Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States

Abstract

The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle. IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.

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

“…2A, S1169A) (31,32). We have shown in many prior publications that NS5A phosphorylation occurs similarly in the HEK293T cells and the Huh7.5.1 cells (19)(20)(21)27). In the wild-type NS3-5A-transfected HEK293T cells, we observed a single NS3 band with a correct molecular weight of about 67 kDa (Fig.…”

“…Thus, even though S2205 phosphorylation plays a key role in NS5A sequential phosphorylation, it is not absolutely required. In contrast, an alanine mutation at S2201 abolishes, whereas a phosphorylation-mimicking aspartate mutation rescues, NS5A sequential phosphorylation (19,24) in the absence of S2205 phosphorylation (20). Therefore, S2201 phosphorylation could bypass S2205 phosphorylation to regulate NS5A sequential phosphorylation.…”

“…Both states are critical to the viral life cycle (14)(15)(16)(17)(18). Recent studies have shown that NS5A transits between hypo-and hyperphosphorylated states via the phosphorylation of a cluster of highly conserved serine residues, i.e., S2201, S2205, S2208, S2211, and S2214, in a sequential manner (19)(20)(21). NS5A transition between the two states is thought to regulate its intracellular distribution in large and small structures required for a complete viral life cycle (20,22).…”

“…Recent studies have shown that NS5A transits between hypo-and hyperphosphorylated states via the phosphorylation of a cluster of highly conserved serine residues, i.e., S2201, S2205, S2208, S2211, and S2214, in a sequential manner (19)(20)(21). NS5A transition between the two states is thought to regulate its intracellular distribution in large and small structures required for a complete viral life cycle (20,22). Of the serine residues, S2205 was shown to undergo phosphorylation and dephosphorylation cycles that fire the casein kinase I ␣-mediated sequential S2208/S2211/S2214 phosphorylation cascade, leading to the hyperphosphorylated state (20,23).…”

“…NS5A transition between the two states is thought to regulate its intracellular distribution in large and small structures required for a complete viral life cycle (20,22). Of the serine residues, S2205 was shown to undergo phosphorylation and dephosphorylation cycles that fire the casein kinase I ␣-mediated sequential S2208/S2211/S2214 phosphorylation cascade, leading to the hyperphosphorylated state (20,23). However, a phosphorylation-ablating alanine mutation at S2205 did not completely abolish sequential phosphorylation (19).…”