Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are
            <i>trans</i>
            -Packaged In Vitro To Generate Infectious Defective Particles

Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCV⌬E). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCV⌬E production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCV⌬E could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.Hepatitis C virus (HCV) is a major etiological agent of severe liver diseases, including liver cirrhosis and hepatocellular carcinoma, with an estimated 170 million people infected worldwide (2). No vaccine is available to prevent HCV infection, and the sole therapeutic treatment available, based on interferon, does not always lead to cure and is often associated with significant side effects (12). HCV is an enveloped plusstrand RNA virus belonging to the family Flaviviridae. The 9.6-kb viral genome encodes a single polyprotein that is co-or posttranslationally cleaved into structural (core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (4). The structural proteins encapsidate the viral genome into infectious particles and mediate the entry of the virus into permissive cells; the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B are the viral components of the membrane-bound replication complexes that catalyze genomic RNA replication (18).HCV envelope glycoproteins E1 and E2 are processed by cellular signal peptidases in the endoplasmic reticulum (ER), are highly glycosylated in the amino-terminal ectodomains, and are anchored to the membrane by the carboxyl-terminal transmembrane domains to form a stable noncovalent heterodimer complex. This oligomer is thought to be the prebudding form of the functional HCV glycoproteins and is essential for interaction with the receptor during HCV entry (52). According to the current model for HCV assembly and secretion, HCV core particles containing the genome assemble in the lipid droplets and acquire the viral envelope by budding into the ER (43), during which time the E1 and E2 proteins are inserted i...

Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 99%

Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

Yan

et al. 2011

“…This early expression from input RNA was not seen with Jc1/GLuc2A (Fig. 1), which could reflect the interference of HCV translation by the viral structural genes (11,56,67,70). Since the NS4A F48A replication defect was initially identified in Jc1/GLuc2A, we tested whether the NS4B I7F suppressor could restore replication of a full-length HCV genome.…”

Section: Resultsmentioning

confidence: 99%

“…To better clarify the role of NS4A in virus assembly, we tried to identify second-site mutations that suppressed these defects. We took advantage of the fact that HCV subgenomes can be packaged in trans (1,56,63) to select for trans-packaged replicons containing mutations of interest. This strategy yielded a mutation in NS3 (Q221L) that fully restored assembly of the NS4A K41 mutant in cis and in trans, but did not suppress the assembly defects of other NS4A mutants.…”

Section: Discussionmentioning

confidence: 99%

The Acidic Domain of Hepatitis C Virus NS4A Contributes to RNA Replication and Virus Particle Assembly

Phan

Kohlway

Dimberu

et al. 2011

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. Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus classified within the genus Hepacivirus in the familyFlaviviridae (21). The nonsegmented HCV genome is 9.6-kb and encodes a long polyprotein that is cleaved by cellular and viral proteases into at least 10 distinct products (47,54). The N-terminal region of this polyprotein encodes the viral structural proteins: core and two envelope glycoproteins, E1 and E2. The remainder of the polyprotein encodes nonstructural (NS) proteins: p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Together, the NS proteins regulate intracellular aspects of the viral life cycle, including RNA replication, virus assembly, and viral subversion of host antiviral responses.NS3-4A is an essential enzyme complex of the viral replication machinery and a major target for HCV-specific drug development. The N-terminal domain of NS3 encodes a chymotrypsin-like serine protease that requires NS4A for full activity and is responsible for the cleavage of the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions (47). In addition, the NS3-4A serine protease antagonizes host innate antiviral responses by cleaving the cellular messenger IPS-1 (also known as MAVS, Cardif, and VISA) (43,44,49,53). The C-terminal region of NS3 encodes a superfamily 2 helicase/NTPase domain, which is essential for virus genome replication (36, 41). The serine protease and RNA helicase activities are coordinated, such that the helicase domain can influence the protease activity, and the protease domain can influence RNA helicase activity (7,8,24,40). More recently, a number of genetic studies have implicated the NS3 helicase domain in virus partic...

“…Experiments to elucidate the role of mannose trimming of N-glycans in the HCV life cycle are currently under way. It has recently been demonstrated that subgenomic replicons or defective genomes of HCV that have the potential of translation and self-replication can be encapsidated into infectious viruslike particles by trans-complementation of the viral structural proteins (1,17,32,41,44). In these studies, the viral RNAs were generally generated by in vitro transcription from linearized corresponding plasmids, followed by electroporation into the cells.…”

Section: Discussionmentioning

confidence: 99%

Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription

Masaki

Suzuki

Saeed

et al. 2010

In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up-and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.Over 170 million people worldwide have been infected with hepatitis C virus (HCV) (22,33,37), and persistence of HCV infection is one of the leading causes of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (16,25,38). The HCV genome is an uncapped 9.6-kb positivestrand RNA sequence consisting of a 5Ј untranslated region (UTR), an open reading frame encoding at least 10 viral proteins (Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), and a 3ЈUTR (46). The structural proteins (Core, E1, and E2) reside in the N-terminal region.The best available treatment for HCV infection, which is pegylated alpha interferon (IFN-␣) combined with ribavirin, is effective in only about half of patients and is often difficult to tolerate (25). To date, a prophylactic or therapeutic vaccine is not available. There is an urgent need to develop more effective and better tolerated therapies for HCV infection. Recently, a robust system for HCV production and infection in cultured cells has been developed. The discovery that some HCV isolates can replicate in cell cultures and release infectious particles has allowed the complete viral life cycle to be studied (23,49,53). The most robust system for HCV production involves transfection of Huh-7 cells with genomic HCV RNA of the JFH-1 strain by electroporation. However, using this RNA transfection system, the amount of secreted infectious viruses often fluctuate and mutations emerge in HCV genome with multiple passages for an extended period of time (54), which limits its usefulness for antiviral screening and vaccine development.DNA-based expression systems for HCV replication and virion production have also been examined (5, 15, 21). With DNA-based expression systems, transcriptio...