Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.     

Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.     

The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase

We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B(570), shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B(552), NS5B(544), NS5B(536) or NS5B(531), with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B(544), NS5B(536) and NS5B(531) showed markedly higher RdRp activities compared to the activities of NS5B(570) or NS5B(552). Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B(570) and NS5B(552) were changed to alanine, their activities were higher than that of the original NS5B(570). The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.     

De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.     

Enzymatic characterization of the full-length and C-terminally truncated hepatitis C virus RNA polymerases: function of the last 21 amino acids of the C terminus in template binding and RNA synthesis

The nonstructural protein NS5B of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp), which plays a central role in viral replication. Most of the reported studies on HCV polymerase in vitro have used a truncated form of the enzyme lacking the C-terminal 21 amino acids (DeltaC(21)-NS5B). In this study, we compared the enzymatic properties of the full-length NS5B (FL-NS5B) and this truncated form. Removal of the C(21) domain enhanced the enzyme stability. Both enzymes are capable of performing de novo and primer-dependent RNA syntheses, but each possesses a unique set of biochemical requirements for optimal RdRp activity. Whereas RNA synthesis by FL-NS5B remained relatively constant at 12-100 mM KCl, synthesis by DeltaC(21)-NS5B rapidly decreased at KCl concentrations greater than 12 mM. The different salt requirement for overall RNA synthesis by these two polymerases can in part be explained by the effect of monovalent ion concentration at the step of template binding, where binding by DeltaC(21)-NS5B but not FL-NS5B decreased proportionally as the KCl concentration increased from 25 to 200 mM. Thus, the C(21) domain appears to contribute to NS5B-RNA template binding, probably through the hydrophobic stacking interaction between its aromatic amino acids and the nucleotide bases of the RNA. This interpretation was supported by the observation that the C(21) polypeptide by itself could also bind to RNA to form binary complexes that were resistant to changes in the KCl concentration. Though both enzymes exhibited similar K(s) values for each of the four NTPs (1-5 microM), DeltaC(21)-NS5B generally required lower NTP concentrations than FL-NS5B for optimal synthesis. Interestingly, DeltaC(21)-NS5B became severely inhibited at elevated NTP concentrations, which most likely is due to competitive binding of the noncomplementary nucleotide to the polymerase catalytic center. Finally, the terminal transferase activity of DeltaC(21)-NS5B was found to be distinct from that of FL-NS5B on several different RNA templates. Together, these findings indicated that the HCV NS5B C(21) domain, in addition to being a membrane anchor, functions in template binding, NTP substrate selection, and modulation of terminal transferase activity.     

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

This study identified specific and avid RNA aptamers consisting of 2′-hydroxyl- or 2′-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2′-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2′-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2′-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2′-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.     

Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.

The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.     

An Uniquely Purified HCV NS3 Protease and NS4A21–34 Peptide Form a Highly Active Serine Protease Complex in Peptide Hydrolysis

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4°C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys–Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k_cat/K_M) of 205 and 196,000 M⁻¹ s⁻¹, respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease–NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k_cat/K_M of 5180 ± 670 M⁻¹ s⁻¹). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Broad, multispecific CD4⁺ and CD8⁺ T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8⁺ T-cell responses but low CD4⁺ T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4⁺ T helper responses but no CD8⁺ T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4⁺ T helper responses but no CD8⁺ T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4⁺ and CD8⁺ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.     

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V_HH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V_HH from a humanized-camel VH/V_HH display library. VH/V_HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V_HH hallmark and were designated V_HH6 and V_HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V_HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V_HH6 and V_HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V_HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.     

Functional characterization of fingers subdomain-specific monoclonal antibodies inhibiting the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.     

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world''s population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4⁺ and CD8⁺ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV⁺ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8⁻ Jurkat cells and CD4⁺ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.     

Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.     

Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.     

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3' end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.     

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.     

A comprehensive structure-function comparison of hepatitis C virus strain JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes

The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.