The vaccinia virus A33R protein provides a chaperone function for viral membrane localization and tyrosine phosphorylation of the A36R protein

The products of the A33R and A36R genes of vaccinia virus are incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with (H3PO4 )-P-32 were immunoprecipitated with antibodies against the A33R protein, tw o prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins, respectivel y. The immunoprecipitated complex contained disulfide-bonded dimers of A33R protein that were noncovalently linked to A36R protein. Biochemical analys is indicated that the two proteins were phosphorylated predominantly on ser ine residues, with lesser amounts on threonines. The A36R protein was also phosphorylated on tyrosine, as determined by specific binding to an anti-ph osphotyrosine antibody. Serine phosphorylation and A33R-A36R protein comple x formation occurred even when virus assembly was blocked at an early stage with the drug rifampin. Tyrosine phosphorylation was selectively reduced i n cells infected with F13L or A34R gene deletion mutants that were impaired in the membrane-wrapping step of IEV formation. In addition, tyrosine phos phorylation was specifically inhibited in cells infected with an A33R delet ion mutant that still formed IEV. Immunofluorescence and immunoelectron mic roscopy indicated that in the absence of the A33R protein, the A36R protein was localized in Golgi membranes but not in IEV. In the absence of the A36 R protein, however, the A33R protein still localized to IEV membranes. Thes e studies together with others suggest that the A33R protein guides the A36 R protein to the IEV membrane, where it subsequently becomes tyrosine phosp horylated as a signal for actin tail formation.