Transcriptional regulation of the human cytomegalovirus US11 early gene

The human cytomegalovirus (HCMV) US11 early gene encodes a protein involved in the down-regulation of major histocompatibility complex class I cell su rface expression in HCMV-infected cells. Consequently, this gene is thought to play an important role in HCMV evasion of immune recognition. In this s tudy, we examined the transcriptional regulation of US11 gene expression. A nalysis of deletions within the US11 promoter suggests that two sequence el ements are important for activation by the viral immediate-early (IE) prote ins. Deletion of a CREB site located at -83 relative to the cap site result ed in a reduction in promoter activity to 50% of the wild-type level. Delet ion of an additional ATF site immediately upstream of the TATA box resulted in abrogation of responsiveness to the IE proteins. To confirm the role of the CREB and ATF sites within the US11 promoter, mutagenesis of these two sites, both individually and in combination, was carried out. Results indic ate that both the CREB element and the ATF site were required for full prom oter activity, with the ATF site critical for US11 promoter activation. Tbe loss of transcriptional activation correlated with a loss of cellular prot eins binding to the mutated US11 promoter elements. In combination with the viral IE proteins, the HCMV tegument protein pp71 (UL82) was found to up-r egulate the US11 promoter by six- to sevenfold in transient assays. These r esults suggest that pp71 may contribute to the activation of the US11 promo ter at early times after infection, Up-regulation by pp71 required the pres ence of the CREB and ATF sites within the US11 promoter for full activation . The role of the ATF and CREB elements in regulating US11 gene expression during viral infection was then assessed, The US11 gene is not required for replication of HCMV in tissue culture. This property was exploited to gene rate US11 promoter mutants regulating expression of the endogenous US11 gen e in the natural genomic context. We generated recombinant HCMV that contai ned the US11 promoter with mutations in either the CREB or ATF element or b oth regulating the expression of the endogenous US11 gene. Northern blot an alysis of infected cell mRNA revealed that mutation of the CREB element red uced US11 mRNA expression to approximately 25% of that of the wild-type pro moter, with identical kinetics of expression. Mutation of the ATF site alon e reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mR NA detectable only at 8 h after infection. Mutation of both the CREB and AT F elements in the US11 promoter reduced US11 gene expression to undetectabl e levels. These results demonstrate that the CREB and ATF sites cooperate t o regulate the US11 promoter in HCMV-infected cells.