Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRF1) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type.
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PMID:The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. 216 95

Although the detection of antibodies to a specific pathogen is used initially as the assay of choice, direct detection of human retroviruses is difficult. First, only a small fraction of cells are infected in the peripheral blood and lymphatic tissue may serve as a reservoir for infection. Second, infected cells may harbor only a small number of copies of the viral sequences. Third, a latent infection marked by transcriptional dormancy is often established thereby obviating the use of proteins or RNA to detect the viruses. Fourth, closely related but distinct members of the onco-and lenti-virus families may complicate specific detection of a particular virus. An additional hurdle is viral heterogeneity. HIV variants, for example, have been identified within and among individuals harboring this virus. Accordingly, sensitive and specific detection of the human retroviruses seemingly requires specific amplification of viral DNA sequences prior to detection. In this regard, an in vitro DNA amplification procedure using DNA polymerase and termed the polymerase chain reaction (PCR) initially applied to human genetic diseases has been successfully applied to human retroviruses. A PCR-based assay has demonstrated utility for detecting infection: (1) prior to the generation of detectable antibodies, (2) in individuals with ambiguous or indeterminate serological status, (3) for neonatal screening, (4) by a specific type or multiple viruses, and (5) in therapeutic trials to allow the monitoring of infected cell load and viremia. It is also unlikely that the viruses identified to date represent all of the retroviruses responsible for human disease. Lymphatic disorders, in general, and immunodeficiencies, in particular, merit closer scrutiny for a retroviral etiologic agent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The polymerase chain reaction (PCR): a valuable method for retroviral detection. 217 Jul 79

The current progress in antiviral therapy is related to our better understanding of the viral multiplication, with potential targets for specific antiviral action at each step of the multiplication cycle inside the infected cell. Amantadine and Rimantadine are anti-influenza A drugs interfering with the penetration and the release of the virus. Most of the other antiviral drugs which are clinically available have the same target in common, namely the viral DNA polymerase. This holds true for modified nucleosides such as Acycloguanosine (Acyclovir), DHPG, Adenine-Arabinoside, Azidothymidine as well as pyrophosphate derivatives such as phosphonoformic acid. Unfortunately the antiviral chemotherapy must confront 3 obstacles: 1) a possible interference with the normal cellular metabolism, leading to residual cytotoxic side effects; 2) the genetic variability of the viruses, producing drug-resistant mutants and 3) the inability of any antiviral chemotherapeutic agent known to date to eradicate latent viral infection. A new approach of the control of latent infection is suggested with anti sense oligonucleotides of hybridons.
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PMID:Perspectives in antiviral chemotherapy. 221 May 92

Following infection of cells by herpes simplex virus, the cell nucleus is subverted for transcription and replication of the viral genome and assembly of progeny nucleocapsids. The transition from host to viral transcription involves viral proteins that influence the ability of the cellular RNA polymerase II to transcribe a series of viral genes. The regulation of RNA polymerase II activity by viral gene products seems to occur by several different mechanisms: (1) viral proteins complex with cellular proteins and alter their transcription-promoting activity (e.g., alpha TIF), (2) viral proteins bind to specific DNA sequences and alter transcription (e.g., ICP4), and (3) viral proteins affect the posttranslational modification of viral or cellular transcriptional regulatory proteins (e.g., possibly ICP27). Thus, HSV may utilize several different approaches to influence the ability of host-cell RNA polymerase II to transcribe viral genes. Although it is known that viral transcription uses the host-cell polymerase II, it is not known whether viral infection causes a change in the structural elements of the nucleus that promote transcription. In contrast, HSV encodes a new DNA polymerase and accessory proteins that complex with and reorganize cellular proteins to form new structures where viral DNA replication takes place. HSV may encode a large number of DNA replication proteins, including a new polymerase, because it replicates in resting cells where these cellular gene products would never be expressed. However, it imitates the host cell in that it localizes viral DNA replication proteins to discrete compartments of the nucleus where viral DNA synthesis takes place. Furthermore, there is evidence that at least one specific viral gene protein can play a role in organizing the assembly of the DNA replication structures. Further work in this system may determine whether assembly of these structures is essential for efficient viral DNA replication and if so, why assembly of these structures is necessary. Thus, the study of the localization and assembly of HSV DNA replication proteins provides a system to examine the mechanisms involved in morphogenesis of the cell nucleus. Therefore, several critical principles are apparent from these discussions of the metabolism of HSV transcription and DNA replication. First, there are many ways in which the activity of RNA polymerase II can be regulated, and HSV proteins exploit several of these in controlling the transcription of a single DNA molecule. Second, the interplay of these multiple regulatory pathways is likely to control the progress of the lytic cycle and may play a role in determining the lytic versus latent infection decision.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The role of viral and cellular nuclear proteins in herpes simplex virus replication. 255 60

Viral functions essential for the establishment of latent infection in murine sensory neurons in vivo were investigated by employing a herpes simplex virus type 1 (HSV-1) variant (KD6/B11) deleted for expression of the ICP4 gene and therefore unable to replicate. Since the viral DNA persisted in these cells, the latency-associated transcripts were expressed for prolonged periods of time, and the variant was biologically retrievable by superinfection with an ICP4-competent agent, we concluded that a latent infection had been established. In situ hybridization experiments designed to investigate gene expression during the acute phase of infection with the variant revealed a highly restricted pattern compared to that of the wild-type parent virus HSV-1 KOS(M). While latency-associated transcripts were detected in a large number of infected neurons, expression of other virus genes was limited to a subset of immediate-early and early genes (ICP0, ICP8, ICP27, and HSV-1 DNA polymerase genes). Expression was further limited to a small proportion of the infected neurons (approximately 1% of neurons expressing latency-associated transcripts). No hybridization was detected with probes specific for the viral TK gene and late genes VP5 and gC. Quantitative assays of viral DNA during the acute phase of infection indicated that the input viral DNA did not replicate. From these results we conclude that HSV-1 latent infection can be established in murine sensory neurons under conditions in which viral genetic expression and DNA replication are severely restricted.
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PMID:Latent infection can be established with drastically restricted transcription and replication of the HSV-1 genome. 838 Jun 70

Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV.
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PMID:Evidence for a novel regulatory pathway for herpes simplex virus gene expression in trigeminal ganglion neurons. 839 54

Herpes simplex virus establishes a latent infection in peripheral neurons. We examined viral gene expression in rat peripheral neurons in vitro and determined that viral gene expression is attenuated and delayed in these neurons compared with that in Vero cells. In addition, using pharmacologic and genetic blocks to viral DNA synthesis, we found that viral alpha and beta gene expression was upregulated by viral DNA synthesis. Although maximal gene expression in neurons requires viral DNA synthetic activity, activation of viral gene expression was seen even in the presence of herpes simplex virus DNA polymerase inhibitors, but not in the absence of the origin-binding protein. Initiation of viral DNA synthesis is apparently a key regulatory event in the balance between the lytic and latent pathways in peripheral neurons.
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PMID:Herpes simplex virus gene expression in neurons: viral DNA synthesis is a critical regulatory event in the branch point between the lytic and latent pathways. 876 59

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.
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PMID:Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection. 952 97

A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.
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PMID:Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice. 1133 74

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.
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PMID:The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas). 1503 69


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