The human cytomegalovirus major immediate-early enhancer determines the efficiency of immediate-early gene transcription and viral replication in permissive cells at low multiplicity of infection

To determine the effect of the human cytomegalovirus (CMV) major immediate-early (MIE) enhancer or promoter on the efficiency of viral replication in permissive human cells, we constructed recombinant viruses with their human MIE promoter, enhancer, and promoter plus enhancer replaced with the murine CMV components. After a low multiplicity of infection (MOI) (0.01 PFU/cell), recombinant human CMV with the murine CMV promoter replicated like the wild type but recombinant virus with the murine enhancer replicated less efficiently. Immediate-early (IE) viral protein pIE72 (UL123), early viral protein (UL44), and viral DNA synthesis were significantly decreased. The effect of the human CMV enhancer substitution with the murine CMV enhancer was also demonstrated in different cell types by using recombinant virus with the UL127 promoter, driving the expression of green fluorescent protein (GFP). After an MOI of 1, GFP expression was high with the human CMV enhancer and significantly lower with the murine CMV enhancer. Even though at a high MOI (10 PFU/cell), the murine CMV enhancer was as efficient as the human CMV enhancer for the transcription of IE genes in human foreskin fibroblast cells, at lower MOIs, the murine CMV enhancer was less efficient. Proximal and distal chimeras of the human and murine enhancers also replicated less efficiently at a low MOI and expressed lower levels of GFP from the UL127 promoter. These experiments demonstrate that the entire human CMV enhancer has evolved for the efficient expression of the viral IE and early genes in human cells. Possible functions of the human CMV enhancer and promoter at a low MOI are discussed.