Splicing and the regulation of viral gene expression.
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Adenoviridae↗
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Results for “Gene Expression Regulation, Viral”
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The regulation of HIV-1 gene expression: the emerging role of chromatin.
Host and viral factors that regulate the expression of the human immunodeficiency virus type 1 (HIV-1) 5' long terminal repeat (LTR) promoter have been studied since the recognition that HIV is the cause of the acquired immunodeficiency syndrome (AIDS). However, complex modifications of nucleosomes within chromatin has been recently recognized as an important mechanism of gene regulation. Nucleosome remodelling can alter the accessibility of DNA to specific activators or repressors, general transcription factors, and RNA polymerase. Emerging data now suggests that dynamic regulation of chromatin structure in the vicinity of the LTR promoter adds an additional level of complexity to the regulation of HIV expression. A better understanding of the role of chromatin in the regulation of HIV expression could lead to much-needed therapies against proviral genomes that are being actively transcribed, and those that are quiescent and persistent.
Chromatin↗
Optimization of the Tet-On system for regulated gene expression through viral evolution.
The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.
Anti-Bacterial Agents↗
Equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression.
EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.
Animals↗
HIV-1 infection and the developing nervous system: lineage-specific regulation of viral gene expression and replication in distinct neuronal precursors.
Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. Microglia and macrophages are major HIV-1 targets in the brain, whereas HIV-1 infected neurons or glial cells have been rarely reported. This suggests that indirect mechanisms may account for the severe neuronal damage observed in these patients. Nevertheless, immature, mitotically active neuronal and glial cells, which are present during fetal development, are susceptible to HIV-1 infection and replication in vitro, suggesting that HIV-1 infection during organ development may present unique features. To better characterize virus-host cells interactions in the developing CNS, we have examined the susceptibility of embryologically and biochemically distinct neuronal cell lines to HIV-1 infection. Here we show that mitotically active, immature neurons of distinct lineages, have different susceptibilities to HIV-1 infection and replication and different abilities to support viral gene expression. Mutational analysis of HIV-1 LTR reveals that a region of the viral promoter between nucleotide -255 to -166 is responsible for most quantitative and qualitative differences in viral transactivation among different neuroblasts. This suggests that specific regions of the viral promoter and cellular factors, either lineage- or differentiation-dependent, which bind to those regions, may contribute to control the levels of virus replication and possibly restrict the viral tropism in the developing brain. This may contribute to the establishment of a virus reservoir in the immature CNS and participate by either direct or indirect mechanisms to the severity of the AIDS-related pediatric neurological dysfunction.
Cell Line↗
Genome sequence and gene expression of Bacillus anthracis bacteriophage Fah.
Fah, a lytic bacteriophage of Bacillus anthracis, is used widely in the former Soviet Union to identify anthrax bacteria. Here, we present the analysis of a 37,974 bp sequence of the Fah genome and examine gene expression of the phage in a model host, Bacillus cereus. Half of the Fah genome contains genes coding for structural proteins and host lysis functions in an arrangement typical of Syphoviridae. The other half of the genome contains genes coding for enzymes of viral genome replication and for numerous predicted transcription factors that are likely to regulate viral gene expression. Primer extension, in vitro transcription assays, and gene array analysis identified temporal classes of Fah genes and allowed location of viral promoters. Fah does not execute host transcription shut-off and relies on host RNA polymerase (RNAP) sigma(A) holoenzyme for transcription of its early and late genes. In addition, Fah encodes a sigma factor, sigma(Fah), a close relative of Bacillus sporulation factor sigma(F) that directs bacterial RNAP to at least one late viral promoter. sigma(Fah) is negatively regulated by host SpoIIAB, an anti-sigma factor that controls sporulation. Thus, sigma(Fah) may link phage gene expression to sporulation of the host.
Bacillus Phages↗
Human T-cell leukemia virus type 1 expressing nonoverlapping tax and rex genes replicates and immortalizes primary human T lymphocytes but fails to replicate and persist in vivo.
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated primarily with adult T-cell leukemia and neurological disease. HTLV-1 encodes the positive trans-regulatory proteins Tax and Rex, both of which are essential for viral replication. Tax activates transcription initiation from the viral long terminal repeat and modulates the transcription or activity of a number of cellular genes. Rex regulates gene expression posttranscriptionally by facilitating the cytoplasmic expression of incompletely spliced viral mRNAs. Tax and Rex mutants have been identified that have defective activities or impaired biochemical properties associated with their function. To ultimately determine the contribution of specific protein activities on viral replication and cellular transformation of primary T cells, mutants need to be characterized in the context of an infectious molecular clone. Since the tax and rex genes are in partially overlapping reading frames, mutation in one gene frequently disrupts the other, confounding interpretation of mutational analyses in the context of the virus. Here we generated and characterized a unique proviral clone (H1IT) in which the tax and rex genes were separated by expressing Tax from an internal ribosome entry site. We showed that H1IT expresses both functional Tax and Rex. In short- and long-term coculture assays, H1IT was competent to infect and immortalize primary human T cells similar to wild-type HTLV-1. In contrast, H1IT failed to efficiently replicate and persist in inoculated rabbits, thus emphasizing the importance of temporal and quantitative regulation of specific mRNA for viral survival in vivo.
Cell Transformation, Viral↗
Late temporal gene expression from the human cytomegalovirus pp28US (UL99) promoter when integrated into the host cell chromosome.
Toward understanding the temporal regulation of human cytomegalovirus (HCMV) late genes, we studied the regulation of the late gene promoter (pp28US, UL99) when outside the context of the viral genome and its response to the immediate early (IE) proteins. Expression of the luciferase reporter gene, regulated by the pp28US promoter, was synchronous with that of the endogenous viral pp28 gene, independently of whether the reporter was episomal or integrated into the glioblastoma cell line U373MG. Cotransfection of the reporter with expression vectors for each of the three major IE genes, IE72, IE86 and IE55, indicated that only IE86 transactivated the pp28US promoter. However, the magnitude of the promoter activation upon HCMV infection suggested that additional factors are also required for higher promoter activity. The promoter activation was specific to HCMV, as herpes simplex virus type 1 infection did not induce luciferase expression.
Chromosomes↗
Viral genes and methylation.
Epigenetics represents a new frontier in cancer research. Methylation is the best studied of the epigenetic mechanisms that regulate gene expression. Regulation of gene expression by means of methylation has been reported for tumor suppressor genes, oncogenes, viral promoters, and age-related genes. In this review, the regulation of viral gene expression by methylation is discussed, with particular emphasis on: (1) the virus-specific factors that bind to promoter regions; (2) the implications of this knowledge for designing viral vectors that can be used to deliver genes for the purpose of gene therapy; and (3) the use of this knowledge for the early detection and prevention of cancer. Since methylation can be reversed by a variety of exogenous agents, great potential exists to develop interventions that target cancer-associated aberrant methylation in an effort to reverse or prevent carcinogenesis.
Animals↗
Human papillomavirus-16 and genital cancer: are tests for the viral gene expression in vitro indicators for risk factors in vivo?
Human papillomaviruses are found in up to 90% of all cervical carcinomas and are considered to play a causal role in the etiology of this malignancy. The genome of human papillomaviruses consists of a single circular DNA molecule with a size of approximately 8000 basepairs. 90% of this genome encodes proteins involved in functions such as neoplastic transformation of the host cell or formation of the viral capsid. The remaining 10% of the genome, which is termed upstream regulatory region (URR), harbours elements to control expression of the viral genes. We have identified in the URR DNA elements that regulate viral gene expression in the presence of glucocorticoid hormones or tumour promoting substances. This was done by DNase I protection experiments and functional analysis of fusion genes. Our data predict that the transforming potential of the virus might be stimulated by certain steroid hormones, polypeptide hormones and tumour promoting chemicals.
Cloning, Molecular↗
Thioredoxin-mediated redox control of human T cell lymphotropic virus type I (HTLV-I) gene expression.
Thioredoxin (TRX) is a small ubiquitous protein with multiple biological functions, including the thiol-mediated redox-regulation of gene expression. We have previously demonstrated that human TRX is overexpressed as a major protein oxidoreductase in human T cell lymphotropic virus type I (HTLV-I)-infected cells. In the present study, we investigated the relationship between TRX and viral gene expression in HTLV-I infection. To study the mechanism that causes overexpression of TRX in HTLV-I-infected cells, we first examined the effect of the HTLV-I transactivator, Tax, on TRX expression. Induction of HTLV-I Tax protein increased the expression of TRX protein in a Tax-transfected Jurkat cell line, JPX-9. Moreover, chloramphenicol acetyltransferase (CAT) analysis with a reporter gene containing the TRX promoter revealed that Tax activates the transcription of TRX gene. To study the role of overexpressed TRX in HTLV-I infection, we next examined the effect of TRX on HTLV-I long terminal repeat (LTR)-mediated transcription using CAT analysis. In an HTLV-I-infected human T cell line MT-2, the HTLV-I LTR transactivation was suppressed by the overexpression of wild-type TRX, but activated by the introduction of inactive mutant TRX. Moreover, in HTLV-I negative Jurkat T cells, the HTLV-I LTR transactivation induced by Tax was also repressed by overexpression of wild-type TRX. Because cellular redox changes were shown to affect the HTLV-I gene expression, it is likely that TRX modulates the HTLV-I gene expression by regulating cellular redox state. Taken together, these findings suggest that overexpressed TRX, which is induced by HTLV-I Tax, may play an important role in HTLV-I infection through the negative regulation of viral gene expression.
Gene Expression Regulation, Viral↗
Baculovirus--insect cell interactions.
Baculovirus interactions with host cells range from the physical interactions that occur during viral binding and entry, to the complex and subtle mechanisms that regulate host gene expression and modify and regulate cellular and organismal physiology and defenses. Fundamental studies of baculovirus biochemistry and molecular biology have yielded many interesting and important discoveries on the mechanisms of these virus-host interactions. Information from such studies has also resulted in exciting new strategies for environmentally sound insect pest control, and in the development and improvement of a valuable eukaryotic expression vector system. In addition a number of important and valuable model biological systems have emerged from studies of baculoviruses. These include robust systems for studies of eukaryotic transcription, viral DNA replication, membrane fusion, and apoptosis. Because functions have been identified for only a small number of baculovirus genes, we can expect many exciting new discoveries in the future and an unfolding of the complex and intricate relationship between baculoviruses and insect cells.
Animals↗
Construction and characterization of a stably transformed HeLa cell line in which the expression of bovine herpesvirus 1 ICP0 (BICP0) is induced by tetracycline.
To explore the effects BICP0 (a principal transactivator of BHV-1 gene expression) on viral promoter elements, we established a cell line in which the expression of BICP0 is regulated by tetracycline. A hybrid promoter containing reiterated copies of the tet-operator (tet-O) and a minimal herpesviral alpha gene transinducing factor (alpha TIF) responsive element (minimal human cytomegalovirus immediate early promoter) was fused to the BICP0 gene and used to transform a HeLa cell line which expressed a fusion protein consisting of the repressor of the tet-O and the transactivating domain of alpha TIF. Simultaneously, the hygromycin resistance gene was transfected to select cells in media containing either hygromycin alone or both hygromycin and tetracycline. Immunofluorescent assays indicated that BICP0 was synthesised in the transformed cell lines solely upon induction of the gene by tetracycline removal. Only cells which had been kept constantly in medium containing tetracycline were able to synthesise BICP0 upon induction. Induced cell lines transactivated the native BICP0 promoter as well as the herpes simplex virus thymidine kinase promoter and the long terminal repeat sequences of human immunodeficiency virus in a dose dependent manner. These cell lines may help to further explore the functions of BICP0 as well as to investigate the molecular basis of interactions between herpes- and retroviruses.
Cell Line, Transformed↗
Overview of the Leishmaniavirus endoribonuclease and functions of other endoribonucleases affecting viral gene expression.
Leishmaniaviruses (LRV) are double-stranded RNA viruses that persistently infect some strains of the protozoan parasite Leishmania. The identification of a short viral RNA transcript led to our discovery of an endoribonuclease activity of the LRV capsid protein. Other known endoribonucleases serve a variety of diverse roles in the regulated balance of processing and degradation of both cellular and viral RNAs, thus determining the amount and functionality of specific RNA molecules in a cell at any given time. The consequence of LRV RNA cleavage on the LRV life cycle has not yet been determined. Here we review the LRV endoribonuclease and discuss potential roles for this endoribonuclease activity in the context of the involvement of other endoribonucleases in regulating viral gene expression and replicative capacity.
Animals↗
DNA methylation, chromatin structure and the regulation of gene expression.
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Animals↗
Herpes simplex virus vectors and gene transfer to brain.
Experiments are in progress to engineer herpes simplex virus type 1 as a gene transfer vector for the nervous system. This virus is well suited for this purpose due to its ability to persist for long periods in a latent state in neurons wherein lytic genes are inactive and a unique viral promoter is capable of inducing transcription from the latent viral genome. The virus can be highly cytotoxic for neurons, however, and thus genes which initiate the lytic cycle must be deleted from the genome in order to force the virus into latency. Candidate genes include the immediately early functions ICP0, ICP27, ICP4 and the virus host shut-off function. The design of a suitable promoter regulator capable of expressing foreign genes during latency must also be developed. Many promoters including strong viral promoters and neuronal-specific cellular promoters have proved to be inadequate due to the transient nature of their activity from the viral vector genome and the viral latency promoter region appears to be weakly active in brain. Current efforts concern the development of auto-regulatable promoters which can remain active even if the viral genome is bound by chromatin.
Base Sequence↗
Regulation of HIV expression by viral genes and cytokines.
The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS), a leading cause of death among young individuals. As a complex pathogenic retrovirus, HIV is characterized in vitro and in vivo by the ability to establish either a latent or productive infection of CD4+ T lymphocytes and mononuclear phagocytes (MPs). Viral latency and expression are under the control of both viral genes and several host-related factors. Among these latter, several proinflammatory and immunoregulatory cytokines profoundly affect HIV replication in T lymphocytes and MPs in vitro. Because many of these cytokines have been found to be elevated in HIV-infected individuals, it is likely that they act as regulators of viral expression in vivo. Both viral genes and cytokines regulate transcriptional and posttranscriptional steps in HIV replication. Thus, it is likely that common regulatory pathways between these two distinct classes of regulator molecules will be identified in the near future and will provide potential targets for pharmacologic intervention and treatment of HIV disease.
Cytokines↗
Regulation of viral and cellular gene expression in sensory neurons.
Herpes simplex virus (HSV) establishes latent infections of neuronal cells but replicates readily in other cell types. This effect is dependent upon the presence of the inhibitory cellular transcription factor Oct-2 in neuronal cells. This factor inhibits the viral immediate-early gene promoters by at least two distinct mechanisms. The significance of Oct-2 in regulating viral and cellular gene expression in sensory neurons is discussed.
Animals↗