Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 ؋ 10 8 50% tissue culture infective doses (TCID 50 )/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus that has only recently been adapted to tissue culture (22,41,46). The full-length genome of isolate JFH-1 was demonstrated to be competent for viral particle production in tissue culture (22,41,46) by using Huh-7-derived cell lines that are permissive to HCV infection and replication (2,20). Several of these HCV cell culture (HCVcc) systems have been described, the most robust of which are based on chimeric J6/JFH-1 viruses or tissue culture-adapted strains of JFH-1 (1,3,4,17,18,26,36,47). However, the quantity of infectious virions these systems can produce is limited, presumably due to currently unidentified constraints on infectious virus particle production in tissue culture (5,16,26,47). After passage of tissue culture-grown J6/JFH-1 virus in animals, the resultant viruses exhibited a higher specific infectivity (23). Similarly, passage of HCV in primary human hepatocytes yielded virus with higher specific infectivity (33). Although Huh-7 cells are currently the most efficient system for culturing of HCV, these data suggest that virus particle production is suboptimal in these cells compared to that in bona fide hepatocytes.Serial passage of HCV in cell culture has yielded virus isolates with increased viral titers (9,10,32,37,44). Interestingly, these ...

Section: Discussionmentioning

confidence: 99%

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Pokrovskii

Bush

Beran

et al. 2011

“…Since previous reports have indicated that NS2 is a relatively short-lived protein (t 1/2 of approximately 3 h [35,46]), mutations destabilizing NS2 could lead to diminished levels and loss of function and explain the observed decreased virus titers. While the D2A, Q8A, W35A, W36A, F77A, K81A, W82A, and L92A mutants did display lower viral protein levels (Fig.…”

Section: Initial Characterization Of the N-terminal Region Of Ns2 By mentioning

confidence: 99%

“…A number of cell culture-adaptive mutations and compensatory mutations that enhance or restore virus production have been mapped to the NS2 N-terminal region (9,28,(31)(32)(33)(34). Likewise, mutagenesis of select residues within the N-terminal domain has implicated the involvement of NS2 in the early steps of the assembly process (9,25,28,35,36). Sequence alignment of several HCV genotypes demonstrates high conservation in the protease domain (aa 94 to 217), and the crystal structure of this domain indicates that NS2 forms a dimer containing a composite active site analogous to those of cysteine proteases (37).…”

mentioning

confidence: 99%

Genetic and Functional Characterization of the N-Terminal Region of the Hepatitis C Virus NS2 Protein

Fuente¹,

Goodman²,

Rice³

2013

The hepatitis C virus (HCV) NS2 protein has dual roles within the HCV life cycle. While well characterized as an autoprotease that cleaves the NS2/NS3 junction, NS2, primarily via its N-terminal region, is also involved in virion morphogenesis. In order to map the determinants necessary for infectious virus production and gain further insight into the multiple points at which NS2 may impact this process, a detailed mutational analysis of residues spanning amino acids (aa) 1 to 92 was performed. Initial block mutagenesis (5 or 7 amino acid residues) in both bicistronic and monocistronic HCV cell culture-based (HCVcc) genomes revealed that all but two blocks had various levels of impaired infectious virus production. None of these mutations affected RNA replication, indicating that the N-terminal region of NS2 is not required for NS2-3 processing and replicase assembly. Fine mapping identified 29 critical residues that, when mutated, yielded at least a 1 log decrease in infectious virus titers. These mutants were characterized further with respect to release of extracellular HCV RNA and core, intracellular infectivity, thermal stability of virus particles, and NS2 interactions. While the most severely debilitated mutants were impaired early in the assembly process, which is in agreement with previous reports, others targeted later steps of virus production, most notably egress. Thus, in addition to participating in early steps in virion assembly, this comprehensive mutagenesis study suggests yet another role for NS2 in later steps in virus production. Hepatitis C virus (HCV) is a positive-strand RNA virus of 9.6 kb from the Flaviviridae family. An estimated 170 million individuals worldwide have been infected (1-3). Of these, 50% to 80% are chronic carriers and have an increased risk of developing hepatosteatosis, cirrhosis, hepatocellular carcinoma, and liver-related death. Phylogenetic analysis of various isolates has resulted in the classification of six main genotypes (gt) containing a number of subtypes (e.g., 1a, 1b, 1c, etc.) and the recent addition of a novel seventh genotype (1, 4). Due to the large sequence diversity of HCV and limited animal models (5), the development of broadly successful treatments such as with direct-acting antivirals (DAA) or prophylactic vaccine has been difficult. Multidrug combinations, similar to highly active antiretroviral therapy (HAART) for HIV infection, will likely be necessary to target different aspects of infection in order to reduce the possibility of viral resistance. The full scope of each viral protein's contribution to the HCV life cycle is still being determined, and such insight may provide additional targets to exploit.With the establishment of an HCV cell culture-based infection system (HCVcc) dependent on the Japanese JFH-1 (gt 2a) strain, a more complete picture of the viral life cycle has begun to emerge, especially for processes such as virus entry and assembly (3, 6, 7). The HCV genome contains one large open reading frame (ORF) encoding structural and no...

“…Several cell culture-adaptive mutations that increase virus yields have been mapped to NS2 (16,21,25,60,76), and mutagenesis of NS2 identified several residues that are important for infectious virus production (10,23,55,71,77). We previously identified second-site mutations in E1 (A78T), E2 (I360T), NS3 (Q221L), and NS4A (E42G) that suppressed defects in virus particle assembly caused by specific NS2 mutations (55).…”

mentioning

confidence: 99%

Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

Stapleford

Lindenbach

2011

122

132

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 Ϸ520-kDa and Ϸ680-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.