The present study provides evidence that the conserved regions in the NTRs of the HCV genome contain genotype-specific signals regulating the initiation of RNA synthesis. This observation combined with the development of efficient intergenotypic replicon chimeras allowed us for the first time to analyze which proteins are involved in the initiation of HCV negative- and positive-strand syntheses. Our results indicate that helicase, NS5A, and NS5B participate in the initiation of progeny positive-strand RNA synthesis and provide a starting point to characterize the interaction between these proteins in more detail.
Analysis of HCV replication in cell culture has long been focused on genotype 1 isolates due to the lack of isolates from other genotypes efficiently replicating in cell culture. A common feature of genotype 1 genomes is the need for adaptive mutations enhancing RNA replication to detectable levels (6
). In striking contrast, the genotype 2a isolate JFH1 supports HCV replication in cell culture to even higher levels than adapted genotype 1 isolates without requiring prior adaptation (24
). The Con1 replicon which was used in the present study contained three cell culture-adaptive mutations (E1202G, T1280I, and K1846T), which stimulated replication in cell culture cooperatively (36
). The mechanism underlying cell culture adaptation is still unknown for NS3 mutations (E1202G and T1280I), while the mutation in NS4B (K1846T) reduces the level of NS5A hyperphosphorylation (2
), which might be responsible for the adaptive phenotype (12
). The effects of adaptive mutations on RNA replication in the context of intergenotypic chimeras between Con1 and JFH1 were hardly predictable, especially since the design of some chimeras required the swapping of the helicase (including mutation T1280I) and NS5A. Therefore, the overall replication efficiencies of particular intergenotypic chimeras as shown in Fig. might be influenced by the presence or absence of the Con1-adaptive mutations. However, given the multiple interactions between all HCV NS proteins, the efficiency of chimeric replication complexes will be determined by a variety of parameters and thus must be interpreted with caution.
Our results indicate that one major difference between JFH1 and Con1 lies in the efficiency of the initiation of RNA synthesis and that the RdRp of JFH1 might have properties that are critical for this performance. This theory is based on several observations. (i) The time required for a complete RNA replication cycle of a subgenomic replicon seems to be 8 to 12 h for JFH1 and Con1, as indicated by the first increase of luciferase activity over background (Fig. ). However, within the next 12 h, JFH1 reaches up to 1,000-fold of input luciferase levels, whereas Con1 gains less than 10-fold. Therefore, JFH1 either generates a higher number of active replication complexes within the initial 8 to 12 h that will, however, not yet initiate RNA synthesis or produces more progeny RNA per replication complex. (ii) The replacement of the 5′ NTR and X region of JFH1 with their Con1 counterparts dramatically reduces JFH1 replication efficiency by more than 2 orders of magnitude (Fig. ), whereas the equivalent manipulation had less severe effects on Con1, resulting in replicons with almost comparable replication efficiencies (Fig. ). We have shown that these changes directly impair the initiation of RNA synthesis; therefore, it seems likely that a higher initiation rate of RNA synthesis is a critical component of the extraordinary replication efficiency of JFH1. (iii) Purified RdRp of JFH1 has a 5- to 10-fold-higher specific activity than Con1 RdRp, although elongation rates were comparable for both enzymes (V. Lohmann, unpublished observations), arguing that JFH1 NS5B is more efficient at the initiation of RNA synthesis. (iv) An intergenotypic replicase chimera based on NS3 to NS5A of JFH1 and NS5B of Con1 was not viable (Fig. ). This is well in line with previous observations demonstrating that even the intragenotypic exchange of NS5B resulted in a reduced replication efficiency (63
), whereas in another study, NS5B of a genotype 2b isolate in the background of genotype 1b NS3 to NS5A was barely replication competent and required compensatory mutations for efficient replication (17
). In contrast, our intergenotypic replicon chimeras harboring NS3 to NS5A from Con1 and NS5B of JFH1 replicated with high efficiencies (Fig. ), again indicating that NS5B of JFH1 might have special properties that account to some extent for the unique properties of this HCV isolate. Further biochemical and structural analysis of NS5B will be required to better understand the contribution of the RdRp to the efficient replication of JFH1.
Genetic studies of HCV RNA-protein interactions have been complicated by the fact that all RNA and protein functions in HCV RNA replication in cell culture have to be provided in cis
, with the exception of certain NS5A functions (1
). Therefore, it has not been possible to dissect the interaction of individual proteins and RNA elements in a cell-based replication assay like it has been for, e.g., alphaviruses (33
). The identification of genotype-specific signals in the NTR in combination with intergenotypic replicase chimeras as described in this study allows us for the first time to study the determinants for the initiation and regulation of negative- and positive-strand HCV RNA syntheses in a genetic system. Chimeras between closely related enteroviruses have already been successfully used to define determinants in the polymerase important for strand-specific RNA synthesis (10
). Similar strategies might also be applicable to other pairs of closely related positive-strand RNA viruses, such as West Nile virus/Kunjin virus, different dengue virus subtypes, and bovine viral diarrhea virus/classical swine fever virus.
Little is known about the actual mechanism of the initiation of HCV negative-strand RNA synthesis. Our results indicate that one step in this process involves an interaction between NS5B and the apical part of the stem of SLI in the X region (Fig. ). This is in line with a previous study showing that the sequence and structure of this part of SLI is very critical for HCV RNA replication (73
). The slight preference of purified NS5B for an RNA template of the same genotype (Fig. ) suggests that the genotype specificity in the case of SLI is mediated by a protein-RNA interaction. However, we cannot formally exclude that an RNA structure within NS5B or in the variable region of the 3′ NTR directs genotype specificity to SLI, analogous to the kissing-loop interaction that was found between SL3.2 in the NS5B coding sequence and SLII in the X region (14
). This possibility also holds true for other, yet unknown, cis
-acting RNA elements in the NS protein-coding region that could be involved in long-term RNA-RNA interactions with the 3′ end of the negative strand and thereby mediate genotype specificity. One obvious limitation of our system is the dependence on genotype diversity in a particular region. Therefore, only SLI is accessible to our analysis within the X region, because the consensus sequences of SLII and SLIII are invariant among all genotypes. Previous in vitro studies have shown specific binding of NS5B to stem-loop structures in its own coding region (32
) and to SLII in the X tail (45
). This might indicate either that NS5B binds sequentially to different regions of the 3′ NTR or that multiple copies of NS5B simultaneously interact with this region to form a higher-ordered complex. The latter hypothesis is in line with in vitro studies demonstrating that NS5B oligomerizes and displays cooperativity (18
). It is also possible that other viral proteins such as NS3 and NS5A are involved in the initiation of negative-strand RNA synthesis but were not identified in our analysis due to their binding to the invariant or the more variable parts of the 3′ NTR. For example, it has been shown that the NS3 helicase, NS5A, and NS5B all efficiently bind to poly(U) (19
), suggesting a functional role for the poly(U/UC) tract in the initiation of RNA synthesis. In addition, specific binding to the 3′ NTR was shown for the NS3 helicase (3
) but not for NS5A (21
). Potential interactions of helicase and NS5A with the variable region of the 3′ NTR could be identified with intergenotypic replicase chimeras but were not in the scope of the present study.
In this study, we have shown that replicons harboring a 5′ NTR of a heterologous HCV genotype are directly impaired in progeny positive-strand synthesis (Fig. ), thereby providing the first experimental system to study this process in cell culture. Using this approach, we were able to define those viral proteins that are involved in the recognition of the signals regulating positive-strand RNA synthesis, namely, the NS3 helicase, NS5A, and NS5B. These signals mapped primarily to the first half of the 5′ NTR (Fig. ), corresponding to the three 3′-terminal stem-loops of the HCV negative strand, which were recently defined in two independent studies (54
). Surprisingly, we were not able to further map genotype-specific signals in this region (Fig. ), probably because the effects of the individual mutations were below the detection limit of our assays. Alternatively, the binding of helicase, NS5A, and NS5B or several copies of one of these proteins to different sites might be stabilized by protein-protein interactions; in this case, the presence of one specific binding region might be strong enough for the docking of the whole protein complex. In line with this hypothesis, it has been shown that purified helicase binds to the 3′-terminal stem-loop of the negative-strand RNA (3
), whereas NS5B seems to bind further upstream (23
), and in addition, several potential binding sites could exist in this region for NS5A (21
). Additional mapping analyses implying different intergenotypic replicase chimeras might help to identify distinct binding regions for the individual nonstructural proteins in the 3′ NTR of the HCV negative strand and to define the protein-protein interaction sites between helicase and NS5A.
Our data imply that helicase, NS5A, and NS5B are part of an initiation complex for RNA synthesis. Previous studies already suggested interactions of NS3 and NS5B (46
) and N5A and NS5B (56
) and several other interactions among the nonstructural proteins (11
). These hints, together with results obtained with purified replication complexes showing equal stoichiometries of the NS proteins in these structures (47
), indicate that the nonstructural proteins altogether act as a complex supporting different functions in the viral life cycle: regulation of translation and replication, induction of membrane alterations, positive- and negative-strand RNA syntheses, and probably retrieval of RNA for packaging into viral particles. It is getting clearer that NS5A plays a central role in the regulation of several of these processes. NS5A is a hotspot for cell culture-adaptive mutations, resulting in an altered phosphorylation pattern (2
) and increased RNA replication efficiency. In turn, these adaptive mutations seem to interfere with HCV virion formation (66
), suggesting that the phosphorylation state of NS5A might regulate RNA replication and packaging, probably via host cell proteins such as human VAP-A (12
) and the cellular kinase CKI (43
). The finding that NS5A indeed binds to RNA (21
) and the crystal structure of domain I of NS5A (62
), showing that an NS5A dimer creates a deep basic groove that could bind single- and eventually also double-stranded RNA, already suggested a direct involvement of NS5A in RNA synthesis, which is further confirmed by our results. The actual role of NS5A and the precise composition of the initiation complex for HCV RNA synthesis have yet to be defined.
In conclusion, intergenotypic replicase chimeras will be a useful tool for the mapping of protein-RNA and protein-protein interactions among viral nonstructural proteins, in particular the newly identified interplay between helicase, NS5A, and NS5B, and will provide deeper insight into the mechanisms governing the initiation and regulation of HCV RNA synthesis.