Identification and analysis of three myristylated vaccinia virus late proteins.

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.     

Molecular-biological study of vaccinia virus genome. III. Identification of the late gene product protein 36K from Hind-III-P-fragment of vaccinia virus strain L-IVP

Vaccinia virus gene encoding 36K protein was cloned in pUR290 bacterial expressing vector and resulted in the synthesis of a chimeric protein in E. coli. The chimeric protein consists of beta-galactosidase and virus protein in C-termini. It has virus antigen specificity. By monospecific antibody 36K protein of vaccinia virus was determined to be non-virion. It is localized in the cytoplasm of infected cells.     

A tandemly-oriented late gene cluster within the vaccinia virus genome.

The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome.