Characterization of the myxoma virus M118L protein: A novel essential poxvirus IMV-associated protein

Citation
Jx. Cao et G. Mcfadden, Characterization of the myxoma virus M118L protein: A novel essential poxvirus IMV-associated protein, VIRUS GENES, 23(3), 2001, pp. 303-313
Citations number
27
Categorie Soggetti
Molecular Biology & Genetics
Journal title
VIRUS GENES
ISSN journal
09208569 → ACNP
Volume
23
Issue
3
Year of publication
2001
Pages
303 - 313
Database
ISI
SICI code
0920-8569(2001)23:3<303:COTMVM>2.0.ZU;2-E
Abstract
Myxoma M118L ORF has the capacity to encode a 76 amino acid protein that is highly conserved in other vertebrate poxviruses including vaccinia (A30L), molluscum contagiosum (MC136L), yaba tumour virus (D13L) and fowlpox virus (FPV 194). The time course analysis by Western blotting using M118L antibo dy showed that the M118L ORF is expressed as a typical poxvirus late gene. The M118L protein can be detected in both the virus infected cytosolic and membrane fractions, even though the M118L protein does not possess a predic ted transmembrane domain. The protein was found to be associated with the s ucrose gradient purified myxoma intracellular mature virus (IMV) as determi ned by Western blotting with M118L antibody. Furthermore, the M118L protein associated with the IMV can be surface labeled with water-soluble biotin a nd is released from the purified IMV with treatment of nonionic detergent N P-40, indicating that the M118L protein is associated with the outer membra ne of myxoma IMV. Unexpectedly, an IMV-associated M118L protein isoform was observed to bind tightly to Streptavidin beads, unlike the six other detec table myxoma IMV surface proteins, suggesting an unusual post-translational modification, such as biotinylation. Extensive attempts to generate the M1 18L deletion mutant using standard homologous recombination technique with E. coli gpt gene as a positive selection marker were unsuccessful. Although PCR analysis clearly indicated the presence of the correctly targeted M118 L deletion mutants in mixed recombinant virus plaques selected with mycophe nolic acid (MPA), repeated passages and plaquing failed to segregate the pu re M118L deletion mutant from either single crossover recombinants or regen erated wild type parental viruses. Taken together, our data strongly indica te that the M118L is a novel poxvirus IMV associated protein that is essent ial for virus viability.