The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells

Human cytomegalovirus (HCMV) infection of permissive cells has been reporte d to induce a cell cycle halt. One or more viral proteins may be involved i n halting progression at different stages of the cell cycle. We investigate d how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant v irus that expresses the green fluorescent protein (GFP) to determine the ef fects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP al lowed us to select for only productively infected cells. Replication-defect ive adenovirus vectors expressing the IE72 or IE86 protein were also used t o efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative auto regulation of the major Immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and mild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection Induced the e ntry of U373 cells into the S phase. All permissive cells infected with HCM V were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of al l permissive or nonpermissive cell types in the S phase increased significa ntly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cel l line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycl e progression are discussed.