Unique amongst the DNA viruses, poxviruses complete their replicative cycles entirely within the cytoplasmic compartment of infected cells. This property requires that these viruses be able to express, activate, localize and concentrate their encoded gene products at the correct intracellular sites to enable the assembly of infectious progeny. The process becomes more complicated due to the fact that poxiviruses produce a number of different virion forms (IMV, intracellular mature virus; IEV, intracellular enveloped virus: CEV, cell-associated enveloped virus; and EEV, extacellular enveloped virus), all of which may play different roles in vivo. Furthermore, some poxviruses (e.g. Cowpox) also produce yet another form of virion which is occluded in an inclusion body, presumably to facilitate the stability and dissemination of infectious particles in nature. To help direct viral protein traffic, poxviruses such as vaccinia virus have adopted many of the same protein modification, activation and targeting pathways used by their cellular hosts including glycosylation, phosphorylation, proteolytic processing, and in particular, acylation. During the previous grant period, our laboratory identified more than a dozen vaccinia acylproteins, both myristylproteins and palmitylproteins and demonstrated that they have key roles in the assembly of vaccinia virions. In the experiments detailed for the upcoming grant period, we now propose to focus and extend our studies in this area by concentrating on three vaccinia virus acyl protein which play central roles in VV replication processes: L1R(a 25 kDa N-terminally myristylated protein involved in IMV maturation and/or penetration), F13L(a 37 kDa palmitylated protein required for EEV envelopment), and the ATI gene product (a 92kDa protein homologue of the CPV 160 kDa ATI protein involved in the occlusion process, which contains a novel internal myristylation modification). For each protein we will use a combination of genetic, biochemical and molecular biological approaches: i) to study the nature, site and enzymology of the modification reaction; (ii) to examine what the function of the acyl modification is relative to the location and activity of the protein; and iii) to determine what biological role these proteins play during the VV replicative process itself.
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