-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV. [ABSTRACT FROM AUTHOR]

Background & Aims: Modulation of the host innate immune response is an attractive means of inhibiting hepatitis C virus (HCV) replication. Having previously determined that expression of the interferon-sensitive gene (ISG)15 protease USP18 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-alfa therapy, we hypothesized that USP18 might hinder the ability of IFN to inhibit HCV replication. Methods: The role of USP18 in IFN antiviral activity was examined using an in vitro model of HCV replication that reproduces the full viral life cycle. USP18 was silenced specifically using small inhibitory RNAs (siRNAs), and the dose response of HCV replication and infectious virus production to IFN-alfa was measured. Results: The siRNA knockdown of USP18 in human cells consistently potentiated the ability of IFN to inhibit HCV-RNA replication and infectious virus particle production by a factor of 1–2 log10. USP18 knockdown also resulted in a number of cellular changes consistent with increased sensitivity to IFN. Decreasing USP18 expression led to increased cellular protein ISGylation in response to exogenous IFN-alfa, prolonged tyrosine phosphorylation of signal transducer and activation of transcription (STAT1), and a general enhancement of IFN-stimulated gene expression. Conclusions: These data suggest that USP18 modulates the anti-HCV type I IFN response, and is a possible therapeutic target for the treatment of HCV infection. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle. [ABSTRACT FROM AUTHOR]

Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials. [ABSTRACT FROM AUTHOR]

Many aspects of the hepatitis C virus (HCV) life cycle have notbeen reproduced in cell culture, which has slowed research progress onthis important human pathogen. Here, we describe a full-length HCVgenome that replicates and produces virus particles that are infectiousin cell culture (HCVcc). Replication of HCVcc was robust, producingnearly 10[sup 5] infectious units per milliliter within 48 hours. Virusparticles were filterable and neutralized with a monoclonal antibodyagainst the viral glycoprotein E2. Viral entry was dependent on cellularexpression of a putative HCV receptor, CD81. HCVcc replication wasinhibited by interferon-α and by several HCV-specific antiviralcompounds, suggesting that this in vitro system will aid in the searchfor improved antivirals. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is an important human pathogen with a high chronicity rate. An estimated 71 million people worldwide are living with chronic hepatitis C (CHC) infection, which carries the risk of progression to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Similar to other RNA viruses, HCV has a high rate of genetic variability generated by its high mutation rate and the actions of evolutionary forces over time. There are two levels of HCV genetic variability: intra-host variability, characterized by the distribution of HCV mutant genomes present in an infected individual, and inter-host variability, represented by the globally circulating viruses that give rise to different HCV genotypes and subtypes. HCV genetic diversity has important implications for virus persistence, pathogenesis, immune responses, transmission, and the development of successful vaccines and antiviral strategies. Here we will discuss how HCV genetic heterogeneity impacts viral spread and therapeutic control. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a positive-strand RNA virus that remains one of the main contributors to chronic liver disease worldwide. Studies over the last 30 years have demonstrated that HCV contains a highly structured RNA genome and many of these structures play essential roles in the HCV life cycle. Despite the importance of riboregulation in this virus, most of the HCV RNA genome remains functionally unstudied. Here, we report a complete secondary structure map of the HCV RNA genome in vivo, which was studied in parallel with the secondary structure of the same RNA obtained in vitro. Our results show that HCV is folded extensively in the cellular context. By performing comprehensive structural analyses on both in vivo data and in vitro data, we identify compact and conserved secondary and tertiary structures throughout the genome. Genetic and evolutionary functional analyses demonstrate that many of these elements play important roles in the virus life cycle. In addition to providing a comprehensive map of RNA structures and riboregulatory elements in HCV, this work provides a resource for future studies aimed at identifying therapeutic targets and conducting further mechanistic studies on this important human pathogen. [ABSTRACT FROM AUTHOR]

Summary The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design. Graphical Abstract Highlights • Ubiquitin signaling factors interact with NS2 and play a role in HCV infection • MARCH8 mediates K63-linked ubiquitination of HCV NS2 and ESCRT recruitment • MARCH8 is required for Flaviviridae infection and intracellular envelopment of HCV • MARCH8 is a potential target for host-targeted antiviral strategies The mechanisms that regulate intracellular viral envelopment are unknown. Kumar et al. report that MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural 2 protein and subsequently ESCRT recruitment and HCV envelopment. MARCH8 is required for infection with other Flaviviridae family members, thereby representing a potential host target for antiviral strategies. [ABSTRACT FROM AUTHOR]

Hepatitis C virus genome replication occurs in endoplasmic reticulum-derived membrane compartments, but it is unknown how these structures arise. In this issue of Cell Host & Microbe, show that the virus recruits a specific lipid kinase to replication sites, stimulates its kinase activity, and alters the phospholipid profile of replication compartments. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity. [ABSTRACT FROM AUTHOR]

Summary RNA is a versatile macromolecule that accommodates functional information in primary sequence and secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, comparative sequence analysis, and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus’ life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a “code within the code” for controlling biological processes. [ABSTRACT FROM AUTHOR]

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but inmost cases, we do not know themolecularmechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane whereHCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposedmechanismincludes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3- 4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipidmetabolism. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro. Thus, we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than for modulating the enzymatic functions of NS3-4A. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly. [ABSTRACT FROM AUTHOR]

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37°C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7 mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells. [ABSTRACT FROM AUTHOR]

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly. [ABSTRACT FROM AUTHOR]