Cellular DNA Polymerases Research Articles

Inhibition of HIV-1 reverse transcriptase by 5′-triphosphates of 5-substituted uridine analogs

The 5′-triphosphates of some 5-substituted 2′-deoxyuridine analogs were investigated for their effects on purified recombinant reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) as well as cellular DNA polymerase α. The triphosphates were competitive inhibitors of the viral enzyme with dTTP as the variable substrate and poly(rA)oligo(dT) as template, and preferentially inhibited the viral polymerase. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the reverse transcriptase, gave nPrearaUTP > nPrdUTP > EtdUTP > nPredUTP > HMdUTP > CEdUTP. Although nPredUTP was less inhibitory than nPrearaUTP under conditions of competitive inhibition, nPredUTP caused a time- and concentration-dependent inactivation of reverse transcriptase activity when preincubated with template. This inactivation was not reversed by excess dTTP. The decrease in template-primer activity did not occur with nPrearaUTP, but was shown with the chain-terminating 5′-triphosphates of 3′-fluoro- and 3′-azidothymidine. As nPredUTP, but not nPrearaUTP, was an alternative substrate, shown by the ability to support DNA synthesis in absence of competing substrate, the incorporation of nPredUTP into the primer-template apparently leads to increased inhibition of the enzyme.

Analogs of 3'-amino-3'-deoxyadenosine inhibit HIV-1 replication.

Several different nucleoside analogs have been demonstrated to inhibit retroviral RNA-dependent DNA polymerase activity in preference to cellular DNA-dependent DNA polymerases. 3'-Amino derivatives of 3-deoxyadenosine was analyzed for their antiviral activity toward HIV-1 and for their host cell toxicity. Puromycin aminonucleoside (PANS), PANS 5'-monophosphate, and 3'-amino-3'-deoxyadenosine triphosphate all inhibited HIV-1 replication in acutely infected cells. No significant antiviral effects of PANS were demonstrated in chronically infected cells. The effect of PANS was demonstrated at an early step in HIV-1 replication, most likely reverse transcription. 3'-Aminonucleoside analogs are a novel class of inhibitors of HIV-1 replication that require further analysis in cell culture and animal studies.

Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.

Phosphorylation of 3'-azido-2',3'-dideoxyuridine and preferential inhibition of human and simian immunodeficiency virus reverse transcriptases by its 5'-triphosphate

3'-Azido-2',3'-dideoxyuridine-5'-triphosphate was found to be a potent and highly selective inhibitor of human immunodeficiency virus type 1 and simian immunodeficiency virus reverse transcriptases. The affinity of 3'-azido-2',3'-dideoxyuridine-5'-triphosphate for the reverse transcriptases was similar to that observed for 3'-azido-3'-deoxythymidine-5'-triphosphate. Both compounds were competitive inhibitors with respect to the normal substrate dTTP and served at least 100 times better as substrates than did dTTP. In contrast, cellular DNA polymerase alpha showed an about 60-times-higher preference for dTTP as substrate than for either inhibitor. The phosphorylation of thymidine in human peripheral blood mononuclear cell extracts was inhibited in a competitive manner by both 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine, with apparent inhibition constants of 290 and 3.4 microM, respectively. The Michaelis-Menten constant, Km, for thymidine was 7.0 microM. 3'-Azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine both served as substrates, with apparent Km values of 67 and 1.4 microM, respectively. The maximal rates of phosphorylation with 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine were 40 and 30%, respectively, of the rate with thymidine. The different affinities of 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine for the thymidine kinase and the Km values observed with these compounds as substrates may explain the difference in effects on human immunodeficiency virus type 1 replication in infected peripheral blood mononuclear cells observed when equimolar concentrations of the two compounds are compared.

Design of some nucleic acid antimetabolites: expectations and reality.

3'-Azido-2'3'-dideoxythymidine (AZT), a thymidine analogue with potent antiretroviral activity against human immunodeficiency virus (HIV) in vitro, has been shown to confer a clinical benefit in patients with advanced acquired immune deficiency syndrome (AIDS). Other 2',3'-dideoxynucleosides, e.g., 2',3'-dideoxy-cytidine and 2',3'-dideoxyadenosine, block the infectivity of HIV against helper/inducer T cells in vitro and protect the cell against the cytopathic effect of the virus. The majority of these antiretroviral agents were synthesized more than twenty years ago as analogues of physiologically important deoxynucleosides in the quest of a more effective cancer chemotherapy. None of the synthetic analogues manifested significant activity when screened against L1210 leukemia in BDF mice despite the fact that the phosphorylation reactions, crucial to the activation of the 2',3'-dideoxynucleosides, are catalyzed by kinases of appropriate target cells. However, the 2',3'-dideoxynucleoside triphosphates, relative to corresponding deoxynucleotides, have a reduced affinity for DNA polymerase alpha, an enzyme that has key DNA synthetic and repair functions in the life of a cell. In contrast, HIV reverse transcriptase, like host cellular DNA polymerase beta (a repair enzyme) and gamma (a mitochondrial enzyme), is much more susceptible to the inhibitory effects of the dideoxynucleotides. This would explain the activity of the fradulent nucleotides at low concentrations against pathogenic retroviruses vis à vis the low cytoxic activity observed with these agents as anti-leukemia drugs.

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Fidelity of Two Retroviral Reverse Transcriptases during DNA-Dependent DNA Synthesis In Vitro

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases.

The inhibition of human immunodeficiency virus (HIV) reverse transcriptase by certain antibiotics and related compounds was studied in comparison with that of avian myeloblastosis virus (AMV) reverse transcriptase and cellular DNA polymerases alpha and beta. In general, compounds that inhibited HIV reverse transcriptase also inhibited AMV reverse transcriptase. For example, 10 micrograms/ml of the isoquinoline quinones used in this study inhibited approximately 80% of the activity of reverse transcriptases of HIV and AMV, but did not inhibit the activity of DNA polymerases alpha and beta even at 50 micrograms/ml. AMV enzyme was more sensitive than HIV enzyme to colistin, enduracidins A and B, janiemycin, glysperin A, and thielavins A and B. The streptonigrin alkyl esters, however, inhibited HIV reverse transcriptase only. Sakyomicin A, luzopeptins, ellagic acid and suramine inhibited the activities of reverse transcriptases and cellular DNA polymerases.

Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid

The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide‐synthesizing enzymes were examined. Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases α, β, γ, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase. ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase α. Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template · primer for the same binding site of the enzyme. On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template · primers or nucleoside 5′ ‐triphosphate substrates. The inhibition constants for ATA were, in general, smaller than those for Evans blue.

Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid

The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide-synthesizing enzymes were examined. Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases alpha, beta, gamma, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase. ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase alpha. Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template.primer for the same binding site of the enzyme. On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template.primers or nucleoside 5'-triphosphate substrates. The inhibition constants for ATA were, in general, smaller than those for Evans blue.

The Role of Low Intracellular or Extracellular pH in Sensitization to Hyperthermic Radiosensitization

Previous work showed that intracellular pH (pHi) and not extracellular pH (pHe) was the determinant in the low pH sensitization of hyperthermic killing. The present studies show that the same is true for heat-induced radiosensitization and loss of cellular DNA polymerase activities. Chinese hamster ovary cells after they had adapted to low pH (6.7) had an increase in pHi which rendered cells partially resistant to the low pH sensitization of heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities. These results were quantified by plotting versus pHe, both the thermal enhancement ratio (TER), defined as the ratio of the X-ray dose without heat to the X-ray dose with heat to give an isosurvival value of 0.01, and the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to the D0 of the radiation survival curve for heat plus radiation. Both the TER and TEF were higher for the unadapted cells than for the adapted cells, i.e., 1.3-1.4 fold higher at a pHe of 6.3. However, the TER or TEF plotted versus pHi was identical for the two cell types. Finally, heat-induced loss of cellular DNA polymerase activities correlated with pHi and not pHe. Therefore, we conclude that pHi and not pHe is responsible for the increase by acid in heat-induced radio-sensitization and loss of cellular DNA polymerase activities.

Inhibition of varicella-zoster virus-induced DNA polymerase by a new guanosine analog, 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine triphosphate.

The triphosphates of the antiherpesvirus acyclic guanosine analogs 9-[4-hydroxy-2(hydroxymethyl)butyl] guanine (2HM-HBG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]), and 9-(3,4-dihydroxybutyl)guanine (buciclovir) were examined for their effects on partially purified varicella-zoster virus (VZV) DNA polymerase as well as cellular DNA polymerase alpha. The triphosphate of 2HM-HBG competitively inhibited the incorporation of dGMP into DNA catalyzed by the VZV DNA polymerase. 2HM-HBG-triphosphate (2HM-HBG-TP) had a higher affinity for the dGTP-binding site on the VZV DNA polymerase than did dGTP; apparent Km and Ki values of dGTP and 2HM-HBG-TP were 0.64 and 0.034 microM, respectively. ACV-triphosphate (ACV-TP) was found to be the most potent inhibitor of VZV DNA polymerase. ACV-TP had a 14 and 464 times better direct inhibitory effect than 2HM-HBG-TP and buciclovir-triphosphate, respectively. The cellular (human embryonic lung fibroblast) DNA polymerase alpha inhibition was related to viral polymerase inhibition as efficacy ratios: 2HM-HBG-TP had a ratio of more than 1,000, which appeared to be similar to that of ACV-TP.

Antiviral activity and mechanism of action of ganciclovir.

Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) is a potent inhibitor of viruses of the herpes family, including cytomegalovirus (CMV), that are pathogenic for humans and animals. The primary mechanism of ganciclovir action against CMV is inhibition of the replication of viral DNA by ganciclovir-5'-triphosphate (ganciclovir-TP). This inhibition includes a selective and potent inhibition of the viral DNA polymerase. Ganciclovir is metabolized to the triphosphate form by primarily three cellular enzymes: (1) a deoxyguanosine kinase induced by CMV-infected cells; (2) guanylate kinase; and (3) phosphoglycerate kinase. Other nucleotide-metabolizing enzymes may be involved as well. The selective antiviral response associated with ganciclovir treatment is achieved because of the much weaker inhibition of cellular DNA polymerases by ganciclovir-TP. Activity and selectivity are also amplified by the accumulation of ganciclovir-TP in CMV-infected cells.

Dual biological activity of apurinic acid on human lymphocytes: induction of interferon-γ and protection from human immunodeficiency virus infection in vitro

The chemically modified DNA, apurinic acid (APA), is cytotoxic for human lymphocytes at concentrations above 100 μg/ml. At low concentrations (0.05–1 μg/ml) APA acts as an inducer of interferon γ (IFN-γ) in lymphocytes in vitro; the maximum interferon titer of 50 units/ml was reached at 0.4 μg/ml. When added to the cells in combination with phytohemagglutinin A (PHA), APA displays a significant synergistic interferon-inducing ability; the maximum titer of 940 units/ml was obtained with 10 μg/ml of APA and 6.25 μg/ml of PHA. APA also proved to be an effective inhibitor of human immunodeficiency virus (HIV-1) replication in H9 cells. At a concentration of 10 μg/ml, APA causes a 49% inhibition of virus growth, while 20 μg/ml of APA are required to inhibit expression of HIV-1 p17 and p24 gag proteins by 60%. The mechanism of anti HIV-1 activity of APA likely occurs at the level of viral reverse transcriptase. This enzyme is inhibited by APA in a noncompetitive way with a K i of 0.39 μM, while the cellular DNA polymerases α, β and γ are 140- to 300-fold less sensitive to APA.

Inhibition of HIV-1 RNA-dependent DNA polymerase and cellular DNA polymerases α, β and γ by phosphonoformic acid and other drugs

Potential specific inhibitors of HIV RNA-dependent DNA polymerase (RDDP) were examined in an in-vitro test system containing purified HIV-1 RDDP, or functionally purified cellular DNA-dependent DNA polymerases alpha, beta and gamma. A wide variety of drugs were tested for their specific inhibitory activity against the viral enzyme to identify substances which, at the same concentration, did not inhibit the cellular DNA polymerases. Phosphonoformic acid and derivatives were found to be the most specific inhibitors, followed by chlortetracycline.

Effect of Cycloheximide on Heat-Induced Cell Killing, Radiosensitization, and Loss of Cellular DNA Polymerase Activities in Chinese Hamster Ovary Cells

A whole-cell assay technique for DNA polymerase alpha and beta was used to measure the activities of both enzymes in Chinese hamster ovary cells after hyperthermic treatment at 43 degrees C in the presence or absence of 10 micrograms/ml cycloheximide (CHM). In the same experiments, the effect of CHM on heat killing and heat radiosensitization was also investigated. CHM treatment before and during heating protected the cells for all three end points, i.e., heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities.

Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5′-triphosphate in comparison to cellular DNA polymerases α and β

The sugar-modified dTTP analogues 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-triphosphate (ddeTTP), 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP), 3′-fluorothymidine 5′-triphosphate (FdTTP), and 3′-azidothymidine 5′-triphosphate (N 3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID so values of the compounds for the HIV-RT (ranging from 0.03 μM for ddeTTP to 0.1 μM for ddTTP) and the cellular DNA polymerase α (> 200 μM). DNA polymerase β is partially affected by N 3dTTP (ID so=31 μM) and by the other analogues (ID so=1–2.2 μM). FdTTP has proved as effective as N 3dTTP (ID so=0.05 μM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K 1 values (about 0.05 μM).

Reverse transcriptase activity in post-transfusion non-A, non-B hepatitis: I. Characterisation and association with retrovirus-like particles in serum

Reverse transcriptase activity was detected in the serum of 16 out of 23 patients with post-transfusion non-A, non-B hepatitis, which was absent before transfusion. Density gradient analysis of positive sera revealed a single peak of reverse transcriptase (RT) activity at 1·15 g ml −1 and 70–90 nm particles which resembled retroviruses were seen in the positive fraction by electron microscopy. Particle-associated RT activity was shown to be dependent upon presence of all four deoxynucleotide triphosphates and was inhibited by pre-treatment with RNase. The product of the endogenous reaction was sensitive to DNase but not to RNase. Actinomycin D partially reduced incorporation of 3H-dTTP. Addition of poly(rA) oligo(dT) strongly enhanced the reaction whereas the enzyme was inactive with poly(dA) oligo(dT). These results indicate that the enzyme detected is a reverse transcriptase with distinct properties from cellular DNA polymerase. Analysis by activity gel assay further confirmed this conclusion, showing co-migration in the gel of RT activity in non-A, non-B hepatitis sera at the same level of purified RT from Rous-associated virus-2. These data strongly indicate the presence of retrovirus-like particles in the sera of patients with post-transfusion non-A, non-B hepatitis.

Specific immune serum to the Epstein-Barr virus DNA polymerase.

Epstein-Barr virus (EBV) DNA polymerase was released from phorbol ester-treated tamarin (Saguinus oedipus) cells (B95-8) and prepared for use as an antigen by sequential column chromatography with DEAE-Sephadex A-25, DEAE-cellulose, phosphocellulose, and single-stranded DNA cellulose. Proteins from single-stranded DNA cellulose with DNA polymerase activity in 100 mM ammonium sulfate were mixed with complete Freund adjuvant and injected intradermally into rats and rabbits. Immune sera that were screened for specific antibody by indirect immunofluorescence procedures reacted with approximately 3% of the cells in EBV-producer cultures (B95-8 and P3HR-1) but not with EBV genome-negative cells (BJAB). In functional enzyme assays, immune sera or the immunoglobulin fraction inhibited the activity of purified EBV DNA polymerase 90%. Inhibition of enzyme activity was not affected by absorption of immune sera with insoluble matrices of proteins prepared with tamarin and human cells which lacked the EBV genome. Cellular DNA polymerase alpha was not inhibited by immune sera to the EBV enzyme.

Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.