Characterization of the human cytomegalovirus UL34 gene.

UL34 encodes the transcriptional repressor of the human cytomegalovirus immune evasion gene, US3, and is essential for viral replication in tissue culture. Two different monocistronic transcripts originate from UL34 at early and late times postinfection and encode two predominant proteins and a third, minor protein. The UL34 proteins are differentially expressed throughout the viral replication cycle, with both proteins localizing to the nucleus and repressing expression of the US3 gene.

Identification of a novel transcriptional repressor encoded by human cytomegalovirus.

The expression of human cytomegalovirus (HCMV) genes during viral replication is precisely regulated, with the interactions of both transcriptional activators and repressors determining the level of gene expression. One gene of HCMV, the US3 gene, is transcriptionally repressed early in infection. Repression of US3 expression requires viral infection and protein synthesis and is mediated through a DNA sequence, the transcriptional repressive element. In this report, we identify the protein that represses US3 transcription as the product of the HCMV UL34 open reading frame. The protein encoded by UL34 (pUL34) binds to the US3 transcriptional repressive element in yeast and in vitro. pUL34 localizes to the nucleus and alone is sufficient for repression of US3 expression. The data presented here, along with earlier data (B. J. Biegalke, J. Virol. 72:5457-5463, 1998), suggests that pUL34 binding of the transcriptional repressive element prevents transcription initiation complex formation.

Characterization of a cluster of late genes of guinea pig cytomegalovirus.

Human cytomegalovirus (HCMV) is the most common congenital infection, and is associated with a high rate of morbidity and mortality in the newborn infant. Guinea pig cytomegalovirus (GPCMV) is transmitted through the placenta with resulting fetal infection, and provides an excellent model for the study of fetal cytomegalovirus infection. We have characterized a cluster of late GPCMV genes, identifying GPCMV homologs of the HCMV G protein-coupled receptor gene, UL33; the transcriptional repressor gene, UL34 and two genes encoding tegument proteins, UL32 and UL35. We also identified the GPCMV homolog of UL37, an antiapoptotic gene. Surprisingly, no GPCMV homolog to HCMV UL36 was identified in the same genomic region. Furthermore, two of the predicted GPCMV proteins share greater identity with HHV-6 and/or HHV-7 homologs than with other cytomegalovirus homologs. The identification of GPCMV homologs of conserved viral genes, particularly genes involved in pathogenicity such as the G protein-coupled receptors, will facilitate future analysis of the role of these genes in infections.

Human cytomegalovirus US3 gene expression is regulated by a complex network of positive and negative regulators.

One immediate early gene of human cytomegalovirus, the US3 gene, causes retention of major histocompatibility locus class I heavy chain proteins in the endoplasmic reticulum and is postulated to have a role in viral pathogenicity. Expression of the US3 gene is regulated by a number of cis-acting elements. In addition, numerous viral proteins are involved in regulating US3 gene expression. US3 transcription was activated modestly by a virion protein, ppUL82. The immediate early proteins encoded by UL122-123 (IE1 and IE2) further activate US3 expression, with the activation enhanced by expression of pTRS1. Other proteins, the immediate early protein encoded by UL37ex1/UL38 and the early protein, pUL84, inhibited IE1 and IE2 activation of US3 expression. US3 transcription is regulated both positively and negatively by a complex network of viral proteins, the interaction of which contributes to precise regulation of US3 gene expression. The ability of pUL37ex1/UL38 to repress expression of the immediate early US3 gene while activating early gene expression suggests that pUL37ex1/UL38 may function to switch viral gene expression from immediate early to early genes.

Characterization of the transcriptional repressive element of the human cytomegalovirus immediate-early US3 gene.

Transcriptional repression is utilized by human cytomegalovirus to regulate expression of the immediate-early US3 gene. Sequences located 3' of the US3 TATA box are required for down regulation of expression. Mutagenesis of US3 sequences identified a 10-nucleotide region that is essential for transcriptional repression. In addition to the 10-nucleotide element, an additional region, which includes the US3 initiator element, was needed to confer repression on a heterologous promoter. Thus, a 19-nucleotide element (-18 to +1 relative to the transcription start site) functioned as a transcriptional repressive element (tre). The tre repressed transcription in a position-dependent but orientation-independent manner. In vivo footprinting experiments demonstrated that transcriptional repression is associated with a decrease in protein interactions with the US3 promoter and surrounding sequences. The data presented here suggest that the association of an as yet unidentified repressor protein with the tre represses transcription by inhibiting assembly of the transcription initiation complex on the US3 promoter.

IE2 protein is insufficient for transcriptional repression of the human cytomegalovirus US3 promoter.

Expression of the human cytomegalovirus (HCMV) US3 gene is regulated in part by transcriptional repression mediated through a cis-repressive region located between the TATA box and the transcriptional start site. The US3 cis-repressive element has extensive sequence identity with a similar repressive element in UL122-123 (the major immediate-early gene complex). Repression of UL122-123 is mediated through the interaction of the IE2 protein with cis-repressive sequences. In spite of the similarity of the two repressive elements, IE2 activated rather than repressed transcription from the US3 promoter. Additionally, IE1 or IE1 and IE2 in combination also activated US3 expression. These data demonstrate that regulation of HCMV immediate-early genes is quite complex and involves a number of proteins.

Regulation of human cytomegalovirus US3 gene transcription by a cis-repressive sequence.

Regulation of immediate-early gene expression in human cytomegalovirus is subject to complex controls. The major immediate-early (mIE) gene is regulated by both positive and negative regulatory signals, including autoregulation mediated by a cis-repressive sequence. A second immediate-early gene, the US3 gene, is transcribed with kinetic similar to those of the mIE gene. I have identified an element present in the US3 gene located from -1 to -13 (relative to the start site of transcription) that mediates a decrease in US3 transcription. The US3 element resembles the cis-repressive element of the mIE gene in sequence, position, and function. The common theme of negative regulation of immediate-early genes shortly after infection suggests that a decrease in the level of immediate-early proteins may be critical for viral replication.

Sequence requirements for activation of the HIV-1 LTR by human cytomegalovirus.

Human cytomegalovirus (CMV) alters the growth and expression of human immunodeficiency virus-1 (HIV-1) in cell culture and may accelerate the course of AIDS in HIV-1 infected patients. CMV infection or the expression of the CMV immediate early genes has been shown to activate gene expression directed by the HIV-1 LTR. However, the cis-acting elements within the HIV-1 LTR that confer responsiveness to CMV have not been clearly delineated. We report on investigations in human fibroblasts designed to precisely map this signal. Our studies demonstrate that more than one nonoverlapping region of the HIV-1 promoter is capable of responding to CMV. Sequences 3' from -19(relative to the start of transcription) are dispensable for CMV responsiveness. We also show that in addition to immediate early region 2, immediate early region 1 is able to activate HIV-1 LTR-directed gene expression.

Translational inhibition by cytomegalovirus transcript leaders.

The regulation of human cytomegalovirus gene expression depends on both transcriptional and post-transcriptional controls. Previous studies revealed that either of two AUG codons contained in the 5'leader of a beta gene (2.7 beta) transcript inhibited translation of a downstream reading frame. We investigated the regulatory effects of 5' leader sequences from the cytomegalovirus DNA polymerase and pp150 genes, each of which also contains upstream AUG codons. Surprisingly, these two leaders did not affect expression of the downstream open reading frame. Detailed analyses were carried out to examine the role of the AUG codons within the pp150 leader. These upstream AUG codons allowed efficient downstream translation, despite the predictions of the scanning model of eukaryotic translation. Further studies of the 2.7 beta leader revealed that an upstream AUG codon, although necessary, was not sufficient to inhibit downstream translation. These results reveal that translational inhibition by CMV transcript leaders requires an AUG codon and additional leader sequences.

FH3, a v-myc avian retrovirus with limited transforming ability.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.

MC29 deletion mutants which fail to transform chicken macrophages are competent for transformation of quail macrophages.

A number of MC29 mutants with deleted myc genes have been previously characterized. Many of these mutants have been found to be defective for transformation of chicken macrophages in vitro and for tumor induction in chickens. Such mutants are capable of transforming Japanese quail macrophages in vitro and inducing a high incidence of tumors in Japanese quail. Thus, Japanese quail may contain a factor(s) capable of complementing the defective transforming proteins encoded by some deleted v-myc genes.

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.

RNA and protein encoded by MH2 virus: evidence for subgenomic expression of v-myc.

MH2 and MC29 are highly related myc-containing avian retroviruses. We found that MH2, unlike MC29, synthesizes a 2.6-kilobase subgenomic mRNA containing myc sequences as well as sequences from the 5' end of the genome. A 57-kilodalton protein containing myc, but not gag, sequences (p57myc) was detected by hybrid selection and in vitro translation of RNA from MH2-transformed cells. Gradient separation of MH2 intracellular RNAs indicated that p57myc is encoded by the subgenomic RNA. A highly oncogenic MH2 virus variant (MH2YS3) (M. Linial, Virology 119:382-391, 1982) was shown to encode only p57myc and not P100, the previously described MH2-encoded polyprotein (Hu et al., Virology, 89:162-178, 1978). Cells transformed by subclones of this virus synthesized predominantly the 2.6-kilobase RNA rather than genomic 5.4-kilobase RNA. These results suggest that only p57myc is required for maintenance of the transformed state after MH2 infection.