RNA-dependent DNA Polymerase Activity Research Articles

Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme

A system for the expression of recombinant human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in Escherichia coli has been developed, which allows purification of the heterodimeric form of the enzyme as well as the separate purification of the two subunits. It is shown that equilibrium formation between monomeric and homodimeric forms of the recombinant 66- and 51-kDa subunits is considerably more rapid than in the case of the corresponding homodimeric forms of HIV-1 RT. In accordance with our previously published studies on HIV-1 RT (Restle, T., Müller, B., and Goody, R.S. (1990) J. Biol. Chem. 265, 8986-8988) RNA-dependent DNA polymerase activity of the HIV-2 RT preparations can be exactly correlated to their dimer content. No significant heterodimer formation can be observed upon coexpression of the 66-kDa subunit of HIV-2 RT with the 51-kDa subunit of HIV-1 RT in the same cell, indicating differences in the dimerization domains of the two proteins. Recombinant HIV-2 RT is not recognized by a set of 23 monoclonal antibodies raised against HIV-1 RT, although it shows weak cross-reactivity with sera from HIV-1-infected patients.

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.

An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed by tetrahydroimidazo-[4,5,1-jk] [1,4]benzodiazepin-2 (1H)-one and -thione derivatives.

Screening of pharmacologically acceptable prototype compounds has recently led to the discovery of a series of ultraselective inhibitors of human immunodeficiency virus (HIV)-1 replication, the tetrahydroimidazo[4,5,1-jk] [1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives. The TIBO compounds completely suppress the formation of proviral DNA in acutely infected cells, as revealed by polymerase chain reaction (PCR) analysis. TIBO derivatives are inhibitory to the reverse transcriptase (RT) of HIV-1 but not that of HIV-2 or other retroviruses. The inhibition is most effective with poly(C)-oligo(dG) as the template/primer, and it is selectively directed against the RNA-dependent DNA polymerase activity and not the accompanying DNA-dependent DNA polymerase and ribonuclease H activity of HIV-1 RT. Kinetic studies point to an uncompetitive inhibition with regard to the template/primer. TIBO compounds are active against HIV-1 replication through a unique interaction with HIV-1 RT. The experimental data indicate the existence of a target on HIV-1 RT that is responsible for the inhibition of replication and a mode of action unrelated to that of previously studied RT inhibitors.

Role of Mg ++ in the mithramycin-DNA interaction: Evidence for two types of mithramycin-Mg ++ complex

Mithramycin(MTR, structure shown in Figure 1) [and the related compound Chromomycin A3(CHRA3)] are antitumor antibiotics which inhibit DNA dependent RNA polymerase activity via reversible interaction with DNA only in the presence of divalent metal ion such as Mg++. In order to understand the role of Mg++ in MTR-DNA interaction, absorbance and CD spectroscopic techniques are employed to study the binding of MTR to Mg++. These studies show: i) the drug alone binds to Mg++ and ii) two different types of drug-Mg++ complexes are formed at low(Complex I) and high(Complex II) ratios of the concentration of Mg++ and MTR. We propose that these two complexes would bind to the same DNA with different affinities and rates. This result suggests that the relative concentration of Mg++ is an important factor to be taken into account to understand the molecular basis of MTR-DNA interaction.

Structure‐Function Studies of HIV Reverse Transcriptasea

The retroviral RT is properly under intensive study as the major target of antiviral therapy. The enzyme exhibits a number of features that make it an attractive target: it is crucial for viral replication; its RNA-dependent DNA polymerase activity is probably unique to viral replication, or if not unique, is generally unimportant in host cell function; its activities are readily monitored; and powerful lead compounds in the form of nucleotide analogues are already in hand. Our laboratory has been involved in studies to elucidate the structure and function of the HIV-1 RT and to develop a formal genetics of the enzyme. Working with constructs expressing RT in bacteria, we been able to use in vitro mutagenesis to localize functions on the molecule; by coupling mutagenesis with high-throughput screening of colonies, we have been able to isolate mutants with specific, rare, phenotypes. We believe that extensions of these efforts will help us to understand the functions of the protein and, coupled to a detailed three-dimensional structure, should facilitate the development of new and better inhibitors.

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

We studied the effect of the natural marine substance illimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 microgram/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6'-hydroxyl group at the ortho position to one of carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity.

Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition.

A new antileishmanial compound, phaseolinone

Inclusion of phaseolinone, a newly described mycotoxin, at 20 μg per ml in a solid culture medium (blood agar overlay) and at 50 μg per ml in a liquid culture (medium 199) inhibited the growth of L . donovani promastigotes. About 90% of the motile promastigotes lost motility after exposure to 50 μg per ml of phaseolinone for 6–7 h and here 3-day-old culture was more sensitive than 7-day-old culture. In an in vitro assay, DNA dependent RNA polymerase activity of 3-day-old promastigotes was considerably inhibited in the presence of this toxin. Therefore, this key enzyme was suggested to be one of the sites of action of phaseolinone.

Cibacron Blue inhibition of prokaryotic and eukaryotic DNA-dependent RNA polymerases

A comparison was drawn between the action of Cibacron Blue F3GA on the enzymic activity of DNA-dependent RNA polymerases from different sources, e.g. Escherichia coli, calf thymus and wheat germ (polymerase II). Sensitivity towards this inhibitor was determined for polymer formation and primed abortive synthesis of trinucleotide UpApU. In case of E. coli polymerase and wheat germ polymerase II the dye inhibits both polymer formation and abortive synthesis. Calf thymus polymerase II is inhibited only in the polymerisation step. The primed initiation reaction was found to be resistant towards the dye. In case of E. coli polymerase and wheat germ polymerase II the sensitive step is the formation of internucleotide bond whereas in case of calf thymus polymerase II the translocation of the enzyme is influenced. An analysis of kinetic data indicates more than one binding site for the dye on RNA polymerase II from calf thymus and wheat germ. Cibacron blue does not inhibit specific transcription catalyzed by RNA polymerase III from human HeLa cells and mouse leukemia L1210 cells.

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.

Further Evidence that Viroplasms Are the Site of Cauliflower Mosaic Virus Genome Replication by Reverse Transcription during Viral Infection

SUMMARY Cauliflower mosaic virus (CaMV) is a DNA plant virus that replicates its genome through an RNA intermediate. The cytoplasmic step of CaMV DNA replication was studied using a fraction consisting of purified viroplasms, which are virus-specific inclusion bodies accumulating in the infected plant cells. The isolated viroplasms retain a DNA polymerase activity able to synthesize CaMV DNA from endogenous templates. A further characterization of the viral DNA sequences produced in the isolated inclusion bodies indicates that newly synthesized DNA, mostly of polarity opposite to that of viral RNA, is single-stranded and partly associated with RNA by base-pairing. In addition to an RNA-dependent DNA polymerase activity, RNA molecules, which presumably originate from the viral RNA template used for reverse transcription, are found to accumulate in the purified inclusion bodies. Furthermore, a small DNA molecule strongly labelled in the purified fraction has been characterized and corresponds to the CaMV reverse transcription intermediate sa-DNA. These results provide further evidence that the reverse transcription of CaMV RNA occurs in the viroplasms. Additional data are presented which suggest that CaMV replication could occur in virion-related particles.

Intrinsic properties of reverse transcriptase in reverse transcription : Associated RNase H is essentially regarded as an endonuclease

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3′-end of the RNA as a model substrate (see Fig. 1). With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3′ termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5′-triphosphorylated termini, 3′-OH, 3′-phosphate, 3′-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.

A Novel in situ Colony Screening Method to Detect Human Immunodeficiency Virus Reverse Transcriptase Activity Expressed in Bacteria: Isolation of Pseudorevertants of Reverse Transcriptase Mutants

An in situ screening procedure is described that permits the detection of RNA-dependent DNA polymerase activity in bacterial colonies expressing the reverse transcriptase of the human immunodeficiency virus. The procedure has been applied to the isolation of rare pseudorevertants of an inactive parental mutant.

Inhibition of moloney murine leukemia virus reverse transcriptase by α-anomeric oligonucleotides

After parallel hybridization to complementary template RNA, α-anomeric oligonucleotides are not primers for Moloney murine leukemia virus reverse transcriptase. As can be expected they are competitors of classical primer oligonucleotides (β-anomeric). They therefore inhibit the RNA dependent DNA polymerase activity of Moloney murine leukemia virus reverse transcriptase with either homopolymeric or heteropolymeric substrates. Non complementary α-oligonucleotides display no inhibitory activity. α-Oligonucleotides are therefore potentiel candidates for inhibition of retroviral reverse transcriptases by interference with the primer binding sites.

Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase

We have described a strain of Escherichia coli that expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes, Proc. Natl. Acad. Sci. USA, 85, 1218–1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced into E. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent DNA polymerase activity.

Absence of significant RNA-dependent DNA polymerase activity in lymphocytes from patients with Kawasaki syndrome

Kawasaki syndrome, an acute febrile multisystem illness of young children, is a panvasculitis with prominent rheumatic features. Arthritis and pancarditis are frequent during the acute stage; coronary artery aneurysms occur in 20% of cases and the disease is now the leading cause of acquired heart disease in childhood. A microbial aetiology is suggested by the acute febrile self-limited character of the disease, the regular occurrence of epidemic outbreaks at intervals of 2-3 years, and the virtual restriction to young children, consistent with the early acquisition of immunity. Reports of elevated DNA polymerase activity (assumed to be RNA-dependent reverse transcriptase) in cultured lymphocytes from patients with acute Kawasaki syndrome suggest that a retrovirus might be the causative agent. We have measured supernatant DNA polymerase activity in lymphocyte cultures from 49 Hawaiian patients in acute and convalescent stages of Kawasaki syndrome and have been unable to demonstrate significant reverse transcriptase activity or other evidence of involvement of a retrovirus in the aetiology of the disease.

Inhibition of human hepatitis B virus DNA polymerase and duck hepatitis B virus DNA polymerase by triphosphates of thymidine analogs and pharmacokinetic properties of the corresponding nucleosides.

Replication of hepadnaviruses involves a viral DNA polymerase containing both a DNA-dependent and an RNA dependent activity. This polymerase is a potential target for chemotherapy against hepatitis B. We have used human hepatitis B virus DNA-dependent DNA polymerase from human serum and duck hepatitis B virus DNA-dependent DNA polymerase from duck serum as well as RNA-dependent DNA polymerase activity from duck hepatitis B-infected duck liver. Triphosphates of thymidine analogs have been synthesized and tested for their inhibitory activities against these enzymes with the intention both to explore differences between these enzymes and structural requirements for inhibitors. The results showed that with the inhibitors tested, hepatitis B virus DNA-dependent DNA polymerase was the most sensitive enzyme and the triphosphate of 5-propenyl-2'-deoxyuridine was the most active inhibitor. In addition, the 5'-triphosphate of 5-propenyl-arabinofuranosyluracil also inhibited the hepadnavirus DNA-dependent DNA polymerases, and was a competitive inhibitor with respect to 2'-deoxythymidine triphosphate as showed by kinetic studies with duck hepatitis B virus DNA-dependent DNA polymerase from serum. Pharmacokinetic analysis showed 5-propenyl-2'-deoxyuridine to be well absorbed orally, but rapidly cleared from plasma. The arabinofuranosyl analog was also well absorbed but cleared less rapidly. Hence, these results indicate the potential of 5-propenyl-2'-deoxyuridine and 5-propenyl-arabinofuransyluracil for chemotherapy of hepatitis B.

α-Anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Nuclease-resistant α-anomeric DNA: β-rna hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A) 12: d[G-G-(T) 12-G-G], or by acid-solubility techniques, using a long substrate, poly(A): poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA: RNA duplexes are the natural substrates, binds to non-physiological α-DNA: RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded α-DNA itself. Unexpectedly, polymerase activity is highly stimulated by α-oligos, analogous in their sequence to the β primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the α: β may occur at different levels.

Specific role of manganese and magnesium on RNA synthesis in rabbit bone marrow erythroid cell nuclei

Specific roles of manganese (Mn) and magnesium (Mg) on the activities of DNA-dependent RNA polymerases I and II isolated from rabbit bone marrow erythroid cell nuclei were investigated. Three main polymerases were separated from the cell nuclei. When RNA polymerase I and Mg were added to the RNA synthesis assay mixture containing erythroid cell DNA as template, RNA transcription activity was highest, but when Mg was replaced with Mn, denatured calf thymus DNA formed a better template than erythroid cell DNA. In contrast, nucleoplasmic DNA from erythroid cell and liver DNA were the best templates to stimulate RNA transcription when RNA polymerase II and Mn were added to the assay mixture. However, if Mn was replaced with Mg, RNA synthesis activity was drastically reduced when the template was nucleoplasmic DNA of erythroid cell. RNA polymerase I and Mg synthesized GC rich RNA, whereas RNA polymerase II and Mn synthesized AU rich RNA. Sedimentation analysis showed that the molecular weights of the RNA produced by polymerase I were larger when the enzyme was activated with Mg than with Mn, whereas those of the RNA produced by polymerase II were larger with Mn than with Mg. Furthermore, RNA produced by polymerase I and Mg using chromatin as a template hybridized better with nucleolar DNA than with nucleoplasmic DNA, whereas that produced by polymerase II and Mn hybridized better with nucleoplasmic DNA than with nucleolar DNA. These results suggest that RNA synthesis is dependent on the activity of specific RNA polymerases and the presence of specific divalent cations and templates, and that the cofactor and template for RNA polymerase I are, respectively, Mg and the nucleolar DNA of cell nuclei, whereas those for RNA polymerase II are Mn and nucleoplasmic DNA.