The human cytomegalovirus gene products essential for late viral gene expression assemble into pre- replication complexes before viral DNA replication

ABSTRACT

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood and these viral proteins could be a target for novel antivirals. HCMV open reading frames (ORFs) UL79, 87, and 95 have homology with late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, 87, or 95. Cells were infected with the recombinant viruses at high and low multiplicity of infection (MOI). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild type viral proteins were expressed in trans. At high MOI, mutation of ORFs UL79, 87, or 95 had no effect on the level of MIE gene expression or viral DNA replication, but late viral gene expression from the UL44, 75 and 99 ORFs was not detected. At low MOI, pre-expression of UL79 or 87 in human fibroblast cells, but not UL95, affected negatively the level of major immediate early (MIE) viral gene expression and viral DNA replication. UL79, 87 and 95 ORFs were expressed as early viral proteins and recruited to pre-replication complexes (pre-RCs) before the initiation of viral DNA replication along with UL44. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The role of UL79, 87, and 95 in pre-RCs for late viral gene expression is discussed.