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Identification and analysis of three myristylated vaccinia virus late proteins
K H Martin1, D W Grosenbach, C A Franke
1Department of Microbiology, Center for Gene Research and Biotechnology, Oregon State University, Corvallis 97331-3804, USA.
Abstract:
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.
Insights
This study identifies new vaccinia virus (VV) myristylated proteins, G9R, A16L, and E7R. These proteins are myristylated on a glycine residue, with A16L and E7R being soluble and L1R membrane-associated.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Vaccinia virus (VV) infection involves numerous viral proteins, some of which undergo post-translational modification.
- Myristoylation, the covalent attachment of myristic acid, is a critical modification for the function of certain viral proteins.
- Previous research identified three VV late proteins labeled with myristic acid, but the identities of other potential myristylated proteins remained unknown.
Purpose of the Study:
- To identify novel vaccinia virus (VV) proteins that are myristylated.
- To confirm the identities of putative myristylated proteins using molecular and biochemical techniques.
- To investigate the subcellular localization of identified myristylated proteins.
Main Methods:
- Time course labeling of VV-infected cells with [3H]myristic acid to identify potential myristylproteins.
- Sequence analysis to preliminarily identify open reading frames (ORFs) encoding putative myristylated proteins.
- Polymerase chain reaction (PCR) amplification and cloning of ORFs into expression vectors.
- In vitro transcription and translation of cloned ORFs using radiolabeled [35S]methionine and [3H]myristic acid.
- Site-directed mutagenesis to create N-terminal glycine-to-alanine mutants.
- Subcellular fractionation of VV-infected cells.
Main Results:
- Six putative myristylproteins were detected in VV-infected cells, with molecular masses of 92, 37, 35, 25, 17, and 14 kDa.
- Sequence analysis and experimental validation identified G9R (37 kDa), A16L (35 kDa), and E7R (17 kDa) as myristylated proteins.
- Mutational analysis confirmed myristoylation occurs on a glycine residue adjacent to the initiating methionine.
- Subcellular fractionation revealed that A16L and E7R are soluble proteins, while L1R (25 kDa) is membrane-associated.
Conclusions:
- The study successfully identified and characterized three novel vaccinia virus (VV) myristylated proteins: G9R, A16L, and E7R.
- Myristoylation of these proteins occurs at a conserved N-terminal glycine residue.
- The distinct subcellular localization of these myristylated proteins (soluble vs. membrane-associated) suggests diverse functional roles within the VV replication cycle.