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Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31
D R Broussard1, L A Guarino, D L Jarvis
1Department of Entomology, Texas A&M University, College Station, Texas 77843, USA.
Abstract:
Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.
Insights
Autographa californica nuclear polyhedrosis virus pp31 protein is phosphorylated by cellular and viral kinases. This dynamic phosphorylation, crucial for viral replication, involves multiple sites and a unique viral kinase.
Area of Science:
- Molecular Virology
- Insect Pathology
- Protein Biochemistry
Background:
- Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein essential for viral replication.
- pp31 accumulates in the virogenic stroma, binds DNA, and functions as a late expression factor.
- Post-translational modification, specifically phosphorylation, can significantly impact protein function.
Purpose of the Study:
- To investigate the phosphorylation and dephosphorylation dynamics of the AcMNPV pp31 protein.
- To identify the kinases and phosphatases involved in pp31 modification.
- To map the phosphorylation sites within the pp31 protein.
Main Methods:
- Analysis of pp31 phosphorylation and dephosphorylation in infected cells.
- Use of aphidicolin to assess the role of late viral gene expression.
- Site-directed mutagenesis of pp31 fusion proteins to identify phosphorylation sites.
- Biochemical assays to characterize kinase and phosphatase activities.
Main Results:
- pp31 is phosphorylated by both cellular and virus-encoded/induced kinases.
- Threonine phosphorylation by a virus-specific kinase requires late viral gene expression.
- pp31 is dephosphorylated by viral phosphatases, indicating dynamic modification.
- At least three phosphorylation sites exist: N-terminal serine (cellular kinase), C-terminal threonine (viral kinase), and C-terminal serine (host or viral kinase).
- Mutagenesis of consensus threonine sites did not abolish viral threonine phosphorylation, suggesting a unique viral kinase recognition site.
Conclusions:
- pp31 phosphorylation is a dynamic, regulated process involving both host and viral factors.
- The viral kinase responsible for threonine phosphorylation is unique and its substrate recognition site is yet to be determined.
- Understanding pp31 phosphorylation is key to elucidating AcMNPV replication mechanisms.