Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication

Abstract

ABSTRACT Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G 0 /G 1 to G 1 /S, and arresting cell cycle progression at the G 1 /S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.