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
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.