MYXOMA VIRUS AND SHOPE FIBROMA VIRUS ENCODE DUAL-SPECIFICITY TYROSINESERINE PHOSPHATASES WHICH ARE ESSENTIAL FOR VIRUS VIABILITY

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SN) led to the discovery of open reading fram es homologous to the vaccinia H1L gene encoding a soluble protein phos phatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were desi gnated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV MIL open reading frames within the well conserv ed central core of the viral genomes closely mirror that of the Orthop oxviruses vaccinia virus and variola virus. The myxoma I1L and SN MIL phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cystei ne, found previously to be essential for phosphotyrosine dephosphoryla tion, The vaccinia H1L phosphatase was originally shown to have the ab ility to dephosphorylate phosphotyrosyl and phosphoseryl residues in v itro. To assess whether this is a common feature of poxvirus phosphata ses, myxoma I1L was expressed as a GST-fusion protein, purified, and s hown to dephosphorylate substrates containing tyrosine and serine phos phorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1 L variant, in which the active site cysteine 110 was mutated to serine , was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate bo th phosphotyrosine and phosphoserine amino acids. In an attempt to asc ertain the biological requirement for the myxoma I1L phosphatase, we c onstructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagat e in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity pho sphatase is an essential factor for virus viability. (C) 1995 Academic Press, Inc.