Biomedical subjects

R M Stenberg

Publications and source records attributed to R M Stenberg.

33 records · Page 2Linked to original sources

PubMed1993

Direct interaction of the human cytomegalovirus IE86 protein with the cis repression signal does not preclude TBP from binding to the TATA box.

The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein, represses the MIEP through a DNA sequence located between the TATA box and the transcription initiation site, designated the cis repression signal (crs). Through mutational analysis, amino acid domains within IE86 responsible for binding the crs element were located at the C terminus. Mutation of the putative zinc finger domain, which precluded IE86 from binding DNA, converted the protein from a repressor of MIEP transcription into an activator. DNase I protection analysis demonstrated that the IE86 footprint overlapped the sequence protected by the TATA-binding protein (TBP). Investigation of whether IE86 was able to displace TBP from DNA revealed that both proteins could bind DNA simultaneously. However, higher concentrations of IE86 were required to obtain protection of the crs element in the presence of prebound TBP. Similarly, higher concentrations of TBP were required to obtain protection in the presence of prebound IE86. These observations indicate that steric hinderance impairs but does not prevent both proteins from binding DNA synchronously.

Base Sequence↗

PubMed1992

An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator.

The major immediate-early promoter (MIEP) of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs. These mRNAs encode nuclear phosphorproteins (IE55, IE72, and IE86), which consist of common and unique amino acid sequences. To date, very little is known of the functional role of the 55-kDa (IE55) protein. Here we present evidence that the IE55 protein is a positive activator of the MIEP. In human fibroblast cells IE55 protein activated the MIEP between 10- and 30-fold. Fusion of IE55 to the GAL4 DNA binding domain resulted in a chimeric protein capable of trans-activating a reporter with GAL4 recognition sequences. These results strongly suggest that IE55 is a bona fide transcriptional activator protein. In addition, the IE55 protein was found not to act synergistically with the IE72 activator protein. The IE55 protein shares the same amino acid sequence as IE86 except for a 154-amino-acid deletion at the C-terminal end of the protein. These proteins were functionally antagonistic; IE55 relieved repression by IE86 and, conversely, IE86 negated IE55 activation. Mutagenesis of the MIEP revealed that the target sequence for activation by IE55 is different from the IE86 autorepressive response element. These experiments suggest that the mechanism of action of the IE55 and IE86 isoforms is distinct. Moreover, from these results it is apparent that the interplay of these factors might be critical in determining the level of HCMV replication in the host.

Base Sequence↗

PubMed1992

An early event in murine cytomegalovirus replication inhibits presentation of cellular antigens to cytotoxic T lymphocytes.

Cytomegalovirus (CMV) infection of simian virus 40 (SV40)-immune mice inhibits priming of SV40-specific helper and cytotoxic T lymphocytes (CTL) in vivo (A. E. Campbell, J. S. Slater, and W. S. Futch, Virology 173:268-275, 1989; J. S. Slater, W. S. Futch, V. J. Cavanaugh and A. E. Campbell, Virology 185:132-139, 1991). We now demonstrate that murine CMV (MCMV) infection of SV40-transformed macrophages and fibroblasts prevents presentation of SV40 T antigen to SV40-specific CTL. MCMV-infected macrophages failed to stimulate SV40-immune CTL precursors in vitro. In addition, MCMV-infected, SV40-transformed macrophage and fibroblast target cells lost their susceptibility to lysis by major histocompatibility complex class I-restricted, SV40-specific CTL clones. MCMV infection did not alter the synthesis of SV40 T antigen in the target cells. MCMV early gene expression was required for inhibition of SV40 T-antigen presentation; immediate-early gene expression was insufficient for this effect. Early viral gene expression also resulted in significant reduction of H-2K and H-2D molecules on the surface of MCMV-infected fibroblasts. However, this reduction occurred independently from suppression of antigen presentation to CTL. The same target cells which were resistant to lysis by SV40 CTL were susceptible to lysis by MCMV-specific CTL. MCMV early gene products therefore interfere with the processing and/or presentation of SV40 T-antigen determinants to CTL independent of alterations in the major histocompatibility complex.

Animals↗

PubMed1992

Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis-acting elements and viral trans-acting proteins necessary for promoter activation.

As a model for analyzing the regulation of human cytomegalovirus late genes, we investigated the 28-kDa phosphoprotein (pp28) gene region. Transcripts of 1.6 and 1.3 kb were expressed in wild-type human cytomegalovirus-infected cells but not in cells infected with a DNA-negative temperature-sensitive mutant (ts66), indicating that DNA replication is absolutely required for pp28 gene expression. Transient promoter activation studies revealed that the pp28 gene region upstream promoter (pp28US) functioned early when expressed independently of the viral genome. However, the promoter was not efficiently activated by immediate-early (IE) proteins but was activated equally well by both wild-type virus and ts66. Deletion analysis of the pp28US promoter indicated that sequences upstream of the CAP site between -107 and -32 were required for activation of the pp28 promoter. Within that region exist a 10-bp sequence at -90 (AGTGAT CGTG) and its inverted repeat at -32 which positively influence pp28 promoter function. Therefore, in the case of the pp28US promoter, viral proteins interact through discrete sequences to facilitate late gene expression.

Base Sequence↗

PubMed1991

A discrete cis element in the human immunodeficiency virus long terminal repeat mediates synergistic trans activation by cytomegalovirus immediate-early proteins.

The major immediate-early (IE) promoter of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs that encode isoformic proteins with apparent molecular masses of 55, 72, and 86 kDa. All of these proteins are potent transcriptional regulatory proteins. We are interested in the collateral interactions between human cytomegalovirus and human immunodeficiency virus (HIV) in the context of dual infection of a cell. The roles of the specific IE protein isoforms and their respective response elements involved in trans activation of the HIV long terminal repeat (LTR) are not known. Here we present evidence that major IE proteins IE86, IE72, and IE55 are capable of trans-activating the HIV LTR in a T-cell line, HUT-78. The IE55 isoform noncooperatively stimulates the HIV LTR in the presence of either isoform IE72 or IE86. Interactions between isoforms IE72 and IE86, however, result in strong synergistic activation of the LTR. Our results suggest that a specific 155-amino-acid protein domain that is unique for the IE86 protein participates in this synergic interaction. Point mutational analysis of the LTR identified a distinct cis-acting target site, located between nucleotide positions -174 and -163, that mediates exclusively synergistic trans activation by the IE72 and IE86 proteins. Finally, this study underscores the role of a cellular intermediate(s) for communicating the synergic interactions between two IE trans activators.

Amino Acid Sequence↗

PubMed1990

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.

Base Sequence↗

PubMed1989

Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products.

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.

Base Sequence↗

PubMed1989

Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region.

Expression of RNA and protein from the human cytomegalovirus immediate-early (IE) gene region (map units 0.732 to 0.751) was analyzed at early and late times after infection. The level of RNA present at late times (48 to 72 h after infection) was significantly higher than that present at IE times (5 h after infection). The profile of IE RNA in the cytoplasm of infected cells was different from that previously reported on polysomes (R. M. Stenberg, P. R. Witte, and M. F. Stinski, J. Virol. 56:665-675, 1985). The data indicate that the 1.95-kilobase (kb) major IE region 1 mRNA, which codes for the 72-kilodalton (kDa) protein, and the 1.7-kb IE region 2 (IE2) spliced mRNA, which codes for the IE2 55-kDa protein, may be preferentially associated with polysomes. However, the IE2 2.2-kb unspliced mRNA, which codes for an 86-kDa protein, may be preferentially excluded. This RNA was abundant in the cytoplasm under IE conditions but was not present on polysomes in significant quantities. This indicates that IE gene products may be involved in translational control of cytomegalovirus RNA. At late times, new transcription takes place within region 2. A 1.5-kb RNA is transcribed from a late promoter in region 2 that apparently does not function in cells infected with DNA-negative mutant ts66. These results demonstrate that the IE gene region is transcribed throughout infection and that multiple levels of regulation exist.

Antigens, Viral↗

PubMed1985

Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1.

Human cytomegalovirus immediate-early (IE) region 2 (0.732 to 0.740 map unit) begins 35 nucleotides downstream of IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). A series of mRNAs that have different splicing patterns are transcribed from region 2. There is an unspliced 1,589-nucleotide exon present in minor amounts and two spliced exons (836 and 289 nucleotides) present in larger amounts. The IE region 2 exons were found to be spliced onto the first three exons of region 1. Therefore, under IE conditions the region 1 promoter-regulatory region can promote transcription of region 2. Promoter sequences (i.e., CAAT and TATA boxes) are found upstream of the 5' end of IE region 2 but presumably function poorly at IE times after infection. The transcriptional regulation of these IE genes and the possible functional roles of the proteins are discussed. We postulate that a series of unique but related proteins are made from the region 2 transcripts. Some of these proteins should contain the same 169 amino-terminal residues as the major IE 72-kilodalton protein encoded by IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). Variations in the amino acid sequences of the region 2 proteins could occur at either the amino terminus, the carboxy terminus, or both termini.

Base Sequence↗

PubMed1985

Autoregulation of the human cytomegalovirus major immediate-early gene.

The gene coding for the human cytomegalovirus major immediate-early 72-kilodalton protein was cloned into simian virus origin of DNA replication plasmid pSVOd. Transfection of this plasmid (pSVCC2) into cells constitutively expressing the simian virus 40 T-antigen resulted in readily detectable levels of immediate-early region 1-specific RNA and protein. Partial restriction enzyme digestion of pSVCC2 was used to generate specific amino acid deletions within the 72-kilodalton protein. Mutant delta S12, which contained a deletion of 145 amino acids at the carboxy terminus of the protein, accumulated at least 10 times more immediate-early region 1 RNA than wild-type pSVCC2 did. In contrast, normal levels of delta S12-specific RNA were detected in cells cotransfected with wild-type pSVCC2. Therefore, the wild-type gene was capable of suppressing transcription of the mutant gene. Our results suggest that the wild-type major immediate-early protein of cytomegalovirus autoregulates transcription of immediate-early region 1 and that one of the regulatory domains is within the carboxy-terminal 145 amino acids of the viral protein.

Animals↗

PubMed1984

Promoter-regulatory region of the major immediate early gene of human cytomegalovirus.

The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.

Base Sequence↗

PubMed1984

Structural analysis of the major immediate early gene of human cytomegalovirus.

The most abundant species of human cytomegalovirus (Towne) immediate early polysome-associated RNA originates from a region of ca. 2.8 kilobases (0.739 to 0.755 map units) within the XbaI-E DNA fragment. These sequences code for a 1.95-kilobase mRNA and are referred to as immediate early coding region one (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). We have utilized the nuclease mapping technique of Berk and Sharp (A. J. Berk and P. A. Sharp, Cell 12:721-732, 1977) to examine this gene in detail. Cloned fragments of human cytomegalovirus DNA, either labeled with 32P in vivo or end labeled in vitro at the 5' or 3' termini, were hybridized to immediate early polysome-associated RNA. The hybrids were treated with single-strand-specific nuclease and subjected to electrophoresis in either neutral or denaturing gels. The major transcript was shown to be a spliced molecule containing a 3' terminal exon of 1,341 nucleotides. Upstream of the major body of the mRNA are three small exon sequences of 185, 88, and 121 nucleotides. The sequence of the exons as well as the locations of the intron-exon splice junctions were determined. Based on the DNA sequence, the viral mRNA molecule has one open reading frame which begins within the second exon and extends for 491 amino acid residues. The predicted molecular weight of the polypeptide originating from this region was estimated to be 64,000. It is hypothesized that this viral gene codes for the major regulatory protein controlling transcription of the viral genome at early times. The properties of the viral gene and its protein product are discussed.

Amino Acid Sequence↗

PubMed1983

Organization and expression of the immediate early genes of human cytomegalovirus.

The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 3' ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription.

Cytomegalovirus↗

PubMed1983

Characterization of a phosphoprotein associated with the SS-B/La nuclear antigen in adenovirus-infected and uninfected KB cells.

We have employed sera from patients with autoimmune disease to characterize the nuclear SS-B/La antigen in uninfected and adenovirus-infected KB cells. A 45,000-dalton phosphorylated polypeptide was specifically precipitated with anti-SS-B sera, and the level of phosphorylation was increased after infection. The increased phosphorylation appears to occur at the same amino acid residues phosphorylated in uninfected cells and results from increased phosphorylating activity rather than from altered enzyme specificity. A competition experiment between infected and uninfected cell extracts indicates that the antigen in the infected cell binds more strongly to SS-B/La antibodies. Fragments of adenovirus-induced virus-associated RNA as well as intact molecules complex with SS-B/La antigen and are immune precipitated with autoimmune sera.

Adenoviridae↗

PubMed1982

Herpes simplex virus-induced changes in cellular and adenovirus RNA metabolism in an adenovirus type 5-transformed human cell line.

We used the viral transcripts (designated Ad-RNA) that accumulated in the cytoplasm of adenovirus type 5-transformed human embryonic kidney cells (cell line 291-31) as models for cellular RNAs to examine how herpes simplex virus modifies cellular RNA metabolism. Infection of 293-31 cells with herpes simplex virus type 1 strain 17 lead to extensive inhibition of Ad-RNA accumulation by 4 h postinfection. The major part of this inhibition was due to an immediate early or alpha gene function, which reduced the rate of transcription of Ad-RNA within the nuclei of the infected cells. In addition, host polyadenylic acid-containing RNA accumulation and rRNA accumulation were affected, but to a lesser extent and at lower rate than Ad-RNA accumulation. In conjunction with previous data, our experimental data allowed us to propose a general scheme for how herpes simplex virus type 1 alters the metabolism of cellular RNA, the possible mechanisms for these changes, and how they correlate with the regulation of herpes simplex virus gene expression.

Adenoviruses, Human↗