Within the context of the infected cell, vaccinia virus (VV) expresses more than 100 virus-encoded gene products during the late (i.e., post-DNA synthesis) phase of the viral replicative cycle. These late functions include both structural proteins and viral enzymes which are packaged into progeny virions. To date, only a limited number of VV late genes have been identified. Furthermore, very little is known concerning either the structure and function of most of the encoded late proteins or the regulatory mechanisms (transcriptional and posttranscriptional) which operate to modulate their activity in order to facilitate their participation in the orderly assembly of infectious progeny virion particles. In order to dissect this complex problem down to a manageable size, we have chosen several representative VV late proteins for detailed study was made to allow us to achieve the dual goals of studying the expression and regulation of VV-encoded late gene products while learning something in general about the phenomena of protein localization, virus- host cell interactions, and the role of posttranslational protein modifications (ADP-ribosylation, and acylation) in protein function. The specific genes to be studied and the experiments to be carried out include:L65(Rifr) -site-directed mutagenesis, chimeric gene fusions, and immunoassays will be used to study the function of this protein, to identify the viral and cellular factors that is associates with in vivo, and the nature of the cis-information which localizes it to the virus factory during assembly;N2 (a-Am1) - directed genetics and PCR-aided sequencing of genomic viral DNA will be used to confirm the """"""""nuclear involvement"""""""" of the N2 protein, immunological reagents will be developed to allow tracking of the N2 protein during infection and to identify potential cellular targets;A (DP-ribosylated)VP1-8 & M(yristylated)VP1-2 - the chemical nature of the prosthetic group will be analyzed by protein chemistry and HPLC techniques, the VV substrate proteins will be identified by cell-free translation and immunoassay methods, peptide mapping will b used to identify sites of modification, and directed genetics used to assess whether modifications are required for the proteins to become functional. The rationale for studying VV are two-fold. First, as a model eukaryotic system the information obtained should be of general relevance with regard to the regulation of gene expression. Furthermore, the ability to easily carry out directed genetics and gene replacement (marker transfer) makes this viral system quite amenable to these type of studies. Second, the results of this work should facilitate the continued development of VV as an eukaryotic cloning and expression vector for use as a vaccine or the production of biopharmaceutical products.
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