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

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

Troels K. H. Scheel, Jannick Prentoe, Thomas H. R. Carlsen, Lotte S. Mikkelsen, Judith M. Gottwein, Jens Bukh^*

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre and Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

Abstract

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

Author Summary

Hepatitis C virus (HCV) is a major public health burden and leads to chronic liver disease, including liver cirrhosis and liver cancer. Understanding the biological functions of the virus is crucial to the development of a vaccine and to improve current therapy through development of directly-acting antiviral compounds. The NS5A protein is a promising antiviral target, but much remains to be understood about its role in the viral life cycle. Great diversity among the seven major HCV genotypes poses challenges for broadly active inhibitors. Here we used infectious cell culture systems for NS5A of the seven major HCV genotypes, and demonstrated that all genotypes depended on the NS5A amphipathic alpha-helix, domain I, low-complexity sequence (LCS) I and domain II for viral replication. Interestingly, effects on replication and virus production by changes in LCSII and domain III varied greatly among NS5A isolates. Furthermore, we found that genotype 2 had evolved genotype-specific residues in domain I of importance for viral function. Thus, the highly diverse sequence of the NS5A protein reflected functional differences between HCV genotypes and isolates. Such differences will be important to consider in understanding HCV biology and for future development of antiviral compounds.

Citation: Scheel TKH, Prentoe J, Carlsen THR, Mikkelsen LS, Gottwein JM, et al. (2012) Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems. PLoS Pathog 8(5): e1002696. doi:10.1371/journal.ppat.1002696
Editor: Aleem Siddiqui, University of California, San Diego, United States of America

Received: February 11, 2011; Accepted: March 27, 2012; Published: May 24, 2012
Copyright: © 2012 Scheel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: TKHS, JP and THRC are the recipients of Ph.D. stipends from Faculty of Health Sciences, University of Copenhagen; TKHS and JP are the recipients of Individual Postdoctoral Stipends from the Danish Council for Independent Research, Medical Sciences. The study was supported by research grants from Lundbeck Foundation (TKHS, JG and JB), The Danish Cancer Society (JMG and JB), Novo Nordisk Foundation (JMG and JB), The Danish Council for Independent Research, Medical Sciences (JB), A. P. Møller and Chastine Mc-Kinney Møllers Medical Research Foundation (TKHS, JMG and JB), Hvidovre Hospital Research Foundation (TKHS and JMG), Aage Thuesen Bruun and Emmy Katy Bruun's memorial foundation (TKHS) and Leo Nielsen and Karen Margethe Nielsens Foundation for Basic Medical Research (TKHS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: jbukh@sund.ku.dk

Discussion Only

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Due to the clinical and biological importance, there has been great interest in the study of HCV genotype-specific functional differences [9]. However, most functional studies of HCV in infectious culture systems have depended on a single HCV isolate (JFH1). The NS5A protein was so far primarily studied in genotype 1 and 2 replicon systems or in the JFH1 genotype 2a cell culture system. Interestingly, replicon-enhancing mutations were not permissible in vivo [32], emphasizing that conclusions from the replicon systems should be drawn with caution. In this study, we used infectious NS5A genotype 1–7 recombinants [12], and demonstrated a universal dependence of viral replication on the NS5A amphipathic alpha-helix, domain I, LCSI and domain II. Thus, it was demonstrated that all HCV genotypes require these domains for replication. Interestingly, isolate-specific effects of mutations in LCSII and of deletions in domain II and III revealed novel functional differences between NS5A isolates. Furthermore, NS5A function was shown to depend on genotype-specific residues in domain I, a finding that could influence the effect of directly acting antiviral compounds directed against this NS5A domain. Thus, functional isolate-specific differences are emerging for HCV [3], [42], which will be of critical importance for our understanding of HCV biology and for development of antiviral strategies that target NS5A and other regions with isolate variability.

We addressed the importance of the various regions of NS5A for replication and virus production, by analyzing more than 80 mutants in culture. We showed that viral replication in context of the complete life-cycle was critically dependent on the NS5A amphipathic-alpha helix, domain I, LCSI and domain II for isolates of genotypes 1a, 1b, 2a, 3a, 4a, 5a, 6a and 7a. The absence of HCV positive cells by immunostaining early after transfection corresponded to observations for knockout mutants of non-structural proteins NS3, NS4A, NS4B, and NS5B, and a mutant with an introduced stop-codon, all expected to abolish replication. We demonstrated that reversion could occur even for such single-residue mutants, potentially due to the high error-rate of the T7 polymerase used for RNA in vitro transcription. Thus, abrogation of replication due to mutations shown to disrupt the hydrophobic face of the amphipathic alpha-helix, mutation of two zinc-binding cysteines in domain I, or due to exchange of a conserved tryptophan in domain II confirmed and extended previous findings in the Con-1(1b) replicon system [16], [22], [23]. Contrarily, dependence on LCSI in the infectious cell culture system was demonstrated by the highly attenuated phenotype caused by the replicon-enhancing mutation S225P [33], which was also not permissible in vivo [32]. Thus, at least for this mutant the infectious NS5A cell culture systems reflected findings in vivo better than replicon systems. Recently, S225P was shown to enhance replication but inhibit HCV Core release of the full-length Con-1(1b) isolate in vitro [46], which was not in agreement with our data from infectious culture systems. In theory, inhibition of virus production but not replication could be specific for the Con-1(1b) isolate, however, the Con-1(1b) and J4(1b) NS5A protein sequences deviate by less than 5%. Replication seemed to depend also on highly conserved residues downstream in LCSI (Figure S1), as deletion of J4(1b) NS5A residues 235–282 (Δ47) in this study abolished replication, while residues 246–308 previously were deleted in J6/JFH1 with no apparent effect on viability [27]. Interestingly, we observed decreased infectivity titers and mutations in the C-terminus of NS5A for domain II Δ250–293 mutants of genotype 1a but not 2a (Figure 2 and [39]). Thus, HCV apparently shows an isolate-specific dependence on the highly variable N-terminal region of NS5A domain II. The genotype 2 protein sequence deviates in this region from most other genotypes (Figure S1), potentially reflected by differences in structure or function and thereby also in the effect of the deletion. With the critical dependence on selected residues or regions of the NS5A amphipathic alpha-helix, domain I, LCSI and domain II for replication, these regions would be obvious targets for antiviral therapy [47].

Great differences in the effect on viral infectivity titers were observed among the NS5A genotype 1–7 mutants when residues 346, 351 and 354 in LCSII were mutated, with J4(1b) and JFH(2a) being least affected. Measurements of intracellular HCV Core showed that most NS5A LCSII mutants had up to 10-fold reduced replication capacities, while ED43(4a) and QC69(7a) mutants were severely attenuated. Greater than 10-fold reductions in intra- and extracellular infectivity for the H77C(1a), S52(3a), SA13(5a) and HK6a(6a) mutants indicated an additional effect on virus production for these isolates. Findings on the JFH1(2a) LCSII mutant were in agreement with previous findings that a Con-1(1b) but not a JFH1(2a) replicon depended on P346 for replication, while mutation of the infectious full-length JFH1 recombinant led to lower levels of infectivity [42]. In line with this previous study, we found that mutation of P346/P351/P354 inhibited replication at an isolate-specific level. Interestingly, replication of the J4(1b) mutant was only slightly decreased, while the Con-1(1b) P346A replicon mutant was severely attenuated [42]. This might be due to differences between the isolates or between the two in vitro systems studied. Our QC69(7a) LCSII mutant lacked 14 nucleotides in the poly-pyrimidine tract (Materials & Methods), however, it is unlikely that this caused the observed attenuation of replication since previous studies demonstrated efficient replication of J6/JFH1 with much shorter poly-pyrimidine tracts [48]. In agreement with previous findings for residue 343 mutants [42], we could not confirm dependence on this position [23] for replication of any of the NS5A recombinants.

Deletion of the highly conserved residues 414-428 led to isolate-specific decrease in infectivity titers and replication (Figure 4). The deletion had very limited effect on the JFH1(2a) mutant, while most other NS5A isolate mutants had reduced replication capacities; most pronounced decreases were observed for H77C(1a), TN(1a) and S52(3a) mutants. Reductions in replication capacities might explain the decrease in intra- and extracellular titers observed for most mutants, however, the less than 2-fold reduction in replication capacity for ED43(4a) and SA13(5a) mutants compared to the greater than 3-fold reduction in produced infectious particles indicated an effect of the 414-428 deletion on assembly of intracellular infectious particles for these recombinants. The finding that a genotype 1a mutant with residues 414-428 replaced by the downstream genotype 2-specific insertion of 20 residues produced infectivity titers comparable to the original NS5A recombinant, indicated that this insertion could compensate for the 414-428 deletion. Others previously studied JFH1 mutants without the 382–428 region [27] or the genotype 2 insertion sequence [49] and found only minor effects on replication, production of infectious particles and co-localization of NS5A and Core on lipid droplets [27]. This correlated with our results with the JFH1(2a) 414-428 deletion mutant. However, deletion of the 408–437 region [29], which covered the 414-428 region and the genotype 2 -specific sequence, or the almost entire domain III (residues 356–439) [27] of genotype 2a, was highly attenuating for viral infectivity but not for replication. Thus, domain III residues outside the 414-428 region appear to be the most important determinants of efficient virus production.

Experiments with H77C(1a) demonstrated that selected LCSII and domain III mutants led to decreased amounts of NS5A normalized to Core protein levels. Assuming that Core stability was not affected by introduced mutations, this suggested a decreased stability of the NS5A protein. Similar observations were recently reported when two or four of the serines S432, S434, S437 and S438 were changed to alanine in a JFH1 recombinant carrying NS5A domain III from H77 [50]. Such effects could possibly be associated with disruption of NS5A co-localization with Core on lipid droplets [27] or disruption of interactions with annexin A2 [51].

The J6/JFH1 S437A mutation was previously shown to significantly decrease infectivity titers 48 hours after transfection [28], however, another study indicated that mutation of at least two of the S432, S434 and S437 residues was required for a significant reduction of particle production and impaired NS5A-Core interaction, and that viral kinetics were attenuated only early after transfection [29]. Similar findings were reported in the JFH1 background with H77 NS5A domain III [50]. This might explain why we observed only a less than 10-fold reduction in infectivity titers on day 3 but not thereafter (Figure 5). Surprisingly, deletion or point mutation of the relatively conserved 363–380 region in domain III (Figure S1) did not significantly affect JFH1(2a) virus production. Similarly, mutation of arginines 356–359 in a putative nuclear localization signal [52] led to only slight reduction of infectivity titers for JFH1(2a). Most interestingly, changing highly conserved residues in NS5A LCSII and domain III of various HCV isolates led to very different effects on replication and virus production. This emphasizes the evolutionary development of functional differences among the HCV genotypes in particular in variable regions of the genome [3]. Investigation of the role of these regions in vivo would be of interest for future studies.

When replacing the entire NS5A gene of J6/JFH1 with that of other isolates [12], or conducting NS5A reverse genetic experiments as done here, putative compensatory mutations were observed outside of NS5A; particularly in p7, NS2 and NS3 (Table 1, [12]). The F26S mutation in p7 provided adaptation to several NS5A recombinants [12], and in this study F26S was acquired by NS5A Δ414-428 deletion mutants. In addition, the p7 mutation also adapted J6/JFH1 recombinants with genotype-specific NS3/4A protease [53]. Interestingly, introduction of F26S into the genetically stable H77C(1a) and TN(1a) NS5A recombinants led to compensatory mutations in NS5A domain III. Moreover, introduction of these NS5A mutations led to mutations in p7, while combining the p7 and NS5A mutations led to stable recombinants. These findings indicated a genetic linkage between NS5A and p7. NS5A interactions might also involve NS3/NS4A, where several Δ414-428 mutants acquired changes, possibly in concert with p7 or other proteins in the Core-NS2 region, since I399T in NS3 (I1425T) acquired by the TN(1a) mutant also adapted a Core-NS2 genotype 1a recombinant [54]. Additionally, a number of potentially compensatory mutations for NS5A deletion mutants in the third transmembrane domain of NS2 (Table 1), for the J4(1b) NS5A recombinant [12], and for Core-NS2 recombinants [7], indicated an interaction between NS5A and NS2. Pull-down, co-localization and reverse genetic experiments demonstrated NS2 interactions with E1, E2, p7, NS3 and NS5A, and studies of compensatory mutations identified the importance of such interactions during production of virus particles [55]–[58]. Furthermore, the p7 F26L mutation was shown to compensate for changes in Core [59], possibly reflecting involvement of these proteins in viral assembly. Unfortunately, useful antibodies targeting p7 are scarce and the protein can not be directly detected in immunostainings. Tagging of p7 was not compatible with efficient virus production (data not shown and [56]), and was therefore not a useful approach for studying protein interactions likely to take place during viral assembly and release. Thus, despite elaborate efforts we failed to establish co-immunoprecipitation or co-localization based evidence for an interaction between p7 and NS5A in the infectious culture system; better reagents are needed for conclusive studies on p7 interactions.

Mutation from genotype 2a to 1a or vice versa of the NS5A genotype 2 or 2a-specific residues 95/97, 140, 151/152, 157 or 165/168/169 led to attenuation in cell culture. This was of particular interest, since the introduced amino acids led to attenuation irrespective of the presence of these amino acid residues at the given position in numerous infectious HCV isolates. This indicated that genotype 2 evolved specific sequence requirements for function of NS5A domain I. Putative genotype-specific compensatory mutations were identified for the JFH1(2a) H124V mutant that changed serine at position 300 (domain II), present in all genotype 2 and only few other isolates, and the JFH1(2a) S151T/W152E mutant that changed threonine at position 122, present only for genotype 2 and 1b isolates. Residues 130 and 188 that changed in the I140C culture are located in close proximity to residue 140 in the NS5A domain I structure [19], suggesting a functional interaction between these residues. Compensatory mutations in domain II and the reduced viability of domain I exchange recombinants suggested important genotype-specific interactions between domain I and other regions of NS5A. Such interactions might in particular be between domain I and II, since a recent study demonstrated that domain III alone could be exchanged between H77 and JFH1 recombinants [50]. To our knowledge this is the first time the function of an HCV protein was found to depend on genotype-specific residues. Since NS5A inhibitors under current development target domain I [14], [47], this may pose challenges for future antiviral therapy.

Although this study significantly increases the number of studied NS5A isolates, only a single isolate was analyzed for most genotypes. Since differences were observed between isolates of the same genotype, e.g. between domain II deletion mutants of H77C(1a) and TN(1a), studies of more isolates will be important to discriminate between isolate- and genotype-specific findings. Since the original NS5A recombinants used in this study all were efficient without requirement for further adaptation, any effects observed for NS5A mutants are likely to be attributed to the particular NS5A isolate. However, it is possible that particular mutations would render NS5A non-functional in the J6/JFH1 genetic background but not in a full-length background of that particular isolate. Thus, it will eventually be of importance to develop full-length cell culture systems for all HCV genotypes.

In conclusion, we demonstrated that all major genotypes depended on the NS5A amphipathic alpha-helix, domain I, LCSI and domain II for viral replication. Interestingly, dependence on LCSII and domain III for HCV RNA replication and virus production varied with the NS5A isolate. Additionally, functional genotype-specific differences of NS5A domain I residues were identified. Our study highlights the emerging evidence of significant functional differences between diverse HCV isolates. Observed differences in NS5A will be important to consider in functional understanding and therapeutic targeting of this protein. Further studies in vitro and in vivo will be important for understanding and targeting this pleiotropic viral protein.

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