Since the identification of adaptive mutations in the HCV subgenomic replicon, it has been noted by several groups that the hyperphosphorylated form of NS5A, p58, is not necessary for replication in cell culture. In this report, we go one step further and demonstrate that high quantity of p58 is not only not necessary but also deleterious for the replication of the Con1 replicon. To demonstrate this hypothesis, we prevent the formation of NS5A p58 by chemical means, without altering the wt sequence of the Con1 HCV genome.
An in vitro assay was set up which enabled us to screen a collection of small molecules for their inhibitory activity on kinase(s) responsible for NS5A phosphorylation. The principle of this in vitro assay is based on the fact that the specific kinase(s) remain stably associated with NS5A after immunoprecipitation. Incubation of this protein complex with [γ-33
P]ATP results in the production of many phosphoproteins (Fig. ), indicating that the polyclonal antibody nonspecifically binds many cellular proteins and kinases under the conditions we used for the immunoprecipitation. However, the phosphorylation of NS5A is the most efficient reaction in the immunoprecipitate prepared from extracts expressing the replicon (Fig. , compare lanes 2 and 3). In replicons containing the A2199T adaptive mutation, p56 and p58 are present in equal amounts (Fig. , lane 2). Nevertheless, in the in vitro assay, radiolabeled phosphate is incorporated predominantly in p56. The formation of p58 depends on the presence of the HCV polyprotein coding from NS3 to NS5A and requires active polyprotein processing (14
). For this reason, the formation of p58 during in vitro labeling is not expected. In addition, one can imagine that the conformation of p58 is different from that of p56 and that phosphorylation sites present in p56 are not exposed or masked by other associated proteins in p58. As a consequence, the in vitro phosphorylation of serine residues in p56 may be more efficient than in p58.
The low stringency of the assay produced a hit rate of about 40%, with a threshold of at least 50% inhibition of NS5A phosphorylation. As a consequence, a large number of compounds showed the same in vitro activity as the three selected compounds but were inactive in cell culture. There are several explanations for the difference between in vitro activity and activity in cell culture. One possibility could be limited penetration of the compounds into the cells. Alternatively, a high potential of the compounds for protein binding or instability in the culture medium or within the cell could limit the access to the compound. All compounds tested were designed to be active-site inhibitors and competitors of ATP. Another possible explanation would be that the affinity of the compound for the kinase is too low to compete with the relatively high concentrations of ATP within the cell.
Retesting the selected compounds in cell culture with the vaccinia T7 infection-transfection system as well as Huh7-68 cells identified three active compounds. The formation of p58 is inhibited in both systems, whereas the basal phosphorylation of p56 remained unchanged (Fig. , lanes 8 to 12). Interestingly, we could not identify any compound that inhibited the basal phosphorylation without affecting the expression of NS5A. NS5A is proteolytically processed from the polyprotein within 15 min (20
). The prevention of basal phosphorylation may destabilize the protein and lead to protein degradation. In this case, any compound which inhibits basal phosphorylation would reduce the total protein amount of NS5A and would have been excluded in our second assay in cell culture.
The identification of three compounds that selectively inhibited NS5A hyperphosphorylation, but not basal phosphorylation, allowed us to verify whether a reduction of the amount of NS5A p58 in the context of the Con1 wt sequence could be sufficient for the activation of subgenomic replication in cell culture. It turned out, in fact, that all three active kinase inhibitors were also active replication inducers (Fig. ).
Compound H479 is the best inhibitor, as it activates replication in more than 50% of the cells. This efficiency is comparable to the S2204A adaptive mutation, resulting in the expression of HCV proteins at levels detectable by Western blotting. Consistent with the inhibition of NS5A hyperphosphorylation, the formation of p58 is significantly reduced (Fig. , lane 16). It is important to stress the point that the only difference which renders the replicon capable of replication is the reduction of p58 formation by a kinase inhibitor without changing the genomic sequence. Thus, an excess amount of p58 seems to be the causative agent responsible for the inhibition of HCV replication in cell culture.
The other two compounds also activated replication (Fig. ); however, HCV proteins and HCV RNA were detectable only in the presence of a synergistic mutation in NS3 (Fig. ). The introduction of mutations may be critical for infectivity, as shown in the case of adaptive mutations. These mutations increase HCV replication efficiency in cell culture, but at the same time, they destroy or significantly attenuate infectivity in vivo (4
). One, therefore, has to carefully choose any change in the wt sequence. In this work, we decided to introduce the mutation E1202G in NS3. This mutation has been shown to increase the replication efficiency of the Con1 subgenome when placed together with the adaptive mutations S2197P, K@2039, S2204I (15
), and S2204A (Fig. ) in NS5A. The advantage of the E1202G mutation is the fact that it has been found as a naturally occurring mutation in an infective clone of the 1A strain, thus suggesting that this mutation does not destroy infectivity in vivo (28
). The E1202G mutation is synergistic when placed together with adaptive mutations at the hyperphosphorylation site of NS5A and is therefore an ideal candidate for synergistic effects in the presence of the kinase inhibitors.
Replicons containing the E1202G substitution in NS3 (m17) do replicate with low efficiency, and HCV proteins cannot be detected by Western blotting (Fig. ). In this genetic background, all three compounds induce replication sufficiently to be detectable by Western blotting and by TaqMan (Fig. ).
The different degrees of replication efficiency induced by compound H479 and compounds A852 and F495 are probably not a consequence of different potency in the inhibition of p58 formation, which seems to be comparable at 5 μM (Fig. ). This apparent difference could be explained by considering the cytotoxicity displayed by compound A852 and especially by compound F495 at concentrations of 8 μM or more (data not shown). During the experiments designed for the detection of inhibition of p58 formation, the cells are exposed to 5 μM compound for several hours, whereas they are exposed for 4 days at 8 μM during the induction of replication. It is therefore possible that inhibition of NS5A hyperphosphorylation is necessary for the induction of replication, but different side effects of the compounds may induce or block other pathways which may have antagonistic effects on the induction of replication.
Interestingly, the compounds we identified potently inhibit phosphorylation of NS5A in vitro (Fig. , lanes 11 to 13), suggesting that they may directly inhibit NS5A-specific kinase(s). However, we cannot exclude the possibility that these compounds act via the inhibition of upstream kinase(s) that may also be present in the immunoprecipitate. The spectrum of cellular kinases inhibited by these agents and whether any of these kinases is directly or indirectly responsible for NS5A hyperphosphorylation needs to be clarified by future work.
Our findings support a role for NS5A p58 in down-regulating HCV RNA replication in cell culture. Based on the present data, we cannot exclude that the compound(s) we identify act via the inhibition of the phosphorylation of cellular protein(s) regulating HCV RNA replication and that inhibition of p58 is not the actual cause of increased replication. However, we believe that the present data are supportive of a direct, causal connection between NS5A hyperphosphorylation and the inability of hepatitis C subgenomic RNA to replicate in cell culture. This is based on two considerations. First, all of the kinase inhibitors that we selected for specific inhibition of NS5A hyperphosphorylation in cell culture were able to activate RNA replication. Second, it has been previously reported that several adaptive mutations found in the NS5A sequence occur at conserved serine residues and greatly impair the hyperphosphorylation of NS5A (2
Figure shows two possible models which try to explain the inhibitory effect of NS5A p58 on HCV replication in cell culture. According to model A, p56 is necessary for active replication. In cell culture, p56 is converted to p58 by cellular kinases and recruits a repressor protein (R) to the replication complex, thereby inhibiting replication. In the presence of the specific kinase inhibitors the conversion to p58 is prevented and the repressor protein R will not be recruited to the replication complex (upper part of Fig. ). There are three types of adaptive mutations which result in active replication in cell culture, and the first occurs exactly at the sites of NS5A hyperphosphorylation (S2197, S2201, and S2204). These mutations prevent the production of p58 and thus result in replication in cell culture. This situation would result in a p56-containing active replication complex, similar to that formed in the presence of the NS5A hyperphosphorylation inhibitor (Fig. , top panel). The second type of adaptive mutations occurs in NS5A, however, at sites different from the hyperphosphorylation sites. As a consequence, p58 is still expressed (as in the case of Huh7-68). This example is shown in the bottom part of Fig. , to the left of each model. The third type of adaptive mutations does not occur in NS5A, but it occurs in other proteins which participate in viral replication, e.g., NS3 or NS5B. This example is shown in the bottom part of Fig. , to the right of each model.
FIG. 5. Working model. Replication in cell culture is inhibited in the presence of p58. (A) p58 recruits a repressor protein R to the replication complex. Upper panel: p56 is converted to the hyperphosphorylated form p58 by cellular kinases, which then binds (more ...)
In the latter two cases, adaptive mutations prevent the association of the repressor molecule and replication can take place even in the presence of p58 (Fig. , bottom part). This model is supported by recent data obtained by Graziani et al. (9
), who demonstrate that coexpression of wt HCV subgenomic replicons together with adapted replicons results in transdominant inhibition of HCV replication in cell culture. Inhibitory cellular proteins may be expressed only in transformed cell lines and be absent in the liver, thus explaining the strict dependence on adaptive mutations for replication in cell culture.
In model B, p58 is not able to interact with an activator protein (A), which is necessary for replication. Also in this model, the kinase inhibitors prevent the conversion of p56 into p58 and binding of NS5A to the activator protein A is maintained (upper part of Fig. ). In this model, one has to assume that the latter two types of adaptive mutations restore protein-protein interactions between NS5A and A, even if NS5A exists as p58. The latter model is supported by recent data obtained by Evans et al. (8a
Production of high levels of p58 may be an artifact of the subgenomic construct. The expression of the nonstructural proteins is driven by the strong encephalomyocarditis virus internal ribosome entry site, and the amount of NS5A p58 during infection in vivo could be significantly lower. Alternatively, the specific kinases and, thus, the formation of p58 are expressed or regulated differently in cell lines with respect to the liver, thus disturbing the correct equilibrium between p56 and p58. Different expression of cellular factors could also explain the strict host dependence of HCV for replication in cell culture. It would be of great interest to identify the kinases which are responsible for the formation of p58. A cellular system in which the expression of this kinase can be regulated would be an ideal system for the study of the replication of HCV containing the original, infective wt sequence.
There is an additional interesting observation we would like to discuss. Figure clearly shows that treatment of cells with compound H479, A852, or F495 results in a substantial reduction of p58 but is not completely abolished. Two lines of evidence indicate that complete prevention of p58 formation eliminates replication. (i) Blight et al. (3
) demonstrated that the mutation S2204I is a very efficient adaptive mutation, whereas the combination of this mutation together with a second adaptive mutation within the hyperphosphorylation sites of NS5A (S2197P) completely abolished replication. (ii) Incubation of cells containing a replicon with the adaptive mutation S2204A with one of our kinase inhibitors also abolishes HCV replication (Fig. , lanes 20 to 25). In both cases, mutation at one hyperphosphorylation site significantly reduces the quantity of p58 and the second event, either a second mutation or inhibition by our compounds, further reduces p58 formation and results in a complete block of replication. This observation suggests, contrary to what has been thought to date, that low amounts of p58 may in fact be necessary for replication and that compounds that completely inhibit NS5A hyperphosphorylation could have antiviral activity in vivo.
In conclusion, we have identified compounds that, by inhibiting protein kinase(s) implicated in NS5A hyperphosphorylation, promote replication of nonadapted HCV sequences in cell culture. This finding points to a role for NS5A p58 in down-regulating HCV RNA replication in cell culture. To date, it is not known whether NS5A p58 has a function in viral replication in vivo or what that function may be. However, compounds that selectively inhibit NS5A hyperphosphorylation could be useful tools for the establishment of a bona fide infection system for HCV in cell culture and could possibly be a starting point for the development of novel antiviral agents.