Regulation of Infectious Genotype 1a Hepatitis C Virus Production. by Domain III of NS5A

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1 JVI Accepts, published online ahead of print on April 0 J. Virol. doi:0./jvi.0-0 Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. JVI0-0 Version 0 Regulation of Infectious Genotype a Hepatitis C Virus Production by Domain III of NSA Seungtaek Kim, Christoph Welsch, MinKyung Yi, and Stanley M. Lemon * Division of Infectious Diseases, Department of Medicine; Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC - USA; Institute for Human Infections and Immunity and the Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX USA Running title: Role of NSA domain III of genotype a HCV * Corresponding author: Inflammatory Diseases Institute,.0 Burnett-Womack CB#, The University of North Carolina at Chapel Hill, Chapel Hill, NC - Tel: () -, Fax: () -0, smlemon@med.unc.edu. Downloaded from on October, 0 by guest

2 ABSTRACT 0 0 Although hepatitis C virus (HCV) assembly remains incompletely understood, recent studies with the genotype a JFH- strain suggest that it is dependent upon phosphorylation of Ser residues near the C-terminus of NSA, a multifunctional nonstructural protein. Since genotype viruses account for most HCV disease yet differ substantially in sequence from JFH-, we studied the role of NSA in production of HS virus. While less efficient than JFH-, genotype a HS RNA produces infectious virus when transfected into permissive Huh- cells. Exchange of complete NSA sequences between these viruses was highly detrimental to replication, while exchanges of the C-terminal domain III sequence (% amino acid sequence identity) were well tolerated with little effect on RNA synthesis. Surprisingly, placing the HS domain III sequence into JFH- resulted in increased virus yields; conversely, HS yields were reduced by the introduction of domain III from JFH-. These changes in infectious virus yield correlated well with changes in NSA abundance in RNA-transfected cells, but not RNA replication or core protein expression levels. Alanine-replacement mutagenesis of selected Ser and Thr residues in the C-terminal domain III sequence revealed no single residue to be essential for infectious HS virus production. However, virus production was eliminated by Ala substitutions at multiple residues, and could be restored by phosphomimetic Asp substitutions at these sites. Thus, despite low overall sequence homology, production of infectious virus is regulated similarly in JFH- and HS viruses by a conserved function associated with a C-terminal Ser/Thr cluster in domain III of NSA. Downloaded from on October, 0 by guest

3 0 0 Infection with hepatitis C virus (HCV) is associated with chronic hepatitis, progressive hepatic fibrosis leading to cirrhosis, and hepatocellular carcinoma (for a review, see ). The virus establishes life-long persistent infection in most infected persons. It is currently thought to infect 0-0 million people worldwide, placing them at risk for potentially life-threatening liver disease. Available interferon-based treatment is limited in its efficacy, and immunization currently is not possible. To overcome these shortcomings in therapeutic and preventive measures, there is a need to increase current understanding of HCV pathogenesis, including the molecular mechanisms involved at various steps in the virus life cycle and the role of virushost interactions in virus persistence and disease progression. HCV is an enveloped, positive-strand RNA virus classified within the genus Hepacivirus of the family Flaviviridae. Its. kb genome encodes a single polyprotein, which is co- and post-translationally processed by both viral and cellular proteases (). The N-terminal segment of the polyprotein comprises the structural proteins (core, E, and E), while the downstream proteins (p, NS, NS, NSA, NSB, NSA, NSB) are nonstructural. Only the segment extending from NS to NSB is required for genome replication (), with NSB, an RNA-dependent RNA polymerase, serving as the catalytic core of a membrane-bound, macromolecular viral replicase complex. p and NS are not required for RNA replication, and accumulating evidence supports important functions for both of these proteins in virus assembly and the release of virus from infected cells (,,,,, ). Emerging data also support essential roles for other nonstructural proteins, in particular NSA (,,,, ) and NS (,, ), in the assembly and release of infectious particles. Downloaded from on October, 0 by guest

4 0 0 The HCV NSA protein is unique, with no known human or viral homologs other than NSA in the closely related GB virus B (GBV-B). It appears to have multiple functions in the virus life cycle and to interact with numerous viral and host proteins (), but little is known about NSA at a mechanistic level. It is required for viral RNA replication and, as mentioned above, plays an essential role in virion production. It is a phosphoprotein (, 0, ), and its phosphorylation status has been suggested to regulate genome replication and virus assembly (, ). It is also a frequent site of cell culture-adaptive mutations that promote the amplification of HCV replicon RNAs in Huh- hepatoma cells (). At a structural level, there appear to be three distinct domains that are separated by segments of the protein that are relatively disordered. The N-terminal domain (domain I) contains a Zn-binding motif and is essential for viral RNA replication (). Two high-resolution structural models have been developed for domain I of NSA based on X-ray crystallography (, ). While they differ significantly, both models suggest a dimeric structure that associates with lipid bilayers in membrane-bound replicase complexes. Importantly, NSA mutants that are deficient in the ability to support viral RNA replication can be trans-complemented in a cell culture system (). The middle domain II, which is involved in antagonizing innate immune responses, and C-terminal domain III of NSA appear to play lesser roles in viral RNA replication (0, 0). Nonetheless, mutations within domain III may reduce the efficiency of genome amplification. The amino acid sequence of domain III is poorly conserved between different viral genotypes and has been considered to be relatively unstructured (). Nonetheless, recent data suggest that it plays an important role in the assembly of infectious virus particles. This aspect of NSA function has emerged from studies of the genotype a JFH- virus, a unique strain of HCV that replicates efficiently in cell culture. Transfection of synthetic JFH- genomic RNA produces Downloaded from on October, 0 by guest

5 0 0 relatively high titers of infectious virus in permissive Huh- hepatoma cells (,, ). During an early step in the process of particle assembly, the JFH- NSA protein is recruited to the surface of cytoplasmic lipid droplets that are decorated with the viral core protein (, ). Genetic evidence also suggests that NSA, in association with NS, acts at a later step in virus production, following intracellular particle assembly but prior to release of infectious virus (). Several recent studies indicate that domain III of the JFH- NSA protein is particularly important for the production of infectious virions, and that phosphorylation of Ser residues within this domain, possibly by cellular casein kinase II (CK II), is required for this process (,,, ). We have found the construction and analysis of inter-genotypic chimeric HCV genomes to be a productive approach to characterizing functional interactions among HCV proteins that are important in the virus life cycle (, ). We adopted a similar approach to investigate the role of NSA in production of infectious virus, generating inter-genotypic NSA chimeras within the background of JFH-, and a genotype a molecular clone, HS, that also produces infectious particles when transfected into cells as RNA (). HS RNA produces substantially fewer infectious particles in transfected cells than JFH- RNA, but like JFH- virus produced in cell culture (), recent studies have shown that virus produced from HS RNA (with an additional adaptive mutation in E) is infectious for chimpanzees as well (unpublished data). We exchanged the entire NSA sequences between these two genotypes, as well as only the domain III sequences, and observed the effects of these exchanges on intracellular genome amplification and infectious virus production. We also carried out a mutational analysis of potential phospho-acceptor sites within the C-terminal domain III sequence of the NSA protein in HS virus, and compared these results with previously published studies of Downloaded from on October, 0 by guest

6 JFH- virus. Despite limited sequence identity, our data indicate that domain III of the genotype a HS NSA protein shares conserved structural and functional elements with the JFH- protein that are essential for production of infectious virions. 0 0 MATERIALS AND METHODS Plasmids. The cell-culture adapted phs infectious molecular clone of genotype a HCV has been described previously (). phs. is a modified version of this plasmid that has enhanced capacity for production of infectious virus in cell culture and that contains an additional ND mutation in E and lacks the Q0R adaptive mutation in NS (). No see m cloning (0) was used to generate NSA chimeras in the phs (), pjfh- (), and chimeric inter-genotypic phj- () plasmid DNAs without altering the original genome sequences outside of NSA or NSA domain III. The SapI restriction enzyme was employed for deletion and insertion of heterologous sequences since its recognition site is nonpalindromic. For Ser/Thr to Ala or Asp substitution mutations, the QuickChange mutagenesis kit (Stratagene) was used. DNA sequencing verified the integrity of the manipulated sequences and the presence of the intended substitution mutations. Cells. Huh-. cells () were kindly provided by Dr. Charles Rice and Apath, L.L.C. The cells were grown in DMEM high glucose medium containing 0% fetal bovine serum and x penicillin/streptomycin at C in a % CO environment. RNA transcription and transfection. Plasmid DNAs were linearized by XbaI restriction digestion. Synthetic RNA was transcribed from linearized DNAs using the MEGAscript kit (Ambion). The concentration and integrity of the transcribed RNAs were confirmed by spectrophotometry and denaturing agarose gel electrophoresis. In-vitro transcribed RNAs Downloaded from on October, 0 by guest

7 (. µg) were transfected into Huh-. cells in six-well culture dishes ( x 0 cells/well) using the TransIT-mRNA transfection reagent (Mirus Bio) as recommended by the manufacturer. Six hrs after transfection, the culture medium was replaced with fresh medium. The cells were split at a : ratio before further incubation at C in a % CO incubator. 0 DNA transfection. For NSA protein expression studies, 0. µg of plasmid DNA was mixed with 0. µg pcmv-gluc plasmid (New England BioLabs) and transfected into Huh-. cells in -well culture dishes ( x 0 cells/well) using TransIT-00 reagent (Mirus Bio) as recommended by the manufacturer. NSA species were quantified in immunoblots using different antibody probes (see text), and the results normalized to the luciferase activity present in supernatant fluids of transfected cultures. Virus titration. Culture supernatants were collected days after transfection with the synthetic RNAs, and the titer of infectious virus determined by inoculation of serial 0-fold dilutions onto naïve Huh-. cells seeded one day previously in -well chamber slides (Lab- Tek) (X0 cells/well). Three days after inoculation, infected cells were fixed with methanol/acetone and labeled for detection of HCV core protein by immunofluorescence microscopy as described (). The titer of infectious virus was determined from the number of foci of infected cells observed at each dilution, and is expressed as focus-forming units (FFU) per ml. To quantify intracellular infectious virus, cell lysates were prepared by multiple freeze/thaw cycles as described () and similarly tested. Downloaded from on October, 0 by guest 0 Immunoblots. A standard immunoblot procedure was employed (). Protein samples transferred to PVDF membranes were probed with the following primary antibodies: anti-core (:,000, Affinity BioReagents, MA-00), E0 (kindly provided by Dr. Charles Rice and Dr. Tim Tellinghuisen), rabbit polyclonal anti-nsa (kindly provided by Dr. Craig Cameron), anti-

8 0 0 NS (Virogen, -A), anti-actin (Sigma, A), anti-myc (Cell Signaling, #), and anti- GAPDH (Ambion, AM00). Proteins were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Southern Biotech, 000, 000) and enhanced chemiluminescence detection (Amersham ECL, GE Healthcare). For quantitative assessment of immobilized protein, proteins were visualized with IRDye 00CW Goat anti-mouse IgG or IRDye 0 Goat anti-rabbit IgG, and images collected on an Odyssey infrared imaging system (LI-COR Biosciences). HCV core ELISA. The amount of soluble HCV core protein released into cell culture media by transfected cells was quantified using the Ortho Diagnostics Trak-C ELISA kit (Ortho-Clinical Diagnostics) according to the manufacturer s instructions. RT-PCR. For quantitative measurement of HCV RNA replication capacity, Huh-. cells were transfected by electroporation () and total cellular RNA isolated at the time of harvest using the RNeasy Mini Kit (Qiagen). Real-time qrt-pcr reactions were carried out with the iq Real-Time PCR Detection System (Bio-Rad) as described (). CK II inhibitor treatment. Cell cultures were split as above hrs after HCV RNA transfection, and refed with media containing -dimethylamino-,,,-tetrabromo-hbenzimidazole (DMAT), a specific CK II inhibitor (Calbiochem). The cells were incubated for hrs followed by refeeding the cells with fresh media (no DMAT). Culture supernatant fluids were collected one day later and assayed for infectious virus as above. Cytotoxic effects of DMAT were assessed using the WST- Cellular Proliferation Assay (Roche Applied Sciences) as recommended by the manufacturer. Downloaded from on October, 0 by guest

9 RESULTS 0 0 Inter-genotypic NSA domain exchanges and genome replication. Genome-length HCV RNAs transcribed from the genotype a pjfh- and a phs plasmids produce infectious virus particles when transfected into Huh- hepatoma cells (,, ). As described above, several prior studies suggest that the C-terminal domain III of the NSA protein is essential for assembly of infectious JFH- virus particles and that phosphorylation of this domain may regulate assembly (,,, ). While it is likely that this is also true for the genotype a HS virus, the genotype a NSA protein has not been similarly investigated. Since genotype infections account for the large majority of infections in most geographic regions (), it is important to study these events in the context of genotype virus. This is especially so since the HS NSA protein shares only % amino acid identity with the JFH- protein overall, and only % within domain III (Fig. A). To determine whether these NSA proteins can function interchangeably, despite the differences in amino acid sequence, we exchanged sequences encoding the entire NSA protein, or NSA domain III only, within the background of these full-length constructs (Fig. B). To avoid any changes in the HCV sequence outside of the exchanged domains, we employed no see m cloning (0) to generate the mutants. The chimeric sequences were verified by sequencing the plasmid DNAs. The impact of these NSA domain swaps on RNA replication was determined following their transfection into Huh-. cells using a real-time, quantitative RT-PCR assay, comparing RNA abundance relative to the abundance of a replication-defective control RNA containing a GND substitution in the GDD motif of NSB (Fig. C). These results demonstrated similar increases in the relative intracellular abundance of the parental HS and JFH- RNAs over Downloaded from on October, 0 by guest

10 0 0 0 the hrs following transfection (Fig. C, left and right panels, respectively). In contrast, both chimeric RNAs with full-length NSA swaps demonstrated significant impairments in RNA replication. The JFH/HA RNA (HS NSA in the JFH- background, Fig. B) showed no increase in abundance when compared to the lethal GND mutant (Fig. C, right panel), while the HS/JA RNA demonstrated a >0-fold reduction in RNA abundance compared with the parental HS RNA by hrs post-transfection (Fig. C, left panel). Consistent with these findings, no core protein expression was observed in cells transfected with either of these fulllength NSA chimeras (Fig. D, lanes and ). These results thus demonstrate that major differences in the sequence and/or structure of the genotype a and a NSA proteins preclude them from functioning interchangeably in support of viral RNA replication. Very different results were obtained with the chimeras in which only domain III of NSA was exchanged: both replicated in a fashion very similar to the related parental RNAs (Fig. C, left and right panels). A modest decrease in accumulation of HS/JAd RNA (HS background with NSA domain III of JFH, Fig. B) relative to the parental HS RNA, evident at hrs only (Fig. C left panel), was matched by a small reduction in core protein expression detectable by immunoblot (Fig. D, lane versus lane ). However, any impact of the domain III swap on viral RNA replication was minimal, and completely undetectable in the JFH- background. This indicates that the major incompatibilities that preclude functional exchange of the full-length NSA proteins with respect to viral RNA replication reside upstream of domain III and in domains I or II. Downloaded from on October, 0 by guest Inter-genotypic NSA domain exchanges and infectious virus production. Infectious virus particles released by the RNA-transfected Huh-. cells were quantified by a fluorescent-focus infectivity assay (, ). Although no overlay is used in this assay, we have

11 0 0 confirmed that there is a linear relationship between the number of focus-forming units (FFU) obtained and the dilution of an HCV inoculum (unpublished data). This correlation indicates that each focus is initiated by a single infectious particle and validates the assay. Both parental RNAs (HS and JFH) produced infectious virus that was detectable in culture supernatant fluids by days post-transfection, although about 0-fold more infectious virus was secreted into the media of cells transfected with JFH- versus HS RNA (Fig. A), as described previously (). Nonetheless, the titer of infectious HS virus produced in these experiments was sufficient to reliably determine the impact of NSA domain exchanges on infectious virus production. No infectious virus was released from cells transfected with the HS/JA or JFH/HA chimeric RNAs even when monitored up to days after transfection. This is consistent with the impairments found in viral RNA replication described above (Fig. A). In contrast, the inter-genotypic exchange of domain III only (HS/JAd and JFH/HAd) resulted in RNAs that could produce infectious virus, despite the low level of amino acid sequence identity shared by these domains (%) (Fig. A and Fig. A). Interestingly, when domain III of HS NSA was placed within the JFH- background, there was a significant increase (variable, but averaging almost 0-fold in replicate experiments) in the infectious virus yield (Fig. A). As discussed above, this occurred without any significant change in intracellular RNA replication (Fig. C, right panel), or intracellular core abundance (Fig. D, lanes and ). The reverse exchange (domain III of JFH- into the HS background) resulted in a ~0% decrease in infectious virus yield at days (Fig. A). While there was no difference in the intracellular abundance of the HS and HS/JAd RNAs at - hrs (Fig. C, left panel), the intracellular core abundance was somewhat reduced in HS/JAd-transfected cells (Fig. D, lanes versus ). Collectively, these Downloaded from on October, 0 by guest

12 0 0 results are consistent with a major role for domain III of NSA in infectious particle assembly, as described previously (,,, ). They also demonstrate that domain III from genotype a and a NSA function interchangeably with respect to virus assembly/release, albeit with varying efficiencies, and that the substantially greater infectious virus yield obtained with JFH- versus HS is not due to greater functionality in this domain of NSA. In fact, somewhat surprisingly, the HS NSA domain functions more efficiently in virus assembly and release than the native JFH- NSA domain. The amount of core protein released into supernatant cell culture fluids has been used as a surrogate measure of infectious JFH- virus production (), but does not correlate well with the release of infectious HS virus from cells (). As shown in Fig. B, core protein was detected by ELISA in media from cultures transfected with each of the chimeric RNAs that were capable of producing infectious virus, but not with the two RNAs with complete NSA swaps and impaired viral RNA replication (Fig. B). However, while the reduction in infectious HS/JAd virus compared to HS was matched by a comparable reduction in core protein released into the supernatant fluids, this was not the case with the JFH/HAd chimera. The reproducible increase in virus yield observed with JFH/HAd versus JFH- RNA (Fig. A) was not matched by an increase in extracellular core abundance (Fig. B). We conclude from these results that the measurement of extracellular core abundance by ELISA is not always a reliable indicator of cell culture-infectious HCV yield, despite its use as such in the past. NSA expression and infectious virus yields. While intracellular core expression was greater in cells transfected with JFH- versus HS RNA (and their related domain III chimeras), immunoblots suggested much larger differences in NSA expression (Fig. D). A much stronger NSA signal, appearing as two distinct bands (presumably hypo- and hyper- Downloaded from on October, 0 by guest

13 0 0 phosphorylated isoforms), was present in immunoblots of lysates of the genotype a JFH- transfected cells compared with lysates from the genotype a HS-transfected cells. Interestingly, there was a rough correlation between NSA abundance and production of infectious virus by the different RNAs. NSA expression was greatest with JFH/HAd, somewhat less in cells transfected with JFH- RNA, and markedly reduced, evident only as a single band and only with prolonged exposure of blots, with HS (Fig. D, compare lanes,, and ). An even fainter, single band was at the extreme limits of detection in lysates of cells transfected with HS/JAd (lane ). Despite the substantial difference in the amino acid sequences of the HS and JFH proteins, this striking difference in the apparent abundance of NSA is unlikely to be due to antigenic variation. The monoclonal antibody used in these blots (E0) was raised to a genotype b (Con strain) NSA protein (), the amino acid sequence of which is 0% identical to HS NSA, but only % to JFH. In addition, similar results were observed with a rabbit polyclonal antibody to genotype b NSA (data not shown). These results are thus more likely to reflect significant differences in absolute NSA abundance. Nonetheless, to confirm that the E0 antibody efficiently detects HS NSA and to ascertain whether there is any bias in its recognition of genotype a vs. a NSA, we ectopically expressed these proteins with an N-terminal Myc tag in Huh-. cells. We then probed immunoblots of cell lysates with E0 and an anti-myc antibody in parallel (Fig. A), quantifying the signal obtained with each antibody. These results revealed that the JFH/HAd chimeric protein bound significantly more E0 relative to anti-myc antibody (explaining the more intense labeling of this protein in Fig. D), but demonstrated that there is no significant difference in the recognition of HS vs. JFH NSA by E0 (Fig. B). This Downloaded from on October, 0 by guest

14 0 0 confirms that the remarkable differences observed in immunoblots of NSA in lysates of cells transfected with HS vs. JFH RNA (Fig. D) reflect absolute differences in protein abundance. In contrast to what was observed in cells transfected with replication-competent viral RNAs (Fig. D), ectopically expressed JFH NSA migrated as a single protein band (Fig. A). A direct comparison indicated that this was the hypo-phosphorylated species, while ectopically-expressed HS NSA appeared to be hyper-phosphorylated (data not shown). Collectively, these results suggest that infectious virus yield might be limited by the abundance of NSA more than viral RNA replication. Since lesser differences were observed in the expression levels of core (and core and NSA are processed from the same polyprotein), the large differences observed in NSA abundance suggest that HS NSA protein may be less stable than JFH- NSA. Immunoblots showed no consistent difference in the abundance of HS vs. JFH NSA when these proteins were expressed ectopically in Huh-. cells (Fig. ), indicating that there is little intrinsic difference in the stability of these proteins. While it remains possible that the stabilities of the phosphorylated proteins differ within infected cells, the low level of NSA expressed by replicating HS RNA precluded a formal comparison of its stability with the JFH- protein. It would not be surprising if differences in the phosphorylation status of NSA also impacted the function of the proteins expressed by genotype a and a RNAs. Differences in the predicted molecular masses of the genotype a, a, and chimeric NSA proteins (see Fig. D) confound a direct comparison of their phosphorylation status by simple gel analysis. Nonetheless, there are clear differences evident in the immunoblot shown in Fig. D. JFH- NSA was expressed as two isoforms of approximately equal abundance, with a modest increase in the hypo-phosphorylated form in cells transfected with the JFH/HAd RNA, Downloaded from on October, 0 by guest

15 0 0 relative to the hyper-phosphorylated isoform (compare lanes and ). In some but not all experiments, we also noted a small amount of a third JFH/HAd band migrating with an apparent molecular mass greater than that of the hyper-phosphorylated form (lane, s ). In contrast, as noted above, only a single NSA band was evident in cells transfected with HS and HS/JAd RNA. While the phosphorylation status of the single HS NSA band was difficult to assess in these experiments, it is interesting to note that it migrated more rapidly in SDS-PAGE than the hypo-phosphorylated JFH/HAd isoform, despite having a greater predicted molecular mass in the absence of post-translational modification (. versus. kda) (Fig. D, compare lanes and ). We were unable to detect HS NSA in these cell lysates following treatment with λ-ppase (data not shown). The apparent molecular mass of the HS protein in SDS-PAGE is addressed in additional detail below. Domain III exchange promotes intracellular assembly of JFH- virus. The enhanced release of infectious virus by cells transfected with the chimeric JFH/HAd RNA versus its JFH- parent could be due either to enhanced assembly of intracellular particles, or facilitation of the release of such particles from the cell. To address this question, we determined the amount of infectious virus present in intracellular and extracellular fractions of RNA-transfected cells. Multiple freeze/thaw cycles were used to prepare intracellular lysates days after transfection, and the titer of infectious virus present in these lysates and the related culture supernatant fluids was determined in the infectious focus assay. While a somewhat lesser (~- fold) increase in extracellular infectious virus yield was observed in this series of experiments than in those described above when cells were transfected with the JFH/HAd chimera versus JFH- RNA, the abundance of intracellular infectious particles was increased ~0-fold (Fig. ). Thus, it is likely that the primary impact of the domain III swap is on enhanced viral Downloaded from on October, 0 by guest

16 assembly within the cell, and that the increase observed in the release of infectious virus is secondary. Of all infectious virus present in these cultures days post-transfection, % of the JFH- virus was present in the extracellular fluids, compared to only % of JFH/HAd virus. 0 0 Since it is possible that the increase in intracellular infectious particles observed with the JFH/HAd RNA could reflect an enhanced interaction between NSA and core protein coating lipid droplets (, ), we compared the intracellular localization of core and NSA in cells transfected with JFH/HAd and JFH- RNAs using laser-scanning confocal microscopy. These studies demonstrated no detectable differences in the cellular localization, or in the degree of co-localization of these two viral proteins (data not shown). As an alternative approach to assessing the role of the structural proteins in the enhanced production of infectious virus by JFH/HAd, we created a similar domain III swap in the background of HJ- virus, an inter-genotypic chimera containing the entire structural protein region (core through NS) from the genotype a HS virus in the background of the genotype a JFH- RNA (, ) (Fig. A). The JFH/HAd construct thus places the HS domain III back in context with the HS core protein. In contrast to the increase in infectious virus yield we observed when domain III from HS was placed into the JFH background (Fig. A), swapping the HS domain III into HJ- caused nearly a ten-fold reduction in infectious virus yield (Fig. B) and 0-fold decrease in core protein secretion (Fig. C). This was associated with a marked decrease in core protein expression, as well as a lower abundance of NSA with loss of most of the hyper-phosphorylated isoform (Fig. D). Downloaded from on October, 0 by guest The NSA proteins encoded by JFH- and HJ- viruses are identical, and the proteins expressed by these two RNAs each show a slight dominance of the faster migrating of the two

17 0 0 major isoforms of NSA (compare Figs. D and D). JFH-/HAd and HJ-/HAd (which share the same chimeric NSA sequences in the background of JFH- nonstructural proteins) were very different in this regard. JFH/HAd was much like JFH-, with only a slight dominance of the fast migrating species, while HJ-/HAd expressed mostly the hypophosphorylated isoform with little of the more slowly migrating species present (compare again Figs. D and D). To ascertain whether the domain III swap influenced RNA replication in the context of the HJ- chimera, we compared RNA replication kinetics in cells transfected with modified genomes in which the envelope-coding regions had been deleted (to prevent any confounding influence from infectious virus production and virus spread in the transfected cell culture). These results revealed that the NSA domain III swap had a substantial, negative effect on RNA replication in the context of the genotype a/a HJ- chimera, resulting in a delay in RNA amplification and a reduction in intracellular viral RNA accumulation of approximately ten-fold (Fig. E). This is different from the absence of any such effect when the HS domain III sequence was placed into JFH- virus (Fig. C, right panel), and explains the reduced core abundance in cells transfected with HJ-/HAd RNA. However, the NSA abundance was well preserved relative to the amount of core protein present in these cells (Fig. D). Collectively, these data provide genetic evidence for interactions between NSA and the upstream structural proteins that influence NSA phosphorylation status and the efficiency of genome amplification. While most previous studies indicate that mutations in domain III have little impact on replication of the viral RNA, recent data from Hughes et al. () are also consistent with domain III playing a role in RNA replication. Downloaded from on October, 0 by guest Virus production mediated by domain III of HS NSA is sensitive to a CK II inhibitor. RNAi-mediated gene silencing and chemical Inhibitor studies reported by

18 0 0 Tellinghuisen et al. () suggest that host CK II phosphorylates Ser- in domain III of the JFH NSA protein, and that this is essential for production of infectious virus. To determine whether the assembly function of domain III in HS is equivalently sensitive to CK II inhibition, we assessed the effect of the CK II inhibitor, -dimethylamino-,,,-tetrabromo- H-benzimidaozle (DMAT) on production of infectious virus by JFH/HAd RNA, which contains the HS domain III in the JFH- background. Cells transfected with JFH- RNA were treated in parallel, and demonstrated a modest decrease in infectious virus yields at µm, and a marked decrease at 0 µm concentration (Fig. A, left panel). While the specificity of a kinase inhibitor is always cause for concern, the data confirm that JFH virus production is inhibited by DMAT (). However, under the conditions we used, virus production appeared to be less sensitive to inhibition by the compound than reported previously. Importantly, JFH/HAd RNA was equally sensitive to inhibition of infectious virus production by DMAT (Fig. A, right panel). NSA and NS protein abundance was moderately reduced at 0 µm, while NS abundance did not seem to be affected (Fig. B). Higher concentrations of DMAT were not tested as 0µM was at the cusp of significant cellular toxicity under the conditions used, as determined by WST- cellular proliferation assays (Fig. C). These results are consistent with the HS and JFH domain III assembly functions being equivalently sensitive to DMAT inhibition, although the effects of the compound, both on virus production and cellular toxicity were less than previously described (). A conserved cluster of Ser residues in domain III of NSA regulates production of infectious HS virus. There is only a limited understanding of which of the many potential phospho-acceptor sites in NSA sequence are actually phosphorylated during virus replication, the functional role of such phosphorylation, or which cellular kinases are Downloaded from on October, 0 by guest

19 0 0 responsible for it. Ser-, near the C-terminus of domain II, has been suggested to be a major site of phosphorylation in the wild-type HS NSA protein (), while mutational analyses suggest that phospho-acceptor sites in domain III are important for basal phosphorylation (). While Ala replacements of these residues generally do not affect RNA replication, Tellinghuisen et al. () suggested that CK II-mediated phosphorylation of Ser- of the JFH- NSA protein (domain III) is essential for intracellular virus assembly. Ser- was predicted to be a site of CK II phosphorylation by the web-based NetPhosK server (). It is located within an ideal sequence context for CK II phosphorylation, just upstream of several acidic residues ( SEED where S is Ser-) (). Although the amino acid sequences of domain III of the JFH- and HS proteins have only % identity, there are two short regions of exact identity: HS residues - (also - in JFH), and - (- in JFH) (Fig. A). Interestingly, the latter of these two regions of identity is immediately upstream of Ser- in the JFH- sequence (Fig. A). This sequence also overlaps a domain identified by Masaki et al. () (residues -) as essential for the recruitment of NSA to core protein decorating lipid droplets, a necessary early step in JFH- virus assembly (). While the results of Tellinghuisen et al. () and Masaki et al. () differ significantly in some respects, in aggregate they suggest that potential Ser phospho-acceptor sites within this region (Ser-, Ser-, and Ser-) are important for JFH- assembly. All three of these Ser residues are conserved in the HS sequence (Ser-, Ser-, and Ser-, Fig. A), and their presence in the HS protein could thus account for the ability of the HS domain III to function in assembly of JFH/HAd virus. However, the HS sequence lacks the CK II SEED motif identified by Tellinghuisen et al. (), and the NetPhos.0 Server predicts instead (with very low probability) CK II phosphorylation of Ser- and Thr- (Fig. A). Downloaded from on October, 0 by guest

20 0 0 0 To ascertain the role of these HS residues in assembly and release of HS virus, we created a series of HS RNA mutants in which Ser-, Ser-, Ser-, Ser-, and Thr- were individually substituted with Ala, and assessed their ability to produce infectious virus following transfection into Huh-. cells. The results of these experiments, repeated with each construct in at least two replicate experiments with independently-transcribed RNAs, are shown in Fig. B. Each of these single-site mutations had an adverse impact on infectious virus production, with the most profound effect associated with SA (~0% reduction in infectious virus yield) and TA (~0%). Alanine substitutions at Ser- and Ser- had only a modest impact (~% decrease), while the SA mutant was least affected (~%). Because Masaki et al. () found cumulative effects of multiple Ala substitutions in this region of the JFH- protein, we also evaluated several HS mutants in which two of the potential Ser/Thr phospho-acceptor sites were substituted with Ala. Two of these demonstrated substantially greater defects in virus production: mutant AWA, containing both SA and SA substitutions (~% reduction), and mutant ASA, containing both SA and TA substitutions (~% reduction) (Fig. B). While none of the double Ala substitutions we evaluated completely abolished infectious virus production, no detectable infectious virus was released from cells transfected with a mutant in which each of the suspect Ser residues was substituted with Ala (mutant SA, Fig. B). Importantly, the mutants demonstrating the greatest defects in infectious virus production were capable of nearly wild-type RNA replication (Fig. C), and cells transfected with these RNAs expressed core at an abundance equal to or only slightly reduced from that of the parental HS RNA (Fig. D, compare lanes with and ). The results of this mutational analysis thus indicate that the assembly and release of infectious genotype a HS virus, like genotype a JFH- virus (), is likely to be dependent upon phosphorylation of several members of a cluster of conserved Ser residues near the Downloaded from on October, 0 by guest

21 extreme C-terminus of domain III. While a role for Thr phosphorylation in NSA has not been identified previously in HCV assembly, Thr- appears to be necessary for efficient production of HS virus, but not by itself sufficient in the absence of two or more of the upstream Ser residues. 0 0 To assess the impact of these mutations on the phosphorylation status of NSA, we subjected lysates of RNA-transfected cells to immunoblot analysis. Lysates of cells containing genotype a (H) and b (HCV-N) replicons (, ) were studied in parallel, as this comparison provided some additional information on the phosphorylation status of NSA expressed by HS RNA. Lysates of HCV-N replicon cells demonstrated two distinct NSA bands, the lower of which accounted for most of the protein present, and the upper of which, while of low abundance, co-migrated in SDS-PAGE with the single NSA band present in either the HS replicon cells or cells transfected with HS RNA (Fig. D, compare lane with lanes and ). Since the genotype a HS and b HCV-N proteins have relatively similar predicted molecular masses in the absence of post-translational modification (. vs.. kd, respectively), this suggests that the single HS band we observed is likely to represent the hyper-phosphorylated form of the protein. Importantly, little NSA protein was detected in lysates of the AWA and SA mutant-transfected cells (Fig. D, lanes and ). A very faint band migrating slightly faster than HS NSA was evident in the AWA lysates with increased exposure time (lane, marked by o ), while no NSA was detected in SA mutanttransfected cell lysates, despite the presence of appreciable core protein. Downloaded from on October, 0 by guest Rescue of virus production with Ala to Asp substitutions in AWA and SA mutants. To more completely document the loss of infectious virus production accompanying the SA mutation in HS virus (Fig. B), we engineered these mutations into phs., a

22 0 0 modified version of phs that produces ~-fold more infectious virus from RNA transfected cells (unpublished data). In replicate experiments, HS. produced over 00 FFU/ml by days post-transfection, while no virus (<0 FFU/ml) was produced by HS./SA (Fig. A), confirming almost a 00-fold reduction in infectious virus production with Ala substitutions at these residues near the C-terminus of NSA. To determine whether replacement of these Ala substitutions with a residue containing a phosphomimetic side-chain, Asp, would rescue production of infectious virus, we constructed a HS./SD mutant as well as HS./DWD in which the Ala residues at and (Fig. B) were replaced with Asp. In sharp contrast to the AWA mutant, HS./DWD produced almost as much infectious virus as HS., while the SD mutant regained the capacity to produce infectious virus, albeit at a level substantially less than HS. (Fig. B). Remarkably, NSA expression, which was much reduced with the AWA and SA mutants despite the absence of any significant reduction in viral RNA synthesis, was largely restored with the DWD and SD mutants (compare Fig. D with Fig. B, bottom panels). Taken collectively, the data shown in Figs. and are strongly suggestive of the need for phosphorylation at or more of the Ser residues located at positions,, and of NSA for production of infectious HS virus. The changes noted in the apparent abundance of NSA in the mutants (Fig. D and A) may be due in part to decreased stability of the mutant proteins. When ectopically expressed as Myc-tagged proteins in Huh-. cells, the abundance of HS/SA ranged from -% that of the wild-type HS protein (depending on whether it was detected in immunoblots with the E0 monoclonal antibody or anti-myc), while HS/SD was.-. fold more abundant (Fig. B). Downloaded from on October, 0 by guest

23 DISCUSSION 0 0 A longstanding limitation in hepatitis C research has been a lack of virus strains that are capable of replicating well both in cell culture and in chimpanzees, still the only available animal model of chronic hepatitis C. While self-amplifying RNA replicons have been successfully established using cloned sequences from multiple HCV strains, very few viral RNAs can be coaxed to produce detectable quantities of infectious virus in cell culture (,, ). In part, this may be due to conflicting effects of cell culture-adaptive mutations on RNA replication and infectious particle production (, ), but many gaps remain in our understanding of the process of virus assembly. Multiple lines of evidence support a role for NSA, and in particular domain III of NSA, in assembly of infectious HCV particles (,,, ). We studied the role of this domain in the genotype a HS virus by creating chimeric viruses in which the entire NSA sequence or only domain III were swapped between it and JFH- virus (genotype a). Both of these HCV RNAs are competent for infectious virus production when transfected as RNA into permissive cells, but they generate quantitatively different yields of virus. Cells transfected with HS RNA release between 0-0 FFU/ml into cell culture supernatant fluids between - hrs after transfection under the conditions used in this study, while JFH RNA produces approximately 0-fold more at these early time points (Fig. A). While exchange of the entire NSA sequence resulted in RNAs that were impaired in their ability to replicate in transfected cells, the domain III chimeric RNAs replicated well and produced infectious progeny, albeit with altered efficiencies. Although the domain III sequences of these two viruses share less than 0% amino acid identity, these experiments demonstrate the existence of a conserved assembly function in domain III that can function in the context of either a genotype a or a virus background. Downloaded from on October, 0 by guest

24 0 0 A rather surprising result to emerge from these studies was the increase we observed in production of infectious JFH- virus when domain III from the HS virus, which produces relatively low virus yields, replaced the related sequence in JFH- (Fig. A). This result shows clearly that the contrasting capacities of the JFH- and HS genomes to produce infectious particles result from differences in the genomes outside of domain III of NSA. Nonetheless, we observed a good correlation between the apparent abundance of the NSA protein and the efficiency of infectious virus production: across both chimeras and the parental genomes, the abundance of NSA and production of infectious virus were similarly ordered (Fig. D). Although matched to some extent by differences in core protein expression, the differences in NSA abundance could not be explained entirely by altered RNA replication efficiency, as the domain III swaps had minimal impact on this (Fig. C). The contrasts we observed in NSA abundance in JFH- vs. HS RNA-transfected cells were not mirrored by similar differences in NSA abundance when these proteins when expressed ectopically in Huh-. cells (Fig. ). While this suggests that the stabilities of JFH- and HS NSA are not intrinsically different, JFH NSA was mostly hypo-phosphorylated when expressed as a solitary protein and it remains possible that there are significant differences in the stability of NSA expressed from replication-competent HS and JFH- RNAs. Within the domain III sequences of JFH- and HS, there are regions with striking amino acid identity that reside near the C-terminus of NSA and are separated by a large insertion in the JFH- sequence (Fig. A). The most C-terminal of these regions of identity contains a number of potential phospho-acceptor sites that have been strongly implicated in the assembly of JFH- virus by Tellinghuisen et al. () and Masaki et al. (). We observed that Ala substitutions at conserved Ser residues in this region had a strongly negative impact Downloaded from on October, 0 by guest

25 0 0 on the production of infectious virus from HS RNA (Fig. B), suggesting that the shared domain III assembly functions of the HS and JFH- proteins may be dependent upon these conserved residues. Ala substitutions at these sites also caused a marked reduction in NSA abundance without similar changes in viral RNA replication efficiency or core protein expression (Fig. C and D). Although direct biochemical investigation of NSA phosphorylation was outside of the scope of the experiments we report in this communication, these latter results, coupled with the positive impact of phosphomimetic substitutions at these residues (Fig. B), support the possibility that these are phospho-acceptor sites. Mutations at these residues cause modest changes in NSA stability, as indicated by differences in protein abundance when HS NSA is expressed ectopically (Fig. B). More pronounced changes in stability could result directly from altered phosphorylation of NSA when expressed from a replication-competent RNA, or from changes in its ability to interact with other viral proteins, including core (), or any of its numerous cellular binding partners (). Unfortunately, NSA expression from HS RNA, and particularly from these NSA mutants, was insufficient for direct pulse-chase measurements of protein stability. The NetPhosK prediction program () suggests that Ser- in JFH- NSA may be phosphorylated by CK II. This is consistent with its location upstream of several acidic residues and with the results of Tellinghuisen et al. (). This residue is also located within the C-terminal region of JFH-/HS sequence identity referred to above (Fig. A), and lies within the Ser cluster identified by Masaki et al. () (residues -) as essential for the recruitment of NSA to core protein associated with lipid droplets during early JFH- virus assembly (). The amino acid sequence of this region of JFH- NSA (SWSTCS, residues -) differs by only a single residue from that in HS (SWSTVS, residues -) (Fig. Downloaded from on October, 0 by guest

26 0 0 A). However, there are fewer acidic residues downstream of the conserved Ser- in HS (homologous to Ser- in JFH-), and only low probability predictions of CK II phosphorylation in this region of domain III by the NetPhos I.0 server at Ser- and Thr-. Although infectious virus production by JFH- and JFH/HAd RNA was inhibited to a similar extent by DMAT, a CK II inhibitor (Fig. ), an Ala substitution at Ser- in domain III of HS caused only a modest decrease in infectious virus yield (Fig. B). The Ser- to Ala mutation was not as inhibitory to virus production as similar Ala substitutions at Ser- or Thr-. Attempts to assess the impact of sirna-mediated knock-down of either or both of the CK II isoforms did not provide a clear answer as to the role of this kinase in HS NSA-mediated production of infectious virus (data not shown). Taken together, however, our results indicate that there is no special role for Ser- phosphoryation in production of infectious HS virus, and suggest instead that virus production is dependent upon multiple Ser residues, and likely Thr- as well, acting in a redundant or possibly cooperative fashion. Phosphorylation at Ser-, or any of the other residues identified by Masaki et al. in JFH- () or by us in HS (Fig. ) could modulate phosphorylation elsewhere in NSA, including at other conserved residues within the C-terminus of domain III. These findings with the HS virus are thus more consistent with the role of the serine cluster identified by Masaki et al. () in JFH- assembly, than with the uniquely important role for Ser- phosphorylation suggested by Tellinghuisen et al. (). While there is no clear explanation for the differences observed in these two prior studies of NSA, it may be important that Masaki et al. () studied assembly of the JFH- virus, while Tellinghuisen et al. () studied a J/JFH- chimera in which the structural proteins were derived from another genotype a virus, J. While both viruses were entirely genotype a in sequence, differences Downloaded from on October, 0 by guest

27 in the structural proteins could well have influenced the requirements for phosphorylation of residues in domain III of NSA. However, it is important to recognize in the context of this discussion that no study, including that described here, has yet directly demonstrated the phosphorylation of any residues in domain III of NSA. 0 0 Although the JFH- virus has provided a very useful model system that recapitulates the entire HCV life cycle in cultured cells (, ), most persons with significant HCV-related liver disease are infected with genotype viruses (). It is important that findings to emerge from the JFH- system are also examined in a genotype background, as we have done here with the role of domain III of NSA in HS assembly. An analysis of almost 00 different HCV sequences shows that the NSA domain III sequence of HS is broadly representative of all genotype a viruses, and similar in many respects to the sequence of genotype b viruses as well (Fig. ). Interestingly, this analysis revealed that while the Thr residue present at position is typical of most genotype a NSA sequences, the Thr residue that aligns with it at position of JFH- is not found in most genotype a viruses. Despite considerable sequence divergence and potential differences in the contribution of specific residues to assembly of infectious virus among different genotypes, our results point to similar mechanisms for the regulation of HCV assembly by domain III of both genotype a and a viruses. Still to be explained is why HS lags so far behind JFH- in the production of infectious virus. ACKNOWLEDGMENTS We thank Yinghong Ma, Yuqiong Liang, and Rhykka Connelly for excellent technical assistance, and Charles Rice, Tim Tellinghuisen and Craig Cameron for making available cell Downloaded from on October, 0 by guest

28 lines and/or antibodies. This study was supported in part by grants from the National Institutes of Health: U-AI000, RO-AI000 and R0-DA0. Downloaded from on October, 0 by guest

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31 0 0 0 Rice. 00. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. ProcNatlAcadSci USA 0:0-0.. Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Bartenschlager.. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science :0-.. Love, R. A., O. Brodsky, M. J. Hickey, P. A. Wells, and C. N. Cronin. 00. Crystal structure of a novel dimeric form of NSA domain I protein from hepatitis C virus. J Virol :-0.. Ma, Y., M. Anantpadma, J. M. Timpe, S. B. Shanmugam, S. Singh, S. M. Lemon, and M. Yi. 00. Hepatitis C Virus NS protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins J Virol In press.. Ma, Y., J. Yates, Y. Liang, S. M. Lemon, and M. Yi. 00. NS helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol :-.. Masaki, T., R. Suzuki, K. Murakami, H. Aizaki, K. Ishii, A. Murayama, T. Date, Y. Matsuura, T. Miyamura, T. Wakita, and T. Suzuki. 00. Interaction of hepatitis C virus nonstructural protein A with core protein is critical for the production of infectious virus particles. J Virol :-.. Miyanari, Y., K. Atsuzawa, N. Usuda, K. Watashi, T. Hishiki, M. Zayas, R. Bartenschlager, T. Wakita, M. Hijikata, and K. Shimotohno. 00. The lipid droplet is an important organelle for hepatitis C virus production. NatCell Biol : Moradpour, D., M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice. 00. Insertion of green fluorescent protein into nonstructural protein A allows direct visualization of functional hepatitis C virus replication complexes. J Virol :00-.. Nainan, O. V., M. J. Alter, D. Kruszon-Moran, F. X. Gao, G. Xia, G. McQuillan, and H. S. Margolis. 00. Hepatitis C virus genotypes and viral concentrations in participants of a general population survey in the United States. Gastroenterology :-.. Phan, T., R. K. Beran, C. Peters, I. C. Lorenz, and B. D. Lindenbach. 00. Hepatitis C virus NS protein contributes to virus particle assembly via opposing epistatic interactions with the E-E glycoprotein and NS-NSA enzyme complexes. J Virol :-.. Pietschmann, T., M. Zayas, P. Meuleman, G. Long, N. Appel, G. Koutsoudakis, S. Kallis, G. Leroux-Roels, V. Lohmann, and R. Bartenschlager. 00. Production of infectious genotype b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog :e000. Downloaded from on October, 0 by guest

32 Reed, K. E., and C. M. Rice.. Identification of the major phosphorylation site of the hepatitis C virus H strain NSA protein as serine. J Biol Chem :0-.. Sambrook, J., and D. W. Russell. 00. Molecular Cloning A Laboratory Manual, rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.. Shimakami, T., C. Welsch, D. Yamane, D. McGivern, M. Yi, S. Zeuzem, and S. M. Lemon. 0. Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus. Gastroenterology. Published on line ahead of print: doi:0.0/j.gastro Steinmann, E., F. Penin, S. Kallis, A. H. Patel, R. Bartenschlager, and T. Pietschmann. 00. Hepatitis C Virus p Protein Is Crucial for Assembly and Release of Infectious Virions. PLoS Pathog :e0.. Tanji, Y., T. Kaneko, S. Satoh, and K. Shimotohno.. Phosphorylation of hepatitis C virus-encoded nonstructural protein NSA. J Virol :0-.. Tellinghuisen, T. L., K. L. Foss, and J. Treadaway. 00. Regulation of hepatitis C virion production via phosphorylation of the NSA protein. PLoS Pathog :e Tellinghuisen, T. L., K. L. Foss, J. C. Treadaway, and C. M. Rice. 00. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NSA protein. J Virol :0-.. Tellinghuisen, T. L., J. Marcotrigiano, A. E. Gorbalenya, and C. M. Rice. 00. The NSA protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem :-.. Tellinghuisen, T. L., J. Marcotrigiano, and C. M. Rice. 00. Structure of the zincbinding domain of an essential component of the hepatitis C virus replicase. Nature :-.. Tellinghuisen, T. L., and C. M. Rice. 00. Interaction between hepatitis C virus proteins and host cell factors. Curr Opin Microbiol :-.. Wakita, T., T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K. Murthy, A. Habermann, H. G. Krausslich, M. Mizokami, R. Bartenschlager, and T. J. Liang. 00. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med :-.. Wozniak, A. L., S. Griffin, D. Rowlands, M. Harris, M. Yi, S. M. Lemon, and S. A. Weinman. 00. Intracellular proton conductance of the hepatitis C virus p protein and its contribution to infectious virus production. PLoS Pathog :e000.. Yi, M., and S. M. Lemon. 00. Adaptive mutations producing efficient replication of genotype a hepatitis C virus RNA in normal Huh cells. J Virol :0-. Downloaded from on October, 0 by guest

33 0. Yi, M., Y. Ma, J. Yates, and S. M. Lemon. 00. Compensatory mutations in E, p, NS, and NS enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol :-.. Yi, M., Y. Ma, J. Yates, and S. M. Lemon. 00. Trans-complementation of an NS defect in a late step in hepatitis C virus (HCV) particle assembly and maturation. PLoS Pathog :e Yi, M., R. A. Villanueva, D. L. Thomas, T. Wakita, and S. M. Lemon. 00. Production of infectious genotype a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 0: Yount, B., M. R. Denison, S. R. Weiss, and R. S. Baric. 00. Systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain A. J Virol :0-.. Zein, N. N Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev :-.. Zhong, J., P. Gastaminza, G. Cheng, S. Kapadia, T. Kato, D. R. Burton, S. F. Wieland, S. L. Uprichard, T. Wakita, and F. V. Chisari. 00. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 0:-. Downloaded from on October, 0 by guest

34 FIGURE LEGENDS 0 0 Figure. Construction and evaluation of replication properties of NSA chimeras. (A) Amino acid sequences of NSA domain III in HS and JFH- viruses. The amino acid sequence identity between these two domains is.%. (B) Schematic diagram of the NSA chimeras constructed for these studies. The entire NSA sequence or NSA domain III was exchanged between HS and JFH- to assess the impact on infectious virus production and viral RNA replication. (C) Replication properties of transfected HS (left panel) and JFH- (right panel) chimeric RNAs. The abundance of in vitro transcribed genomic RNA, relative to that of a replication-defective mutant with a GND mutation in NSB, was determined at various times following transfection using a real-time RT-PCR assay. Shown are mean values ± S.E. of triplicate RT-PCR assays. (D) Immunoblots of lysates of HCV RNA-transfected cells, showing the abundance of core and NSA in comparison to actin and GAPDH loading controls, respectively. Faint bands are indicated to their left as: x, HS NSA; o, HS/JAd NSA; s, additional hyper-phosphorylated NSA species (see text); *, nonspecific band. The predicted molecular masses of the HS, JFH-, and chimeric NSA proteins, prior to any post-translational modification, are shown between the NSA and GAPDH blots. Figure. (A) Virus yields (extracellular culture fluids) from RNA-transfected cells after inter-genotypic (a a and a a) exchanges of NSA or NSA domain III. Culture supernatants obtained at day after transfection were assayed for infectious virus. Shown are the mean yields from each chimera in triplicate transfections, ±S.D. (B) ELISA for HCV core protein present within supernatant culture fluids of RNA-transfected cells. Data shown represent mean values ± S.E from duplicate experiments. Downloaded from on October, 0 by guest

35 0 0 Figure. Ectopically expressed NSA proteins. (A) Expression vectors encoding various NSA molecules, each with an N-terminal Myc tag, were transfected into Huh-. cells along with a second vector expressing Gaussia luciferase as a transfection control. Cell lysates were prepared hrs later and subjected to SDS-PAGE followed by immunoblotting with the E0 monoclonal antibody or an anti-myc antibody. The result shown is representative of replicate experiments. (B) E0 and anti-myc antibodies bound by NSA proteins in parallel immunoblots of cell lysates were detected with an infra-red fluorescent probe and quantified using an Odyssey fluorescent scanner. Results for each antibody were normalized to the amount bound by the JFH protein. The results shown represent the mean ± S.E. in separate transfection experiments. The JFH/HAd protein demonstrated greater binding of E0 relative to anti-myc, but there are no significant differences in the recognition of JFH and HS NSA by these antibodies. Equivalent abundance when the NSA proteins were probed by anti-myc antibody suggests that there are no significant differences in the intrinsic stability of these proteins. Differences in transfection efficiency, monitored by assessing luciferase activity, were minimal. Figure. Infectious virus titer of cell culture supernatant fluids and intracellular lysates of JFH- and JFH/HAd RNA-transfected cells on day after transfection. Lysates were prepared by multiple freeze/thaw cycles. Shown are the means ± S.E. from duplicate transfections. Percentages are the proportion of the total virus yield released into supernatant culture fluids (i.e., virus in supernatant/virus in supernatant + virus in lysate). Downloaded from on October, 0 by guest Figure. Replication and infectious virus yields from the structural protein chimera HJ- with or without exchange of domain III of NSA from HS virus. (A) Schematic showing organization of the HJ- inter-genotypic chimeric RNA and related NSA domain III (d)

36 0 0 swap with HS sequence. HS sequence (core-ns) is shown as open boxes, while JFH- derived sequence (NS-NSB) is shaded; non-coding RNA segments are from JFH-. The asterixes indicate the location of compensatory mutations (E and NS) that promote infectious virus yields from the chimera (). In HJ-/ E-p and E-p/HAd, the E-p sequence has been deleted. (B) Infectious virus production. Shown are the means ± range of infectious virus yields from chimeric RNAs calculated from duplicate transfections. (C) ELISA for core protein secreted by cells transfected with HJ- and HJ-/HAd RNAs. Means ± S.E. were calculated from duplicate experiments. (D) Immunoblots for HCV core protein in lysates of HJ- and HJ-/HAd RNA-transfected cells; actin served as a loading control. Also shown are immunoblots for NSA in lysates of cells transfected with the related E-p mutant RNAs; GAPDH was the loading control. (E) Real-time RT-PCR measurements of HCV RNA replication. The relative HCV RNA copy number represents the copy number for each construct relative to a replication-lethal GND mutant, normalized to the value present hrs after transfection. Results shown represent the mean ± S.E. calculated from triplicate RT-PCR assays. Figure. Inhibition of virus production by DMAT, an inhibitor of CK II. (A) Following transfection of the indicated RNA, cells were treated with the indicated concentration of DMAT for hrs. The media was then replaced with fresh media (no drug), followed hrs later by harvesting of supernatant fluids for virus titration. Means ± S.E. were calculated from duplicate experiments. (B) Immunoblots for NSA, NS, NS, and GAPDH from cell lysates prepared hrs after transfection. (C) Cytotoxic effects of DMAT assessed in a WST- cellular proliferation assay. Means ± S.E. were calculated from triplicate experiments. Downloaded from on October, 0 by guest

37 0 0 Figure. Impact on viral RNA replication and infectious virus production of Ala substitutions at potential Ser/Thr phospho-acceptor site residues in domain III of HS. (A) Possible Ser/Thr phospho-acceptor sites in the C-terminal region of domain III of the NSA proteins of JFH- and HS virus. At the top of the panel, the HS and JFH- sequences are aligned: Ser residues found to be important for the NSA-core interaction and assembly and release of infectious JFH- virus by Masaki et al. () (red box), and Ser-, identified by Tellinghuisen et al. () as a site of CK II phosphorylation (red arrow), are highlighted. Within the related HS sequence, Ser- and Thr-, are possible sites of CK II phosphorylation predicted by the NetPhos.0 server. Below are shown the C-terminal NSA sequences of the single- and multiple-ala substitution mutants studied. Potential Ser/Thr phospho-acceptor sites studied are shown in red, while Ala substitutions ablating these sites in the mutants are shown in bold-face type. (B) Potential phosphorylation sites near the C-terminus of domain III of HS NSA were mutated to Ala to assess their role(s) in infectious virus production. Shown are the mean virus yield following transfection of the mutant RNAs, normalized to that of HS virus (00% = mean of 0 FFU/ml) ± S.E. from two independent transfections. (C) HCV RNA replication compared to the replication-lethal GND mutant, measured by real-time RT-PCR assay. The means ± S.E. were calculated from triplicate RT-PCR assays. (D) Immunoblots for HCV NSA, core and GAPDH in lysates of the RNA-transfected cells. Figure. Phosphomimetic Ala to Asp substitutions restore production of infectious virus by AWA and SA mutant RNAs. Infectious virus yields from cells transfected with (A) HS. and HS./SA RNAs and (B) HS., HS./DWD and HS./SD mutants. Results shown in each are the mean ± S.E. from two transfections with independent RNA transcripts. Downloaded from on October, 0 by guest

38 Below each is shown an immunoblot of NSA and core protein expression with (A) actin or (B) GAPDH loading controls. 0 Figure. Sequence logo depiction of amino acid sequence conservation within domain III of NSA among genotype a ( sequences), b ( sequences), and a ( sequences) strains of HCV from the European HCV Database, euhcvdb (). Sequences were aligned with MUSCLE (0), with minor manual modifications, and logos generated with WebLogo (). The height of each single character amino acid code is proportional to the representation of that amino acid at each position. The sequence extending from Ser to Thr which contains potential phosphoacceptor residues in HS is boxed in red. To show how representative the HS sequence is of other genotype viruses, residues identical to those in HS are colored blue in the genotype a and b sequences, while those that differ from HS are shown in gray. Residues identical to JFH- are colored red in the genotype a sequence. Downloaded from on October, 0 by guest

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