The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization

Abstract

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…2C), suggesting that the mechanism of antiviral action of NS5A inhibitors do not involve the rupture of NS5A-NS5A interactions. While our studies were in progress, a report showed that NS5A inhibitors such as BMS-790052 impair hyperphosphorylation of NS5A and that this activity is mediated by sequences within Domain 1 (38).…”

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

“…HCV nonstructural proteins were expressed from replicon plasmids in BHK-21 cells by using a modified vaccinia virus-T7 (MVA-T7) expression system or following transfection of replicon RNA into Huh7.5 cells as previously described (26,33). Western blot analysis of NS5A and NS3 proteins was performed as previously described (26).…”

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors. [12][13][14] Furthermore, NS5A inhibitors have been shown to induce redistribution of NS5A from endoplasmic reticulum-derived foci, [12][13][14] possibly to lipid droplets, 12 and limit hyperphosphorylation of NS5A, [14][15][16] although these effects may be indirect. Finally, it has been suggested that NS5A inhibitors may disrupt interactions of NS5A with HCV RNA, other viral proteins, and/or host factors that are coopted by NS5A during the HCV life cycle.…”