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...

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. However, less than 5% of BLV-infected cattle will develop lymphoma, suggesting that, in addition to viral infection, host genetic polymorphisms might play a role in disease susceptibility. Bovine leukocyte antigen (BoLA)-DRB3 is a highly polymorphic gene associated with BLV proviral load (PVL) susceptibility. Due to the fact that PVL is positively associated with disease progression, it is believed that controlling PVL can prevent lymphoma development. Thus, many studies have focused on the relationship between PVL and BoLA-DRB3. Despite this, there is little information regarding the relationship between lymphoma and BoLA-DRB3. Furthermore, whether or not PVL-associated BoLA-DRB3 is linked to lymphoma-associated BoLA-DRB3 has not been clarified. Here, we investigated whether or not lymphoma-associated BoLA-DRB3 is correlated with PVL-associated BoLA-DRB3. We demonstrate that two BoLA-DRB3 alleles were specifically associated with lymphoma resistance (*010:01 and *011:01), but no lymphoma-specific susceptibility alleles were found; furthermore, two other alleles, *002:01 and *012:01, were associated with PVL resistance and susceptibility, respectively. In contrast, lymphoma and PVL shared two resistance-associated (DRB3*014:01:01 and *009:02) BoLA-DRB3 alleles. Interestingly, we found that PVL associated alleles, but not lymphoma associated alleles, are related with the anti-BLV gp51 antibody production level in cows. Overall, our study is the first to demonstrate that the BoLA-DRB3 polymorphism confers differential susceptibility to BLV-induced lymphoma and PVL.

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo-and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase I␣, which requires a priming phosphorylation at S235. IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypoand hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.KEYWORDS NS5A, antibody, hepatitis C virus, protein phosphorylation, proteomics H epatitis C virus (HCV) is an enveloped virus with a 9.6-kb single-stranded positivesense RNA genome. It infects about 185 million people worldwide and is a leading cause of liver diseases and related complications (1, 2). The HCV genome encodes a long polyprotein composed of 10 proteins from NH 2 to the COOH terminus: core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. A complete HCV life cycle requires 3