A CIS REPRESSION SEQUENCE ADJACENT TO THE TRANSCRIPTION START SITE OFTHE HUMAN CYTOMEGALOVIRUS US3 GENE IS REQUIRED TO DOWN-REGULATE GENE-EXPRESSION AT EARLY AND LATE TIMES AFTER INFECTION

Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MI E) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was de termined by comparing the levels of transcription from these two enhan cer-containing promoters in recombinant viruses with a,wild-type or mu tant CRS. Upstream of the CRS of the US3 promoter was either the endog enous enhancer (R-2) or silencer (R-1). The downstream US3 gene was re placed with the indicator gene chloramphenicol acetyltransferase (CAT) . Infected permissive human fibroblast cells or nonpermissive, undiffe rentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the,wild-type or mutant CRS. With the,w ild-type CRS, the maximum level of transcription in permissive cells w as detected within 4 to 6 h after infection and then declined. With th e mutant CRS and the R-2 enhancer upstream, expression from the US3 pr omoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS nas mutated. Less expressi on aas obtained when only the R-1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-con taining US3 promoter appears to allow for a short burst of US3 gene ex pression followed by repression at early and late times after infectio n. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of th e CRS and the viral IE86 protein in controlling enhancer-containing pr omoters is discussed.