The roles of post-translational modification of PIV5 P protein in viral gene expression

Open Access
Sun, Dengyun
Graduate Program:
Cell and Developmental Biology
Doctor of Philosophy
Document Type:
Date of Defense:
March 15, 2011
Committee Members:
  • Biao He, Anthony Schmitt, Dissertation Advisor
  • Biao He, Committee Chair
  • Anthony Paul Schmitt, Committee Chair
  • Na Xiong, Committee Member
  • Andrea Marie Mastro, Committee Member
  • Avery August, Committee Member
  • Kinase
  • Virus
  • Post-translational modification
  • Gene expression
  • Apoptosis
  • Immune response
Paramyxovirus RNA synthesis requires the large (L) protein and the phosphoprotein (P). It was initially thought that phosphorylation of the P protein was important for viral gene expression, however there is no direct proof supporting the assumption. Recently, our group reported that phosphorylation at S157 of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlated with decreased viral gene expression. In this study, I have shown that: (1) Polo-like kinase 1 (PLK1) bound the P protein through the S(pS157)P motif; (2) PLK1 inhibitor increased PIV5 gene expression; (3) PLK1 over-expression inhibited PIV5 gene expression; (4) PLK1 inhibitor reduced phosphorylation level of the P protein in PIV5 infected cells; and (5) PLK1 directly phosphorylated the P protein in vitro, indicating that PLK1 down-regulated PIV5 gene expression by phosphorylating the P protein. Furthermore, I have determined the PLK1 phosphorylation site, S308, within the P protein and found that mutation at S308 to alanine increased the minigenome activity, which was not affected by either PLK1 inhibitor or PLK1 over-expression. Mutation at either the binding site or the phosphorylation site of PLK1 in PIV5 increased virus gene expression, which correlated with increased induction of cell death and cytokine induction. The results indicate that PIV5 limits its viral gene expression to avoid induction of innate immune responses. Since the P protein is heavily phosphorylated, we speculate that phosphorylation of the P protein at other sites may play a positive role in PIV5 gene expression. I have identified a phosphorylation site of PIV5 P protein by mass spectrometry, T286, and found that (1) mutation of T286 to alanine reduced PIV5 minigenome activity; (2) P-T286D showed very low activity and P-T286E showed no activity in the minigenome system; (3) rPIV5-P-T286A virus grew slower than PIV5; (4) viral RNA synthesis and protein expression in rPIV5-P-T286A infected cells were delayed. The defects of T286A/D/E were not due to NP-P-L complex formation. I also found that P-T286A/D/E affected viral transcription. It is the first time that the P protein phosphorylation is found to be important for paramyxovirus gene expression in virus infection. Sumoylation is a protein post-translational modification and plays an important role in regulating protein function and signal transduction. I have discovered that the P protein of PIV5 can be sumoylated by SUMO1 in both transfected cells and infected cells. I have identified one sumoylation site within the P protein, K254. Mutation at K254 to arginine (P-K254R) reduced PIV5 minigenome activity. Incorporation of K254R into the genome of the virus (rPIV5-P-K254R) resulted in reduction of viral growth, viral RNA synthesis and viral protein expression, indicating that PIV5 uses host sumoylation system to regulate its growth.