Study of the HCV life cycle in surrogate models of infection, e.g., HCVpp and replicon cell lines, has permitted analysis of the cellular and molecular events that govern viral entry and steady-state HCV RNA replication. Aspects of the viral life cycle not recapitulated in these systems, such as those occurring downstream of endosomal internalization of the infectious particles, leading to primary translation of the viral genome and establishment of the replication complexes, are beginning to be characterized (5
) thanks to the development of cell culture infection systems that recapitulate every step of the HCV life cycle (37
By adaptation of this cell culture system into an unbiased cell-based screening methodology (22
), interrogation of chemical libraries for antiviral molecules against HCV enables discovery of antiviral compounds that interrupt aspects of the infection that were previously uncharacterized. Thus, this methodology has the potential not only to provide novel lead compounds for therapy against this important pathogen but also to identify novel chemical probes to study aspects of HCV infection that have been functionally and biochemically uncharacterized.
Using this methodology, we have identified and characterized a novel family of small molecules that efficiently inhibit viral spread at nanomolar concentrations. These compounds efficiently inhibited HCV infection without altering viral entry, as shown by the lack of antiviral activity against HCVpp (), or interfering with persistent HCV RNA replication and virus particle production in persistently infected cells ().
Time-of-addition experiments indicate that the inhibition occurs at early steps of the infection, since addition of the compounds as early as 2 h after virus inoculation resulted in the virtual loss of antiviral activity (). The results shown in argue against a rapid loss of antiviral activity due to compound instability since the compounds retained full antiviral activity even when they were added to the cultures 6 h before virus inoculation. Since HCVpp entry was unaffected by these compounds, we postulated that they target steps downstream of viral entry. This hypothesis was confirmed by the antiviral activity of the compounds against the initiation of HCV RNA replication by HCV subgenomic replicons that were electroporated into naïve Huh-7 cells ( and B). This effect was dependent on the time of addition of the compounds and was not observed once HCV RNA replication had been initiated (), recapitulating the lack of antiviral activity observed in persistently infected cells. Overall, these observations indicate that compounds 1 and 2 specifically target a very early step in HCV RNA replication without altering steady-state HCV RNA production. These results suggest that the compounds target a transient event that is rate limiting for the initiation of HCV RNA replication, rather than HCV RNA replication per se.
It has recently been shown that the cellular autophagy machinery is required for translation of incoming HCV genomes but not for translation of progeny genomes (12
). Collectively, those observations and the results presented herein suggest that the cellular and viral factors required to initiate viral replication are different from those required to maintain it once replication complexes have formed. Another possible interpretation of our results derives from the fact that expression of HCV proteins, notably NS4B, causes a profound reorganization of cellular membranous compartments to promote the formation of replication complexes in modified ER membranes (5
). It is therefore formally possible that the compounds described above target HCV RNA replication per se
but that virus-induced cellular changes (e.g., alteration of intracellular membranes) reduce the access of the compounds to their molecular target(s) sufficiently once replication complexes have been established to reduce their efficacy, resulting in an apparent lack of antiviral activity.
Selection of virus variants with reduced susceptibility to compound 2 (C2R virus) demonstrates the specific antiviral activity of this compound. We have shown that a point mutation in NS5A (F2004L) identified in the resistant virus variants is sufficient to confer the resistance phenotype to subgenomic replicons (), suggesting that the mechanisms by which these compounds inhibit HCV infection and establishment of the subgenomic-replicon replication are the same. Our results also indicate that mutation F2004L did not result in a significant fitness cost, as the C2R virus grows to titers comparable in magnitude (106 FFU/ml) and kinetics to those of the control virus (). Moreover, introduction of the F2004L point mutation into the subgenomic replicon did not impair baseline replication (), indicating that this mutation does not weaken viral replication even in a wild-type genetic background.
The NS5A residue F28 (F2004 in the polyprotein) is not conserved among all genotypes and strains, e.g., Con1 and J4L6 from genotype 1b display a leucine residue in that position (). However, a Con1-based replicon was susceptible to compound 2 () even at a concentration (2.5 μM) that was inactive against the JFH-1 F28L mutant, suggesting that a leucine residue in position 28 does not confer resistance to compound 2 in the context of genotype 1b. In any case, in the context of JFH-1 (genotype 2a), position F28 is located in a conserved class I proline-rich (PR) motif immediately adjacent to the membrane-anchoring amphipathic alpha helix located at the N terminus of NS5A (57
). Although no specific function has been attributed to this conserved PR motif, PR motifs in NS5A have been proposed as structural elements that determine interaction of NS5A with cellular factors containing Src homology 3 (SH3) domains (57
). This could be demonstrated for the C-terminal proline-rich motif, which mediates interaction with kinases like phosphatidylinositol 3-kinase (PI3K) (56
), Lck, Hck, and Fyn (40
), as well as adaptor proteins such as Grb2 (57
) and amphiphysin II/BinI (63
), but not for the N-terminal motif, in which the mutation conferring resistance to compound 2 (F28L) is included. It is therefore possible that compounds of the chemical family described here interfere with interactions of NS5A via its N-terminal PR motif and that mutation F28L restores such interactions that could be essential for the establishment of HCV RNA replication. Position F28 is located in close proximity to the N-terminal membrane-anchoring domain in NS5A and might contribute to the interaction of this protein with cellular membranes. It is possible that, during the initial phases of the infection, compounds 1 and 2 interfere with docking of NS5A into membranes devoid of viral replication complexes and that this does not occur once replication complexes have been formed. While these speculative scenarios would explain our observations, extensive biochemical studies are required to define the molecular events underlying both antiviral activity and resistance. Studies aimed at determining the composition of the molecular complexes that compounds 1 and 2 may bind in the cell will contribute to understanding the mechanisms underlying the establishment of HCV RNA replication complexes, a process in which NS5A could play a specific role.
NS5A has recently been revealed as a novel target for potent antiviral compounds that inhibit viral RNA replication (e.g., BMS-790052) (17
). Genetic evidence indicates that compound 2 targets NS5A directly or indirectly () but seems to do so by a different, unprecedented mechanism that targets initiation but not steady-state HCV RNA replication. Remarkably, despite this apparent differential mode of action, position F28 in JFH-1 is aligned with position 28 in genotypes 1a and 1b (), where mutations M28T and L28T, respectively, have been reported to confer partial resistance to BMS-790052 (14
). These genetic data indicate that both compounds impose similar selective pressures on the N terminus of NS5A, suggesting that they could target similar functions in NS5A but appear to do so by different molecular mechanisms.
Multiple functions involving different steps in the viral life cycle have been attributed to NS5A. However, no specific enzymatic activity has been ascribed to NS5A, and the molecular mechanisms by which it functions remain elusive. Thus, the inhibitors described herein constitute potentially powerful chemical tools that could facilitate our understanding of the functions that NS5A plays in the viral life cycle and may lead to the development of novel compounds with improved therapeutic potential.