Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.     

The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells

The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.     

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A–NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.     

Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.     

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.Hepatitis C virus (HCV) is an enveloped RNA virus which is estimated to infect some 123 million individuals (24). In the majority of cases the virus establishes a chronic infection that can ultimately result in liver fibrosis, cirrhosis, or hepatocellular carcinoma. Thus, there is great interest in elucidating the mechanisms of viral replication, with a view to developing new chemotherapeutic agents. Since 1999, use of the subgenomic replicon system has led to significant progress in the understanding of the mechanism of viral RNA replication. It has been demonstrated that the five nonstructural proteins—NS3, NS4A, NS4B, NS5A, and NS5B—are necessary and sufficient to replicate an RNA molecule containing the 5′ and 3′ untranslated regions (UTRs) of the viral genome. However, apart from the RNA-dependent RNA polymerase (NS5B), the precise details of the roles of each of the nonstructural proteins in the process of RNA replication remain undefined. One problem associated with the subgenomic replicon system is the observation that the replicon RNA undergoes culture adaptation in which, as a result of the error-prone nature of the polymerase, mutations that confer enhanced replicative capacity are selected for in culture. Importantly, it has been shown using the chimpanzee model that, once engineered back into an infectious clone of the virus, such mutations may be attenuating in vivo (5). Recently, the HCV field has been revolutionized by the development of a cell culture infectious system based on a genotype 2a clone derived from a patient with fulminant hepatitis: the JFH-1 clone (30). JFH-1 is also unique in that subgenomic replicons derived from this clone are able to replicate efficiently without culture adaptation. This observation, as well as the fact that full-length genomes of JFH-1 are able to coordinate the coupling of RNA replication to packaging and release of infectious virus particles in Huh7 cells, points to fundamental differences between the RNA replication machinery of JFH-1 and that of the genotype 1b culture-adapted replicons.The majority of mutations conferring culture adaptation map to the region coding for the NS5A protein. NS5A is a zinc-binding phosphoprotein that, as well as playing a critical role in RNA replication, also interacts with a plethora of cellular proteins (18). The protein has been demonstrated to consist of three domains separated by low-complexity sequences (LCS) (29). Of particular interest is the observation that within LCS2 (between domains II and III) (Fig. ​(Fig.1a),1a), NS5A contains two closely spaced polyproline motifs that are able to bind to the SH3 domains of Src-family tyrosine kinases (16), as well as other SH3 domain containing proteins (e.g., Grb2 and amphiphysin II/Bin1) (23, 25, 33). These motifs, which we have termed PP2.1 and PP2.2 (Fig. ​(Fig.1a),1a), conform to the consensus SH3 binding motif Pro-X-X-Pro-X-Arg/Lys, where X is any amino acid (21). Interestingly, although the C-terminal motif (PP2.2) is absolutely conserved in all HCV genotypes, we and others have shown that it is dispensable for HCV RNA replication because alanine substitution of three prolines in this motif (shown to abolish SH3 domain interactions [16]) within a culture-adapted subgenomic replicon had either no effect (19) or resulted in only a modest reduction in replicative capacity (23, 33). The N-terminal motif (PP2.1), however, is only conserved in genotype 1 isolates, although within this motif a proline at residue 346 in genotype 1b (342 in genotype 2a) is absolutely conserved throughout all genotypes, which suggests it has an important role in virus replication.Open in a separate windowFIG. 1.Polyproline motifs in NS5A. (a) Schematic of the structure of NS5A showing the endoplasmic reticulum-membrane associating amphipathic helix (gray box) (4), the position of the coordinated zinc ion, and the three domains with interlinking LCS (black boxes) (29). The lower part of this figure shows the amino acid sequence of the region from residues 343 to 356. These correspond to polyprotein residues 2315 to 2328 in the genotype 1b infectious clone J4 (31). Note that in JFH-1 the corresponding residues in the polyprotein are 2311 to 2325 (residues 339 to 352 within the NS5A sequence). The prolines and basic residues of the SH3 binding motifs (Pro-X-X-Pro-X-Arg/Lys) are in boldface. The accession numbers for the six sequences are as follows: 1a infectious clone H77 (AF009606), 1b infectious clone J4 (AF054247), FK5.1 culture-adapted subgenomic replicon (AJ242654) (13), Con1 isolate (AJ238799), 2a infectious clone JFH-1 (AB047639), and 3a isolate (D17763). (b) Schematic of mutants constructed in the present study. The wild-type FK5.1 sequence is on the top line, residues mutated to alanine indicated by A in the subsequent lines, hyphens indicate unchanged residues.To shed more light on the role of these proline residues in the viral life cycle we have undertaken a mutagenic analysis both in the context of genotype 1b or 2a subgenomic replicons and the cell culture infectious JFH-1 clone. Our results point to key roles of these prolines in multiple stages of virus replication but highlight a surprising discrepancy between the requirements in the two systems.     

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

trans-Complementation of an NS2 Defect in a Late Step in Hepatitis C Virus (HCV) Particle Assembly and Maturation

Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase–mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.     

Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus

There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction ("H-NS2/NS3-J") and at a site of natural, intergenotypic recombination within NS2 ["H-(NS2)-J"] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.     

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

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.     

Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.     

A Novel Strategy To Develop a Robust Infectious Hepatitis C Virus Cell Culture System Directly from a Clinical Isolate

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10⁵ focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.     

PDlim2 selectively interacts with the PDZ binding motif of highly pathogenic avian H5N1 influenza A virus NS1

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.     

Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

Hepatitis C Virus (HCV) NS4B protein has many roles in HCV genome replication. Recently, our laboratory (Q. Han, J. Aligo, D. Manna, K. Belton, S. V. Chintapalli, Y. Hong, R. L. Patterson, D. B. van Rossum, and K. V. Konan, J. Virol. 85:6464–6479, 2011) and others (D. M. Jones, A. H. Patel, P. Targett-Adams, and J. McLauchlan, J. Virol. 83:2163–2177, 2009; D. Paul, I. Romero-Brey, J. Gouttenoire, S. Stoitsova, J. Krijnse-Locker, D. Moradpour, and R. Bartenschlager, J. Virol. 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.     

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly

Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.     

Species-Specific Contribution of the Four C-Terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence

Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian C-terminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.Interspecies transmission of influenza viruses can lead to the introduction of new subtypes of influenza virus into the human population (31). The emergence of a new influenza virus that is able to spread efficiently between humans can cause a pandemic, as evidenced by the recent introduction of the swine-origin 2009 A/H1N1 virus to humans (10). The spread of avian influenza A viruses from birds to humans could also lead to the introduction of a new viral subtype with pandemic potential (22). Fortunately, the efficient replication of avian influenza A viruses in humans and interhuman transmission are generally limited and require further adaptations of the virus to humans. One determinant of host adaptation lies in the receptor binding specificity of hemagglutinin (HA) (52). In addition, several reports have underlined the role of amino acid 627 of the PB2 polymerase subunit in determining viral host range and virulence (15, 36, 44, 45). Large-scale sequence analyses of viruses isolated from different bird and mammalian species have been performed in order to identify previously unrecognized determinants of host adaptation and virulence (2, 32). Those studies have identified a 4-amino-acid motif in the C-terminal domain of NS1 that could represent a previously unnoticed host adaptation motif. Indeed, the vast majority of avian influenza viruses have an NS1 protein with a C-terminal ESEV domain, while typical human viruses have a conserved RSKV domain. The conservation of these species-specific motifs in the NS1 protein despite important sequence variability in the rest of the protein suggests that these four C-terminal amino acids are under strong selection pressure in their respective natural hosts (3, 5, 25).NS1 is a multifunctional protein implicated in the regulation of viral gene expression and in the inhibition of the host antiviral response (12). In order to test the role of these newly identified NS1 domains, Jackson et al. previously introduced various C-terminal motifs into NS1 of the mouse-adapted human influenza virus A/WSN/33 strain by use of reverse genetics (24). Mice inoculated with a virus containing an avian C-terminal ESEV NS1 domain had high viral loads in the lungs and decreased survival compared to mice inoculated with a virus containing a C-terminal RSKV domain. These results showed that the C-terminal ESEV motif found in avian NS1 proteins increases virulence in mice when introduced into a human strain of influenza virus. Whether this finding also applies to avian influenza viruses remains unknown. Moreover, whether the C-terminal ESEV domain of NS1 increases replication in human cells remains unknown. Finally, how the C-terminal domains of NS1 modulate virulence in nonmammalian hosts, such as birds, is also unknown.Here, we assessed the contribution of the C-terminal domains of NS1 to the pathogenicity of an avian influenza virus. By using reverse genetics, we generated H7N1 viruses containing an NS1 protein with a C-terminal avian ESEV domain or a C-terminal human RSKV domain. The replications of these viruses in human, mouse, and duck cell were compared. In addition, we assessed their pathogenicity in mice and ducks. Our results show that the C-terminal RSKV domain increases the replication of an avian influenza virus in human cells. To our surprise, we observed that the C-terminal RSKV domain increases replication in ducks. In contrast, the C-terminal ESEV domain increases virulence in mice. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.