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)

The recently discovered human herpesvirus-6 (HHV-6) is being associated with an increasing number of conditions in which there is evidence of immunologic dysfunction. A number of widely available antiviral agents have shown little or no activity against the virus. We found that Ampligen [Poly (1): Poly (C12U), a synthetic, mismatched, double-stranded RNA, has potent, previously unexpected antiviral effects. Cells known to allow replication of HHV-6 were infected with the virus and treated with Ampligen under various conditions. When cells were pretreated with Ampligen (concentrations of 100 or 200 micrograms/ml) prior to infection or treated shortly after infection, viral replication was inhibited by 46-98%. At 100 and 200 micrograms/ml, Ampligen also inhibited the DNA polymerase activity of HHV-6 by 42-98%. When lower concentrations of Ampligen (10 and 50 micrograms/ml) were used, only pretreatment of cells, with Ampligen, followed by virus infection and carrying the infected cells with Ampligen, significantly inhibited HHV-6 infection (83.7 and 89.1% respectively). Indirect evidence suggests that Ampligen may inhibit viral attachment to cellular receptors and/or inhibit intracellular maturation of the virus. The above concentrations of Ampligen were not toxic to the cells used in the study. Given these in vitro findings, and the low frequency of toxicity reported with the use of Ampligen, clinical trials of this drug in patients with evidence of reactivated HHV-6 infection would seem to be warranted.
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PMID:Ampligen inhibits human herpesvirus-6 in vitro. 789 86

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.
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PMID:Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products. 820 92

The reasons for acyclovir's activity and selectivity in cells infected with HSV or VZV may be summarized as follows: 1. Activation by a HSV- or VZV-specified TK. 2. Greater sensitivity of viral DNA polymerase than of the cellular polymerases to ACV-TP. 3. Inactivation of the viral DNA polymerase, but not the cellular polymerases, by ACV-TP. 4. Chain termination of viral DNA by incorporation of ACV-MP. For the Epstein-Barr virus, which is also sensitive to acyclovir, there is no selective activation in infected cells [Colby et al., 1981], but the viral polymerase can be inhibited by very low levels of ACV-TP [Datta et al., 1980]. For HCMV, the activation of acyclovir is very poor but the viral polymerase is also more sensitive to ACV-TP than the cellular polymerases. One of the important contributions of acyclovir was the demonstration for the first time that a compound could prevent the DNA replication of a DNA virus at concentrations far below those that affect cellular DNA synthesis. As we all know, in the past 15 years there has been a complete rejuvenation of antiviral chemotherapy. I think it is very fortunate that we changed our outlook on the possibility of making potent and selective antiviral agents in time so that, when the AIDS epidemic came along, we did not feel completely at a loss on ways to attack viral disease.
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PMID:Acyclovir: discovery, mechanism of action, and selectivity. 824 87

A 47-year-old man with acute hepatitis B virus (HBV) infection who had been receiving immunosuppressants after renal transplantation developed progressive liver failure. During the clinical course (approximately 7 months), anti-HBc IgM antibody and HBV-DNA polymerase levels remained high, but the serum alanine aminotransferase (ALT) level was consistently less than 150 K.U. Histopathologic examination of the liver showed submassive hepatic necrosis without significant inflammation accompanied by marked fibrosis. Most hepatocytes showed strong nuclear expression of HBc antigen by immunohistochemical staining and electron microscopy revealed numerous intranuclear core-like particles. Hepatitis B virus infection in immunosuppressed individuals occasionally insidiously progresses, resulting in liver failure. The clinical course of such patients thus merits close scrutiny.
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PMID:Fatal acute hepatitis B virus infection while receiving immunosuppressants after renal transplantation. 828 32

The hepatitis B virus is a member of an unusual family of noncytopathogenic, hepatotropic DNA viruses--the hepadnaviruses. The complete virus comprises a lipoprotein coat, the hepatitis B surface antigen, enveloping a nucleocapsid core that contains a small, circular DNA molecule. Four open reading frames have been identified on the hepatitis B virus DNA genome. They encode seven proteins, including a hepatitis B virus DNA polymerase molecule with reverse transcriptase activity. The replication of the virus resembles that of retroviruses and occurs predominantly but not exclusively in hepatocytes. Virus variants involving genomic mutations have been identified. Testing for hepatitis B surface antigen permits detection of many but not all acutely infected patients. Diagnosis of acute infection rests on the identification of IgM antibodies to the hepatitis B core antigen. Antibody to hepatitis B surface antigen appears in serum during the convalescent phase of hepatitis B virus infection. It is the neutralizing, protective antibody largely responsible for immunity to reinfection. In persistent infection hepatitis B surface antigen is present, antibody to hepatitis B core antigen is predominantly an IgG antibody, antibody to hepatitis B surface antigen is not detectable or is present in very low titers and viral replication may be active. Persistent infection leads to an asymptomatic carrier state, chronic hepatitis, cirrhosis and hepatocellular carcinoma. No specific treatment exists for acute hepatitis B virus infection. Current data indicate that approximately 50% of adults who have chronic infection achieve virologic, biochemical and histologic remission from treatment with alpha-2b-interferon.
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PMID:Hepatitis B today: clinical and diagnostic overview. 832 12

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.
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PMID:Unusual regulation of expression of the herpes simplex virus DNA polymerase gene. 839 56

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.
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PMID:Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. 852 51

Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.
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PMID:The role of ATF in regulating the human cytomegalovirus DNA polymerase (UL54) promoter during viral infection. 903 45

Murine cytomegalovirus (MCMV) has been used extensively as an animal model for human cytomegalovirus (HCMV). Understanding drug resistance and its treatment in MCMV may lead to more effective treatments of HCMV disease. Most ganciclovir-resistant HCMV clinical isolates exhibit a decreased capacity to induce ganciclovir phosphorylation (to its biologically active form) in infected cells. Using an MCMV strain resistant to both ganciclovir and cidofovir, the intracellular metabolism of these drugs was studied to determine if MCMV resistance correlates with decreases in drug phosphorylation. The wild-type (WT) MCMV used for comparison was inhibited in plaque reduction assays, by ganciclovir and cidofovir by 50% at 5.1 and 0.24 microM, respectively; the resistant strain was inhibited at 72 and 2.7 microM, respectively. In uninfected, WT, or resistant virus-infected cells, the extent of metabolism of 10 microM ganciclovir or 1 microM cidofovir to intracellular triphosphorylated species was similar. Phosphorylation and catabolism (following drug removal) rates over time were also similar. Intracellular levels of ganciclovir triphosphate and cidofovir diphosphate increased less than two-fold with increasing multiplicity of virus infection. Because few differences in drug phosphorylation between WT and resistant virus-infected cells were found, virus resistance to ganciclovir and cidofovir apparently is not linked to altered drug phosphorylation. Since the viral DNA polymerase is the antiviral target for these compounds, the resistant MCMV is most likely a DNA polymerase mutant.
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PMID:Metabolism of ganciclovir and cidofovir in cells infected with drug-resistant and wild-type strains of murine cytomegalovirus. 921 45

Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg2+, resistance to N-ethylmaleimide, and inhibition by 3'-azido-2',3'-dideoxythymidine-5'-triphosphate, coumermycin A1, and pyridoxal 5'-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T20 DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T's or C's in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5' AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5'-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.
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PMID:Efficient gap repair catalyzed in vitro by an intrinsic DNA polymerase activity of human immunodeficiency virus type 1 integrase. 949 61


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