Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A

Abstract

Replication of the genotype 2 hepatitis C virus (HCV) requires hyper-phosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyper-phosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyper-phosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in the above sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wildtype NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction, too, abolished NS5A hyper-phosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant that resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyper-phosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated auto-cleavage at the NS3-4A junction is critical to NS5A hyper-phosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyper-phosphorylation could occur in an NS4A-NS4B-NS5A polyprotein. Importance For twenty some years, the HCV protease NS3 was implicated in NS5A hyper-phosphorylation. We now show that it was the NS3-mediated cis-cleavage at the NS3-4A junction that permits NS5A hyper-phosphorylation at serine 2201, 2208, 2211, and 2214, leading to hyper-phosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at the above serine residues right after NS3-4A cleavage and before NS5A was released from the NS4A-5A polyprotein. Our data suggest that the dual functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyper-phosphorylation.

How Viruses Use the VCP/p97 ATPase Molecular Machine

“…Purification of the HCV replicase revealed an association with VCP, and p97-specific inhibitors caused abnormal localization of the NS5A protein [ 86 , 87 ]. NS5A has been shown to be phosphorylated on multiple serines by cellular kinases in the context of a polyprotein precursor [ 88 , 89 ], and its activity is essential for production of the viral replicase complex [ 90 ]. Based on the use of a reporter vector system or in cells expressing a subgenomic replicon, which avoids effects on HCV entry, p97 inhibitors revealed that ATPase activity was required for viral RNA replication [ 87 ].…”

How Viruses Use the VCP/p97 ATPase Molecular Machine

“…Purification of the HCV replicase revealed an association with VCP, and p97-specific inhibitors caused abnormal localization of the NS5A protein [83,84]. NS5A has been shown to be phosphorylated on multiple serines by cellular kinases in the context of a polyprotein precursor [85,86], and its activity is essential for production of the viral replicase complex [87]. Based on the use of a reporter vector system or in cells expressing a subgenomic replicon, which avoids effects on HCV entry, p97 inhibitors revealed that ATPase activity was required for viral RNA replication [84].…”