No abstract

Cell proteins can restrict the replication of viruses. Here, we identify the cellular BclAF1 protein as a human cytomegalovirus restriction factor and describe two independent mechanisms the virus uses to decrease its steady-state levels. Immediately following infection, the viral pp71 and UL35 proteins, which are delivered to cells within virions, direct the proteasomal degradation of BclAF1. Although BclAF1 reaccumulates through the middle stages of infection, it is subsequently down-regulated at late times by miR-UL112-1, a virus-encoded microRNA. In the absence of BclAF1 neutralization, viral gene expression and replication are inhibited. These data identify two temporally and mechanistically distinct functions used by human cytomegalovirus to down-regulate a cellular antiviral protein.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from-128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IRL. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IRI plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.

IFI 16 is a member of a family of interferon-inducible proteins, including the human MNDA (myeloid nuclear differentiation antigen), the recently identified AIM-2 (absent in melanoma), and the homologous murine molecules, p202, p204, and D3. IFI 16 contains a domain at the amino terminus capable of binding double-stranded DNA and a bipartite nuclear localization signal. No molecular or biological function has been assigned to any of the human family members, although a role in transcription regulation has been proposed. In the present study, we show IFI 16 fused to the GAL4 DNA binding domain can function as a transcriptional repressor. IFI 16-mediated repression is not dependent on the position or distance of IFI 16 binding, relative to the site of transcription initiation, and it can significantly repress when only one GAL4 DNA element is present in the promoter. We mapped the transcriptional repression domains to the 200 amino acid repeat regions common to all human and mouse family members. We also demonstrate that wild type IFI 16 can repress transcription of a reporter gene containing the minimal promoter region of the human cytomegalovirus UL54 gene. Thus, IFI 16 is a transcriptional repressor, with a modular structure typical of many known transcription regulators.

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (0-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from-609 to-41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from-40 to +106 are suflicient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.