IDENTIFICATION AND ANALYSIS OF 3 MYRISTYLATED VACCINIA VIRUS LATE PROTEINS

Previous studies have shown that at least three vaccinia virus (VV) la te proteins (with apparent molecular asses of 37, 35, and 25 kDa) labe l with myristic acid, Time course labeling of VV-infected cells with [ H-3] myristic acid reveals at least three additional putative myristyl proteins, with apparent molecular masses of 92, 17, and 14 kDa, The 25 -kDa protein has preriously been identified as that encoded by the LIR open reading frame, Leaving the identities of the remaining proteins to be determined, Sequence analysis led to the preliminary identificat ion of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respec tively, Using synthetic oligonucleotides and PCR techniques, each of t hese open reading frames was amplified by using VV DNA as a template a nd then cloned individually into expression vectors behind T7 promoter s. These plasmid constructs were: then transcribed in vitro, and the r esulting mRNAs sere translated in wheat germ extracts and radiolabeled with either [S-35]methionine or [H-3]myristic acid. Each wild-type Do rypeptide was labeled with [S-35]methionine or [H-3]myristic acid in t he translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [S-35]methionine, not with myristic acid, This result provided strong evidence that: th e open reading frames had been correctly identified and that each prot ein is myristylated on a glycine residue adjacent to the initiating me thionine, Subcellular fractionations of VV-infected cells suggested th at A16L and E7R are soluble, in contrast to L1R, which is a membrane-a ssociated protein.