Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report a case of simultaneous infection with hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) in a 26-year-old Japanese homosexual man. He was admitted to our hospital for acute hepatitis caused by HBV. At that time, HIV-1antibody (Ab) was not detected in his serum. After 6 months, he was readmitted to our hospital for further examination of his liver because of confined liver enzyme abnormalities. Anti-HIV- Ab was detected in his serum by both enzyme immunosorbent assay (EIA) and particle agglutination (PA). His serum HIV-1 RNA level was 50 x 10(4) copies/ml and serum levels of HBV DNA polymerase (DNA-P) and HBV DNA were 6535cpm and 3 plus (>1000 copies/ml). His clinical course and laboratory data suggested progression from acute to chronic hepatitis related to coinfection with HIV-1. The diagnosis was chronic active hepatitis caused by HBV as an opportunistic infection due to coinfection with HIV-1. We began highly active antiretroviral therapy (HAART) because interferon (IFN) therapy was ineffective. HAART was started at an initial dosage of 600 mg zidovudine (AZT), 300 mg lamivudine (3TC), and 2400 mg indinavir (IDV) daily. After 4 weeks, the serum level of HBV DNA-polymerase (p) had decreased markedly to 37cpm and that of HIV-1 RNA had decreased to below the sensitivity threshold, indicating considerable suppression of the replication of these viruses by the treatment. But HBV DNA remained at low levels. Although the incidence of HBV infection in patients with HIV-1 infection has been reported to be high in the United States and Europe, simultaneous HBV and HIV-1 infection leading to persistent HBV infection is rare.
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PMID:Highly active antiretroviral therapy used to treat concurrent hepatitis B and human immunodeficiency virus infections. 1021 32

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.
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PMID:The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases. 1046 85

The effect of the DNA polymerase-beta (beta-pol) deficiency on mitogenic response and cytokine production was studied in spleen lymphocytes from 4-5- and 20-22-month-old beta-pol(-/+) mice and their age-matched wild-type littermates. The proliferative response of lymphocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (interleukin (IL)-2, IL-4, and interferon necrosis factor (IFN)-gamma) was assessed by enzyme-linked immunosorbent assay. There was no significant difference in Con A- or LPS-induced proliferation or cytokine production in young beta-pol(-/+) mice compared with young wild-type littermates or in old beta-pol(-/+) mice compared with old wild-type littermates. However, mitogen-induced proliferation and cytokine production changed significantly with age. The proliferative response to Con A and to LPS, and the IL-2 production was significantly lower, and IL-4 and IFN-gamma levels were significantly higher in lymphocytes from old beta-pol(-/+) mice and old wild-type mice than in lymphocytes from young beta-pol(-/+) mice and young wild-type littermates. In addition, flow cytometric analysis showed no significant differences between young beta-pol(-/+) mice and young wild-type littermates or between old beta-pol(-/+) mice and old wild-type littermates in the proportion of B- and T-cell populations, and T-cell subsets. However, the number of lymphocytes expressing CD4+ phenotype slightly decreased and the proportion of lymphocytes expressing CD44/Pgp-1 (memory) phenotype increased with age. Thus, we found no evidence for alteration in immune function in DNA polymerase-beta deficient mice, although they exhibit a decline in immunologic function with age.
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PMID:Normal immune function in young and old DNA polymerase-beta deficient mice. 1078 76

We compared two commercial assays for quantification of serum hepatitis C virus (HCV) RNA to investigate whether pretreatment levels of serum HCV RNA could predict the outcome of interferon (IFN) therapy. The Amplicor HCV Monitor test is based on a single, combined reverse transcription and amplification reaction carried out by the Tth DNA polymerase using specific primers for the 5' untranslated (UTR) region. The Quantiplex HCV RNA 2.0 assay is based on specific hybridization of viral RNA by synthetic oligonucleotides complementary to the 5'-UTR and core regions of the genome, allowing equal quantification of the six major genotypes. Receiver-operating characteristic (ROC) analysis was employed to identify the best cut-off value (predicting patients who were non-responsive to treatment) with corresponding sensitivity and specificity values. Logistic regression analysis was performed using these cut-off values. We studied 133 consecutive patients with chronic hepatitis C enrolled in a prospecsustained responders was 5322 copies ml-1 by the Monitor test and less than 0.2 million equivalents ml-1 (MEq ml-1) by the Quantiplex assay; for the 115 non-responders/relapsers, the median viraemia was 83,125 copies ml-1 and 1.128 MEq ml-1 for the Monitor test and Quantiplex assay, respectively. Spearman's rank test gave a correlation of 0.63 between assays. The best predicting cut-off values were 22,134 copies ml-1 for the Monitor test and 0.330 MEq ml-1 for the Quantiplex assay; their respective sensitivities and specificities were 72% and 75% for Monitor and 67% and 83% for Quantiplex. By logistic regression analysis, the age and gender-adjusted odds ratios of high vs low HCV RNA levels, defining the risk of non-response, were 10.6 (CI 3.1-35.7) for Monitor and 14.3 (CI 4.3-47.3) for Quantiplex. The two assays had comparable sensitivity for serum HCV RNA but they identified different predictive cut-offs for non-response to therapy.
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PMID:Serum levels of hepatitis C virus RNA predict non-response to interferon therapy: comparison of two commercial assays. 1084 32

In the present study, we employed a plasmid DNA encoding murine interferon (IFN)-beta to assess its antiviral efficacy in an in vitro transfection-infection assay and in an ocular HSV-1 infection model of mice. In the in vitro assay, transfection of mouse fibroblasts with the IFN-beta transgene resulted in a 17-fold or greater reduction in the viral load of HSV-1 at a multiplicity of infection (MOI) of 1 compared to that of those mice treated with the plasmid control. RT-PCR analysis of representative immediate early (ICP27), early (thymidine kinase, TK) and late (VP16) viral genes found no changes in the level of expression comparing the IFN-beta transgene- to the vector-treated control group, suggesting that the IFN-beta transgene may act at the post-transcriptional level of viral replication. In the ocular HSV-1 infection model, topical application of the plasmid DNA encoding murine IFN-beta onto mouse cornea enhanced cumulative survival and significantly reduced the viral load of HSV-1 in the eyes and trigeminal ganglia of mice at both day 3 and 6 post-infection compared with mice treated with the plasmid vector control or normal saline. Neutralizing antibody to IFN-beta blocked the protective effect elicited by the IFN-beta transgene. Unlike the in vitro experiment, viral gene expression was reduced in the trigeminal ganglion of mice pre-treated 24 h with the IFN-beta transgene day 3 (ICP27 and VP16) and day 6 (ICP27, TK, DNA polymerase, and VP16) post-infection in comparison to mice treated with the plasmid vector control as determined by semi-quantitative RT-PCR.
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PMID:A plasmid construct encoding murine interferon beta antagonizes the replication of herpes simplex virus type I in vitro and in vivo. 1090 Mar 42

The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.
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PMID:Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor gene. 1095 64

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.
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PMID:Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53. 1111 11

The hepatitis C virus (HCV) is known to circulate in vivo as a quasispecies, a population of closely related, but genetically nonidentical virions. HCV reverse transcriptase (RT)-(nested) polymerase chain reaction (PCR) strategies are used to study quasispecies diversity at certain important viral genetic loci, predominantly at hypervariable region 1 (HVR 1) of the E2 envelope gene, and the interferon sensitivity determining region (ISDR) of the nonstructural 5a (NS5a) gene. We have found that the choice of DNA polymerase employed in viral PCR has effects on the inferred viral diversity at two distinct loci on the HCV genome. Nested HVR 1 and ISDR PCR was performed with both proofreading (Pwo) and nonproofreading (Taq) DNA polymerases on identical cDNA derived from three separate HCV-positive sera. Amplicons were cloned and sequences determined for 18-20 individual clones per sample. Quasispecies diversity determined from HVR 1 and ISDR PCR products showed that there was a marked effect on the inferred diversity depending on which DNA polymerase was employed in the PCR. The deduced amino acid sequences of the major variants within each specimen were identical for both Taq and Pwo DNA polymerase-mediated PCRs. However, a greater number of minor variants were observed in the Taq-generated amplicons, 80% of which were not observed in the Pwo-generated amplicons. Primer editing in the Pwo-generated amplicons was observed in 19% (20/104) of clones examined. Single-strand conformational polymorphism analysis of multiple replicates of each amplicon revealed good intra-PCR reproducibility in terms of genetic heterogeneity, and that as such the observations were not due to poor PCR reproducibility. The use of nonproofreading DNA polymerases to assess viral diversity can yield an incorrect quasispecies spectrum and affect RT-PCR assay performance. The contribution of Taq-induced errors and lack of adaptability of primers to potentially heterologous template-binding sites indicate that proofreading DNA polymerases should be the enzyme of choice in these systems.
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PMID:Inferred hepatitis C virus quasispecies diversity is influenced by choice of DNA polymerase in reverse transcriptase-polymerase chain reactions. 1116 7

We experienced a 24-year-old Japanese man, who was a hepatitis B virus carrier with nephrotic syndrome. Liver biopsy showed that he was suffering from chronic hepatitis (activity 2, fibrosis 2). Renal biopsy revealed membranous nephropathy(MN) with focal segmental glomerulosclerosis(FGS). Immunofluorescentic findings revealed the presence of HBe antigen along the glomerular capillaries as well as HBe antigenemia in circulation. Therefore, we diagnosed this case as HB virus-related membranous nephropathy associated with FGS lesions. He was treated with interferon(IFN) alpha-2b for over a month and angiotensin converting enzyme inhibitor. These therapies reduced urinary protein excretion from 4-6 g/day to 1-2 g/day, in accordance with a decrease in the titer of HBV DNA polymerase. The second renal biopsy revealed that the histological change from MN to membranoproliferative glomerulonephritis Type III after IFN therapy. These results suggest that IFN therapy might be effective for HB virus-related MN associated with FGS.
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PMID:[Effect of interferon therapy on hepatitis B virus related membranous nephropathy associated with focal segmental glomerulosclerosis]. 1128 Feb 14

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.
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PMID:Molecular virology of Kaposi's sarcoma-associated herpesvirus. 1131 14


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