[Current developments in antiviral chemotherapy. 1: Principles and overview--amantadine].
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Amantadine↗
Biomedical subjects
T Stamminger
Publications and source records attributed to T Stamminger.
UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression.
The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression. Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far. While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses. This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV. Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection. However, these transcripts could not be detected in the presence of cycloheximide. Additional, larger transcripts were present exclusively at late times after infection. To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli. A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection. In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.
Amino Acid Sequence↗
Identification of binding sites for the 86-kilodalton IE2 protein of human cytomegalovirus within an IE2-responsive viral early promoter.
The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) can act as both an activator and a repressor of gene expression. The mechanisms for both of these functions are not well defined. It has recently been demonstrated that this protein has sequence-specific DNA binding properties: it interacts directly with a target sequence that is located between the TATA box and the cap site of its own promoter. This sequence, termed the CRS (cis repression signal) element, is required for negative autoregulation of the IE1/IE2 enhancer/promoter by IE2. We demonstrate now that binding of this protein to DNA is not confined to this site but occurs also within an early promoter of HCMV that has previously been shown to be strongly IE2 responsive. By DNase I protection analysis using a purified, procaryotically expressed IE2 protein, we could identify three binding sites within the region of -290 to -120 of the UL112 promoter of HCMV. Competition in DNase I protection experiments as well as gel retardation experiments showed that the identified binding sites are specific and have high affinity. Deletion of IE2 binding sites from this promoter reduced the level of transactivation; however, the remaining promoter could still be stimulated about 40-fold. Constructs in which IE2 binding sites were fused directly to the TATA box of the UL112 promoter did not reveal a significant contribution of these sequences to transactivation. However, if an IE2 binding site was reinserted upstream of nucleotide -117 of the UL112 promoter, an increase in transactivation by IE2 was obvious, whereas a mutated sequence could not mediate this effect. This finding suggests that DNA-bound IE2 can contribute to transactivation but seems to require the presence of additional transcription factors. Moreover, a comparison of the detected IE2 binding sites could not detect a strong homology, suggesting that this protein may be able to interact with a broad spectrum of different target sequences.
Base Sequence↗
Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.
We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.
Culture Media, Conditioned↗
Analysis of proteins encoded by IE regions 1 and 2 of human cytomegalovirus using monoclonal antibodies generated against recombinant antigens.
The genomic region of human cytomegalovirus (HCMV) encoding the major immediate-early (IE) proteins was cloned as overlapping fragments into a prokaryotic expression vector. Three recombinant polypeptides were used as antigens to generate monoclonal antibodies specifically reactive with the proteins encoded by IE region 1 and IE region 2. At least 10 different antigenic regions were identified on the IE proteins of HCMV. One monoclonal generated against an IE-2 polypeptide of 156 amino acids (termed SMX) was found to react with a viral pentapeptide, which was also a constituent of the beta-chain of human HLA-DR. This peptide was contained in the 40-kDa (p40) protein encoded by IE region 2. This protein appeared to be an abundant product of the major IE region in infected cells at late times after infection. The putative translation initiation site for p40 was mapped to position 3348 of the IE2 sequence using monoclonal antibody SMX. It is therefore proposed that p40 consists of amino acids 242-580 encoded by the IE2 gene of HCMV (strain AD169). Neither this late protein nor any other of the IE1/IE2-encoded proteins was detectable in extracellular virions.
Amino Acid Sequence↗
Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells.
Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it.
Amino Acid Sequence↗
The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter.
The 86-kDa IE-2 protein of human cytomegalovirus is able to autoregulate its own expression via a short nucleotide sequence, termed the cis repression signal (CRS), that is located between the TATA box and the cap site of the IE-1/2 enhancer-promoter. Here we report that the 86-kDa IE-2 protein can interact directly with the CRS, thus demonstrating that IE-2 is a DNA-binding protein. This could be shown by both DNase I protection and gel retardation experiments using a procaryotically expressed IE-2 protein that was purified to near homogeneity. The interaction was sequence specific since a mutated form of the CRS that had previously been reported to be defective in mediating negative regulation could no longer compete for binding in DNase I protection experiments. In addition, an IE-2-reactive monoclonal antibody was able to elicit a supershift in gel retardation experiments, thus proving the presence of IE-2 within the protein-DNA complex. These results suggest that formation of a specific complex between an IE protein and a target sequence located near the cap site of its own gene promoter may be a common mechanism used by both alphaherpesviruses and betaherpesviruses to autoregulate IE gene transcription, although the sequence requirements differ between the two herpesviral subgroups.
Amino Acid Sequence↗
Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB.
Expression of the immediate early 1 and 2 gene (IE-1/2) of human cytomegalovirus, an important pathogen in immunosuppressed patients, is controlled by a strong enhancer/promoter. To define the promoter domain within this large cis-active region of about 550 nucleotides, DNA-protein interactions were studied. DNase I footprinting experiments using procaryotically expressed transcription factor Sp1 revealed an extensive interaction of this transcription factor with both consensus and aberrant recognition elements within the IE-1/2 promoter region. Protection of these Sp1 binding sites could also be observed when nuclear extracts prepared from HeLa cells and permissive human fibroblast cells were used. After in vitro mutagenesis of Sp1 targets and transient expression of mutagenized CAT-expression plasmids, however, no significant reduction in CAT activities was found. By analyzing a series of 5' deletion mutants of the IE-1/2 promoter region, a strong cis-acting element was localized between nucleotides -94 and -78, upstream of sites that interact with Sp1. Gel retardation experiments demonstrated binding of recombinant transcription factor CREB to this motif which reveals it as an aberrant CREB recognition sequence. Thus, this study identifies several previously unknown binding sites for transcription factors Sp1 and CREB within the proximal promoter region of the IE-1/2 gene, which differ markedly in their relevance for constitutive promoter function.
Antigens, Viral↗
Strong trans activation of the human cytomegalovirus major immediate-early enhancer by p40tax of human T-cell leukemia virus type I via two repetitive tax-responsive sequence elements.
The immediate-early 1 and 2 gene locus of human cytomegalovirus (HCMV) that encodes trans-activator proteins with effects on both homologous and heterologous promoters is expressed under control of a complex enhancer/promoter regulatory region. This enhancer contains four types of repetitive sequence elements with 17, 18, 19, and 21 bp that bind cellular transcription factors. Although the HCMV enhancer acts as a powerful stimulator of transcription in most cell types examined, human T cells do not support strong activity. The present study demonstrates that the tax gene product of human T-cell leukemia virus type I trans activates the major enhancer of HCMV more than 60-fold in the T-cell line Jurkat. When a series of chloramphenicol acetyltransferase expression plasmids containing synthetic oligonucleotides with the 17-, 18-, 19-, or 21-bp motif upstream of a minimal immediate-early 1 and 2 gene promoter was tested, two of the four repeat motifs could be identified as Tax-responsive elements. Both the 18- and the 19-bp motifs were able to act as strong Tax-responsive elements even when they were present as single copies. Thus, in addition to interacting with human immunodeficiency virus, HCMV is able to interact with a second retrovirus of clinical importance.
Avian Sarcoma Viruses↗
Comparative sequence analysis of human cytomegalovirus strains.
Three regions of DNA from five low-passage clinical isolates of human cytomegalovirus were amplified by polymerase chain reaction. The DNA sequences as well as the predicted amino acid sequences were compared with those of the laboratory strains AD169 and Towne. The genomic regions consisted of (i) three regions from the major glycoprotein (gp58/116, unique long [UL]55), (ii) three regions from the integral membrane protein (IMP, UL100), and (iii) a region from the major immediate-early 1 and 2 (IE-1/2) enhancer/promoter. Homologies ranged from 75.8 to 100.0% on the nucleotide level and from 47 to 100% on the amino acid level. The following two patterns were observed. (i) There are regions with a high degree of conservation with few scattered point mutations (mainly in the IE-1/2 enhancer/promoter and in the IMP gene). (ii) There are clusters of highly variable regions (parts of the gp58/116 gene and of the IMP gene). Within the areas of high variability, the strains could be classified into a limited number of subtypes.
Amino Acid Sequence↗
An inducible promoter mediates abundant expression from the immediate-early 2 gene region of human cytomegalovirus at late times after infection.
An abundant late transcript of 1.5 kb originates from the immediate-early 2 (IE-2) gene region of human cytomegalovirus (HCMV) at late times after infection. The transcriptional start of this RNA was precisely mapped, and the putative promoter region was cloned in front of the CAT gene as reporter. This region, which comprises 78 nucleotides of IE-2 sequence upstream of the determined cap site, was strongly activated by viral superinfection at late times in the replicative cycle. As shown by RNase protection analyses, the authentic transcription start is used. No activation of this late promoter was observed after cotransfection with an expression plasmid containing the HCMV IE-1 and -2 gene region. This result suggests that, compared with early and early late promoters of HCMV, different or additional viral functions are required for the activation of true late promoters.
Base Sequence↗
Discordant expression of the immediate-early 1 and 2 gene regions of human cytomegalovirus at early times after infection involves posttranscriptional processing events.
Expression of the immediate-early 1 and 2 (IE-1 and IE-2) gene region of human cytomegalovirus (HCMV) was studied during initial phases of the replicative cycle. Accumulation of RNA from IE-1 and -2 was found to be differential. Transcripts from IE-2 reached peak levels very early in infection between 3 and 5 h, whereas IE-1 RNA peak levels were detected later, between 6 and 8 h. A strong decrease in steady-state levels of a 2.2-kb IE-2 RNA was observed at a time when IE-1 transcripts showed a further increase in abundance. Northern (RNA) blot experiments revealed that expression of both the IE-1 RNA and the 2.2-kb IE-2 transcript is controlled by the IE-1 enhancer-promoter. Nuclear run-on experiments demonstrated equal rates of primary transcription for IE-1 and -2 at a time when different steady-state levels of RNA were observed. Concomitant with down-regulation of IE-2 RNAs, a decrease in the size of the IE-1 transcript was detected. At 2 to 5 h after infection the IE-1 transcript migrated at 1.95 kb, whereas later in the replicative cycle the RNA was found at 1.8 kb. RNase H blot analysis revealed that this size discrepancy is due to a shorter poly(A) tail of the IE-1 RNA at early times after infection. These experiments suggest that in addition to transcriptional regulation, specific posttranscriptional mechanisms are involved in controlling expression from the IE-1 and -2 gene region of HCMV.
Antigens, Viral↗
Immediate-early transcription regulation of human cytomegalovirus.
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Antigens, Viral↗
Cell type-specific induction of the major immediate early enhancer of human cytomegalovirus by cyclic AMP.
The major immediate early enhancer of human cytomegalovirus (HCMV) is known to exert a strong constitutive transcription stimulation in a broad spectrum of cells. This basal activity can be augmented considerably by elevated levels of intracellular cAMP in a cell type-specific manner. Cyclic AMP induction was observed in several lymphoid cell lines and in HeLa cells. One of the functionally important enhancer sequence modules, the 19 bp repeat element, mediates this effect as a cAMP-responsive element (CRE). It acts more efficiently than the corresponding sequence from the human chorionic gonadotropin gene. It is suggested that protein kinase C is involved in the pathway which leads to the activation of CRE-containing genes in lymphoid cells. Gel retardation assays indicated that similar, but not identical complexes are formed between nuclear protein extracts and the CREs of HCMV and the gonadotropin gene.
Alkaloids↗
Transcriptional analysis of the eight-kilobase mRNA encoding the major capsid protein of human cytomegalovirus.
The 8-kilobase mRNA coding for the major capsid protein (MCP) of human cytomegalovirus was precisely mapped. Two 5' ends of the transcript were located within HindIII fragment a, 29 and 34 base pairs, respectively, downstream of the sequence TATTAGA. The 3' end was localized within HindIII fragment b of the viral genome. The MCP transcript was synthesized at late times after infection and was not detected before viral DNA replication. In addition, the MCP promoter region could be identified, which strongly responded to viral trans activation at early and late times after infection in a transient expression assay.
Base Sequence↗
The role of a repetitive palindromic sequence element in the human cytomegalovirus major immediate early enhancer.
The major enhancer, extending from nucleotides -530 to -120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for AhaII. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single AhaII site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.
Animals↗
Human cytomegalovirus: recent aspects from molecular biology.
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Base Sequence↗
Organization of terminal reiterations in the virion DNA of herpesvirus saimiri.
The population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. 'light' or L-DNA) linked to direct reiterations of a 1.44 kbp repeat unit of 71% (G + C) (i.e. 'heavy' or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from greater than 30 to 1) at either 'left' or 'right' ends but that all molecular ends are derived by a unique cleavage at a site close to the single ApaI restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the 'right' end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0.8 kbp of the L-DNA sequences at the 'left' H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.
Base Composition↗