The role of NF-B in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-B response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEPenhancer that, although this region is essential for HCMV growth, none of the four NF-B response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.The major immediate-early promoter (MIEP) of human cytomegalovirus (HCMV) is responsive to a multitude of transcription factors and plays a pivotal role in initiating the viral transcription/replication cycle (7, 16; reviewed in references 22 and 23). Regulation of the MIEP has been postulated to be critical in determining HCMV permissiveness and the transition between latent and lytic infection. Thus, deciphering the molecular mechanisms of the MIEP regulation may reveal key control points contributing to HCMV pathogenesis.The MIEP enhancer includes four cognate NF-B recognition sites, and NF-B activates MIEP transcription in transient-transfection assays (20,(25)(26)(27). HCMV infection results in rapid induction of cellular 27,30), and several groups have reported a potential contribution of NF-B to the replication strategy of HCMV through regulation of the MIEP (8, 13). In contrast, we and others have reported a neutral or even a negative role of NF-B activation on HCMV transcription/replication cycle in different cell types (3,4,11,14,15). However, the basis for these experimental discrepancies is currently unclear. Importantly, a direct test of the requirement for the MIEP NF-B binding sites in HCMV transcription/ replication has still not been performed. Here we report on formally assessing the direct requirement of the cognate binding sites for NF-B in contributing to major immediate-early (MIE) transcription and viral growth.As a first step toward understanding NF-B regulation of the HCMV MIEP, we deleted enhancer sequences from Ϫ52 to Ϫ667 (including all NF-B response elements), in HCMV AD169. A parental HCMV bacterial artificial chromosome (BAC) (5, 6) containing the E-GFP open reading frame (ORF) under control of the murine cytomegalovirus (MCMV) MIEP (Fig. 1A, line 1) was used to construct two enhancerless HCMV recombinant mutants. In HCMVdE, MIEP sequences from Ϫ52 to Ϫ667 were removed (Fig. 1A, line 2), and in HCMVdE::Kan, enhancer sequences were replaced with a 1-kbp stuffer region to maintain the genomic spatial integrity of the ie1/ie2 and UL127 promoters (Fig. 1A, line 3). Once the integrity of constructed HCMV genomes was confirmed by restriction analysis (data not shown), they were transfected in MRC-5 fibroblasts. Three days posttransfection, ϳ100 single cells expressing green fluorescent prot...