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Intracellular phosphorylation of the Sendai virus P protein
S Byrappa1, D D Hendricks, Y B Pan
1Department of Immunology/Microbiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Virology
|April 1, 1995
Summary
Sendai virus P protein phosphorylation occurs constitutively at serine sites during infection. Cell-free phosphorylation differs significantly, questioning its relevance for understanding viral protein modification.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- The phosphorylation status of viral proteins is crucial for understanding virus replication and pathogenesis.
- Sendai virus P protein plays a key role in viral RNA synthesis.
- Previous studies have not fully elucidated the phosphorylation patterns of the Sendai virus P protein in infected cells versus cell-free systems.
Purpose of the Study:
- To investigate and compare the phosphorylation sites and patterns of the Sendai virus P protein during virus infection and in cell-free systems.
- To determine the role of cellular phosphatases in regulating P protein phosphorylation.
- To assess the correlation between intracellular and cell-free phosphorylation of the P protein.
Main Methods:
- Analysis of P protein phosphorylation in Sendai virus-infected cells, P/C gene-transfected cells, and purified virions.
- Cell-free phosphorylation assays using virion-associated protein kinase (VAPK).
- Tryptic phosphopeptide mapping to identify phosphorylation sites.
- Inhibition of cellular phosphatases (PP1 and PP2A) using okadaic acid (OA).
Main Results:
- Intracellular P protein (from infected cells, transfected cells, and virions) is phosphorylated exclusively at serine residues, primarily on one peptide (TP1), indicating constitutive phosphorylation.
- Cell-free phosphorylation by VAPK occurs at both threonine and serine residues, with multiple phosphorylation sites.
- Inhibition of phosphatases in infected cells increases P protein phosphorylation at serine sites, but the phosphopeptide map differs from VAPK-mediated phosphorylation.
- Additional phosphorylation does not impede P protein association with nucleocapsids.
Conclusions:
- Sendai virus P protein exhibits constitutive serine phosphorylation at a primary locus in infected cells, with potential for additional phosphorylation sites.
- Cell-free phosphorylation by VAPK shows distinct patterns and sites compared to intracellular phosphorylation.
- The significant differences observed question the validity and relevance of current cell-free phosphorylation models for studying Sendai virus P protein modification.