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

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 antibody 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 predicted transmembrane domain. The protein was found to be associated with the sucrose gradient purified myxoma intracellular mature virus (IMV) as determined by Western blotting with M118L antibody. Furthermore, the M118L protein associated with the IMV can be surface labeled with water-soluble biotin and is released from the purified IMV with treatment of nonionic detergent NP-40, indicating that the M118L protein is associated with the outer membrane of myxoma IMV. Unexpectedly, an IMV-associated M118L protein isoform was observed to bind tightly to Streptavidin beads, unlike the six other detectable myxoma IMV surface proteins, suggesting an unusual post-translational modification, such as biotinylation. Extensive attempts to generate the M118L 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 M118L deletion mutants in mixed recombinant virus plaques selected with mycophenolic acid (MPA), repeated passages and plaquing failed to segregate the pure M118L deletion mutant from either single crossover recombinants or regenerated wild type parental viruses. Taken together, our data strongly indicate that the M118L is a novel poxvirus IMV associated protein that is essential for virus viability.