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The HCV NS5A protein plays multiple roles during viral replication, including viral genome replication and virus particle assembly. The crystal structures of the NS5A N-terminal domain indicated the potential existence of the NS5A dimers formed via at least two or more distinct dimeric interfaces. However, it is unknown whether these different forms of NS5A dimers are involved in its numerous functions. To address this question, we mutated the residues lining the two different NS5A dimer interfaces and determined their effects on NS5A self-interaction, NS5A-cyclophilin A (CypA) interaction, HCV RNA replication and infectious virus production. We found that the mutations targeting either of two dimeric interfaces disrupted the NS5A self-interaction in cells. The NS5A dimer-interrupting mutations also inhibited both viral RNA replication and infectious virus production with some genotypic differences. We also determined that reduced NS5A self-interaction was associated with altered NS5A-CypA interaction, NS5A hyperphosphorylation and NS5A subcellular localization, providing the mechanistic bases for the role of NS5A self-interaction in multiple steps of HCV replication. The NS5A oligomers formed via different interfaces are likely its functional form, since the residues at two different dimeric interfaces played similar roles in different aspects of NS5A functions and, consequently, HCV replication. In conclusion, this study provides novel insight into the functional significance of NS5A self-interaction in different steps of the HCV replication, potentially, in the form of oligomers formed via multiple dimeric interfaces.
Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168. To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.