Functional analysis of human cytomegalovirus pUS28 mutants in infected cells

The human cytomegalovirus (HCMV)-encoded viral G protein-coupled receptor pUS28 contributes to an array of biological effects, including cell migration and proliferation. Using FIX-BAC (bacterial artificial chromosome, derived from the HCMV clinical isolate VR1814) and lambda red recombination techniques, we generated HCMV recombinants expressing amino-terminally FLAG-tagged versions of wild-type pUS28 (FLAG-US28/WT), G-protein coupling deficient pUS28 (FLAG-US28/R129A) and chemokine-binding domain deficient pUS28 (FLAG-US28/DeltaN). Infection with the FLAG-US28/R129A virus failed to induce inositol phosphate accumulation, indicating that G-protein coupling is essential for pUS28 signalling to phospholipase C-beta (PLC-beta) during HCMV infection. The FLAG-US28/DeltaN virus induced about 80 % of the level of PLC-beta signalling induced by the FLAG-US28/WT virus, demonstrating that the N-terminal chemokine-binding domain is not required for pUS28-induced PLC-beta signalling in infected cells. The data presented here are the first to describe the functional analyses of several key pUS28 mutants in HCMV-infected cells. Elucidating the mechanisms by which pUS28 signals during infection will provide important insights into HCMV pathogenesis.