Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication

Abstract

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase I␣ (CKI␣) directly phosphorylated Ser-235 in vitro. Inhibition of CKI␣ reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKI␣ probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.Chronic HCV 2 infection affects 130 -170 million people worldwide (1). The infection is often asymptomatic until development of severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma, making chronic HCV infection the most common cause of liver transplant (2). HCV is an enveloped virus with a positive, single-stranded RNA genome encoding three structural (core, E1, and E2) and seven non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (1). The structural proteins together with the host membranes make up the viral particles, whereas the non-structural proteins are required for a complete life cycle. Already, there are several approved highly efficient HCV antivirals targeting non-structural proteins, including NS3/4A protease inhibitors (boceprevir, telaprevir, and simeprevir) and an NS5B RNA-dependent RNA polymerase inhibitor (sofosbuvir) (3). However, their high costs prohibit their accessibility to most patients (4). New competitive alternatives are desirable.NS5A is a multitasking protein required for the HCV life cycle and thus a good antiviral target (5). It is a phosphoprotein that appears as two bands at 56 and 58 kDa on immunoblots, respectively, referred to as hypophosphorylated (p56) and hyperphosphorylated (p58) NS5A (6). NS5A interacts with many viral and host proteins and participates in various aspects of the viral life cycle (7). For example, NS5A was reported to interact with the hVAP-A protein that takes part in the replication protein complex formation (8 -10). NS5A mutatio...

Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication

“…Aspartate mutations suggest that NS5A phosphorylation occurs in a sequential cascade where phosphorylation of one serine residue leads to phosphorylation of the other serine residues (12,21). In our previous proteomics study (22), we identified phosphorylation at S222, S235, and S238 with high confidence scores. Using genetic mutations, we found that alanine mutation at S235 blunted NS5A hyperphosphorylation and viral replication.…”

“…Using genetic mutations, we found that alanine mutation at S235 blunted NS5A hyperphosphorylation and viral replication. Single-alanine mutation at S222 or S238 did not have apparent effects; however, double mutations at S222 and S238 reduced NS5A hyperphosphorylation and viral replication (22), suggesting that phosphorylation events among these sites are interdependent. In the previous work, we generated an antibody specific to NS5A S235 phosphorylation and used it to show that S235 phosphorylation correlated with viral replication in time and intracellular locations (22).…”

“…Single-alanine mutation at S222 or S238 did not have apparent effects; however, double mutations at S222 and S238 reduced NS5A hyperphosphorylation and viral replication (22), suggesting that phosphorylation events among these sites are interdependent. In the previous work, we generated an antibody specific to NS5A S235 phosphorylation and used it to show that S235 phosphorylation correlated with viral replication in time and intracellular locations (22). Here, we generated two additional antibodies specific to NS5A S222 and S238 phosphorylation to measure their levels of phosphorylation and to study interdependence among S222, S235, and S238 phosphorylation.…”

“…NS5A hyperphosphorylation has been associated with viral replication and assembly (17,18). Many studies, largely based on genetic mutations, have pinpointed several serine residues in the LCS I region responsible for NS5A hyperphosphorylation and functions (17)(18)(19)(20)(21)(22)(23). For example, S225 was reported to participate in the formation of viral replication protein complex (24) as well as viral assembly (18).…”

“…Isoleucine mutation at S232 enhances viral replication in HCV genotype 1 (26); however, alanine mutation at the same site reduces viral replication or assembly in genotype 2 (17,18), leaving S232 a mysterious site for functions in different genotypes. Recently, S235 was shown to be a critical amino acid for HCV replication, likely via regulating replication complex formation (22,23). Thus, genetic mutations that either remove or mimic phosphorylation have been very instrumental in demonstrating the functions of the above-described serine residues.…”