Abstract
Vaccinia virus, a complex dsDNA virus, is unusual in replicating exclusively within the cytoplasm of infected cells. Although this prototypic poxvirus encodes >200 proteins utilized during infection, a significant role for host proteins and cellular architecture is increasingly evident. The viral B1 kinase and H1 phosphatase are known to target cellular proteins as well as viral substrates, but little is known about the cellular substrates of the F10 kinase. F10 is essential for virion morphogenesis, beginning with the poorly understood process of diversion of membranes from the ER for the purpose of virion membrane biogenesis. To better understand the function of F10, we generated a cell line that carries a single, inducible F10 transgene. Using uninduced and induced cells, we performed stable isotope labeling of amino acids in cell culture (SILAC) coupled with phosphopeptide analysis to identify cellular targets of F10-mediated phosphorylation. We identified 27 proteins that showed statistically significant changes in phosphorylation upon the expression of the F10 kinase: 18 proteins showed an increase in phosphorylation whereas 9 proteins showed a decrease in phosphorylation. These proteins participate in several distinct cellular processes including cytoskeleton dynamics, membrane trafficking and cellular metabolism. One of the proteins with the greatest change in phosphorylation was mDia, a member of the formin family of cytoskeleton regulators; F10 induction led to increased phosphorylation on Ser22 Induction of F10 induced a statistically significant decrease in the percentage of cells with actin stress fibers; however, this change was abrogated when an mDia Ser22Ala variant was expressed. Moreover, expression of a Ser22Asp variant leads to a reduction of stress fibers even in cells not expressing F10. In sum, we present the first unbiased screen for cellular targets of F10-mediated phosphorylation, and in so doing describe a heretofore unknown mechanism for regulating stress fiber formation through phosphorylation of mDia. Data are available via ProteomeXchange with identifier PXD005246.
Highlights
From the ‡Departments of Biochemistry & Molecular Biology and Microbiology & Immunology, and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina; §Department of Microbiology & Molecular Genetics and the Biotechnology & Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin
One of the most significant plagues to have affected mankind, smallpox was successfully eradicated through worldwide vaccination with vaccinia virus, a natural attenuated virus that is closely related to variola and serves as the prototypic poxvirus for experimental research
Generation and Characterization of Cell Lines Exogenously Expressing the Vaccinia Virus F10 Kinase—The first lethal phenotype seen upon repression or inactivation of the viral F10 kinase is a dramatic block in the initiation of morphogenesis [19, 20]
Summary
The first lethal phenotype seen in infections with the ts mutant, Cts, which has a single point mutation that renders the F10 kinase inactive at nonpermissive temperatures (39.7 °C), is a dramatic block in morphogenesis [19, 20] Under these conditions, the full repertoire of biochemical events occur unperturbed (DNA replication, three stages of gene transcription and protein synthesis). No evidence of membrane biogenesis or virion assembly can be found within the cytoplasmic areas that have been cleared of organelles in preparation for morphogenesis [20] This block is reversible; within 15 min after cells are shifted to the permissive temperature crescent membranes are seen and by 90 min, mature virions are readily observed [32]. MDia, was investigated further and was confirmed to be phosphorylated in cell culture in an F10-dependent manner. mDia is involved in regulating the actin cytoskeleton, and the consequence of mDia phosphorylation by F10 is a significant loss of stress fiber formation that has consequences for cell structure and function
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