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Phosphorylation of the viral nonstructural protein NS1 during MVMp infection of A9 cells
R Corbau1, N Salom, J Rommelaere
1Applied Tumor Virology Program, Institute National de la Santé et de la Recherche Médicale U375-, Heidelberg, D-69120, Germany.
Abstract:
The major nonstructural protein of parvovirus MVMp, NS1, is an 83-kDa nuclear phosphoprotein which exerts a variety of functions during a viral infection. These multiple tasks range from its major involvement in viral DNA amplification and promoter regulation to the cytotoxic action on the host cell. Since these most divergent functions are exerted in an orderly fashion, it has been proposed that NS1 is regulated by posttranslational modifications, in particular phosphorylation. So far it has been shown that the capacity of NS1 for initiation of replication is regulated in vitro by phosphorylation through members of the protein kinase C family, most likely as a result of control of the DNA unwinding activity (J. P. F. Nüesch et al., 1998, J. Virol. 72, 9966-9977). To substantiate these in vitro findings in vivo, we investigated NS1 phosphorylation during an MVMp infection in a natural host cell, A9 fibroblasts, with reference to characteristic features of the virus cycle. The NS1 phosphorylation pattern was found to change throughout the infection, raising the possibility that distinct tasks of NS1 might be achieved through differential phosphorylation of the polypeptide. In addition, we present in vivo evidence that a phosphorylated form of NS1 is able to initiate viral DNA replication and becomes covalently attached to replicated DNA. Moreover, NS1 was found to be phosphorylated in vivo within the helicase domain, showing alignment with at least one phosphopeptide generated by an "activating" kinase in vitro. These data suggest that phosphorylation-mediated regulation of NS1 for replicative functions as observed in vitro may also take place during a natural virus infection.
Insights
The parvovirus MVMp nonstructural protein 1 (NS1) undergoes phosphorylation changes during infection, suggesting differential regulation of its functions. Phosphorylated NS1 initiates viral DNA replication in vivo.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- The parvovirus MVMp nonstructural protein 1 (NS1) is crucial for viral DNA replication, promoter regulation, and host cell cytotoxicity.
- NS1's diverse functions are proposed to be regulated by post-translational modifications, particularly phosphorylation.
- In vitro studies indicate protein kinase C family members regulate NS1 replication initiation via phosphorylation, likely by controlling DNA unwinding activity.
Purpose of the Study:
- To investigate NS1 phosphorylation in vivo during MVMp infection in A9 fibroblasts.
- To correlate NS1 phosphorylation patterns with the viral life cycle.
- To provide in vivo evidence for phosphorylation-dependent regulation of NS1's replicative functions.
Main Methods:
- Monitoring NS1 phosphorylation patterns in A9 fibroblasts throughout MVMp infection.
- Analyzing the association of phosphorylated NS1 with viral DNA.
- Mapping phosphorylation sites within the NS1 helicase domain.
Main Results:
- NS1 phosphorylation patterns exhibit dynamic changes during the MVMp infection cycle.
- A phosphorylated form of NS1 was observed to initiate viral DNA replication and covalently attach to replicated DNA in vivo.
- NS1 phosphorylation occurs within its helicase domain, consistent with in vitro findings using activating kinases.
Conclusions:
- NS1 phosphorylation is dynamically regulated during MVMp infection in vivo.
- Phosphorylation plays a critical role in NS1-mediated viral DNA replication in a natural host cell.
- These findings support the hypothesis that phosphorylation controls NS1's replicative functions during natural parvovirus infection.