Graduate Thesis Or Dissertation
 

Evidence for the modification of vaccinia virus core proteins by ADP-ribosylation

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  • Vaccinia virus (VV), the prototype member of the orthopoxvirus family, is a large virus of complex morphology which contains a 191 Kbp double-stranded DNA genome whose expression is tightly regulated in a temporal fashion during viral replication. The regulation of gene expression can be exerted at various of levels, including transcriptional, translational, and post-translational points of control. In addition to transcriptional regulatory mechanisms, the occurrence of a variety of post-translational modifications in VV has been demonstrated. In an effort to better understand the role played by post-translational modifications during the viral replication cycle, we chose to focus on one specific modification event, ADP-ribosylation. Experiments were designed to determine whether any VV proteins might be subject to ADP-ribosylation. The ability to metabolically label a subset of viral proteins by growth of the virus in the presence of [3H]adenosine, in addition to the effects of the ADP-ribosylation inhibitor nicotinamide on viral core protein precursor processing and replication, provided evidence that this or some similar modification is an obligatory event during VV replication. Immunological reagents were used to identify several of the modified proteins. Biochemical evidence obtained via labeling with various precursor compounds, boronate affinity chromatography, and reverse phase HPLC analysis confirmed that the proteins were modified by ADP-ribose or a closely related compound. Additional ADP-ribosylation inhibitor studies provided further support for the initial finding that the viral proteins are subject to ADP-ribosylation or some related modification, and the evidence obtained from these experiments supports a model where this modification event might serve a function in either the proteolytic processing of the core protein precursors, or in localization of the mature core proteins to sites of VV replication within infected cells.
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