RESUMO
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.
Assuntos
Citomegalovirus/genética , Citomegalovirus/metabolismo , Regulação Viral da Expressão Gênica , Genes Virais/genética , Proteínas Virais/metabolismo , Sequência de Bases , Humanos , Dados de Sequência Molecular , RNA Viral/análise , RNA Viral/genética , Transcrição Gênica/genética , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Citomegalovirus/genética , Endopeptidases , Proteínas Oncogênicas , Proteínas Repressoras/análise , Proteínas Virais/análise , Células Cultivadas , DNA/metabolismo , Glicoproteínas , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas de Membrana , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Proto-Oncogênicas , Ubiquitina Tiolesterase , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Genes Virais , Família Multigênica , Roseolovirus/genética , Sequência de Aminoácidos , Animais , Fibroblastos , Cobaias , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sintenia , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
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.
Assuntos
Citomegalovirus/fisiologia , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Proteínas Virais/genética , Replicação Viral/genética , Células Cultivadas , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Glicoproteínas , Humanos , Proteínas de MembranaRESUMO
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.
Assuntos
Citomegalovirus/genética , Genes Precoces , Proteínas Imediatamente Precoces/genética , Proteínas Repressoras/fisiologia , Transcrição Gênica , Glicoproteínas , HIV-1/genética , Humanos , Proteínas de Membrana , Regiões Promotoras Genéticas , TATA BoxRESUMO
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.
Assuntos
Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Glicoproteínas de Membrana , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas do Envelope Viral , Proteínas Virais , Citomegalovirus/metabolismo , Genes Precoces , Glicoproteínas , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas de Membrana , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , TATA Box , Transativadores/metabolismoRESUMO
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.
Assuntos
Citomegalovirus/metabolismo , Regulação Viral da Expressão Gênica , Genes Precoces , Proteínas Imediatamente Precoces/biossíntese , Transcrição Gênica , Sequência de Bases , Células Cultivadas , Citomegalovirus/genética , Primers do DNA , Fibroblastos , Genes Virais , Glicoproteínas , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas de Membrana , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Ativação TranscricionalRESUMO
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.
Assuntos
Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV , HIV-1/genética , Proteínas Imediatamente Precoces , Antígenos de Superfície/genética , Antígenos Virais/genética , Células Cultivadas , Citomegalovirus/imunologia , Infecções por Citomegalovirus/genética , Genes Virais , Humanos , Plasmídeos , Regiões Promotoras Genéticas , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
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.
Assuntos
Citomegalovirus/genética , Biossíntese de Proteínas , Transcrição Gênica , Sequência de Bases , Códon/genética , Fibroblastos , Regulação Viral da Expressão Gênica , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Sinais Direcionadores de Proteínas/genética , RNA Viral/genética , Mapeamento por Restrição , Pele , TransfecçãoRESUMO
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.
Assuntos
Vírus da Leucose Aviária/genética , Transformação Celular Neoplásica , Oncogenes , Proteínas Tirosina Quinases/genética , Proteínas Oncogênicas de Retroviridae/genética , Animais , Leucose Aviária/microbiologia , Leucose Aviária/patologia , Sequência de Bases , Divisão Celular , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Galinhas , Genes Virais , Dados de Sequência Molecular , Proteína Oncogênica p55(v-myc) , Mapeamento por Restrição , Vírion/genéticaRESUMO
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.