Sequential S232/S235/S238 phosphorylation of the hepatitis C virus nonstructural protein 5A
Journal
Journal of Virology
Journal Volume
92
Journal Issue
20
Date Issued
2018
Author(s)
Abstract
The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.
SDGs
Other Subjects
alanine; aspartic acid; nonstructural protein 5A; casein kinase Ialpha; CSNK1A1 protein, human; NS-5 protein, hepatitis C virus; serine; viral protein; amino acid sequence; amino terminal sequence; Article; carboxy terminal sequence; cell lysate; controlled study; enzyme inhibition; Hepatitis C virus; human; human cell; immunoblotting; immunoprecipitation; nonhuman; priority journal; protein function; protein phosphorylation; protein RNA binding; virus assembly; virus replication; amino acid substitution; cell line; dna mutational analysis; Hepacivirus; host pathogen interaction; liver cell; metabolism; phosphorylation; physiology; protein processing; virology; Amino Acid Substitution; Casein Kinase Ialpha; Cell Line; DNA Mutational Analysis; Hepacivirus; Hepatocytes; Host-Pathogen Interactions; Humans; Phosphorylation; Protein Processing, Post-Translational; Serine; Viral Nonstructural Proteins
Publisher
American Society for Microbiology
Type
journal article