Date Published: July 23, 2018
Publisher: Public Library of Science
Author(s): Saravanabalaji Shanmugam, Alyssa K. Nichols, Dhanaranjani Saravanabalaji, Christoph Welsch, MinKyung Yi, Glenn Randall.
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.
Hepatitis C virus (HCV) is a main causative agent associated with chronic liver diseases including chronic hepatitis, cirrhosis and hepatocellular carcinoma [1, 2]. It is an enveloped, positive-stranded RNA virus belonging to the genus hepacivirus within the flaviviridae family . A single polyprotein translated from the viral genome encodes structural proteins, including core, E1, and E2 at the N-terminal domain followed by the viral assembly accessory proteins p7 [4, 5] and NS2 [6–10]. The C-terminal domain encodes five different nonstructural proteins including NS3, NS4A, NS4B, NS5A and NS5B, which comprise viral replicase complexes  and regulate viral assembly [12–17].
HCV NS5A is a multifunctional protein involved in both viral RNA replication and virus production [24, 54, 55]. Our study provided mechanistic insights regarding the roles of, crystal structure-defined, NS5A dimer interface residues in these two critical steps in the HCV life cycle. Specifically, our data revealed that these residues regulate NS5A self-interaction, NS5A-CypA interaction, NS5A hyperphosphorylation, NS5A localization to LD and NS5A-core interaction, promoting HCV replication and infectious HCV production.