THE DOMINANT PHOSPHOPROTEIN PP65 (UL83) OF HUMAN CYTOMEGALOVIRUS IS DISPENSABLE FOR GROWTH IN CELL-CULTURE

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses, The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies, Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed, However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the tate gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses, In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.