RNA-directed DNA Polymerase Activity Research Articles

Evidence for Copurification of HERV-K–Related Transcripts and a Reverse Transcriptase Activity in Human Platelets From Patients With Essential Thrombocythemia

AbstractWe have previously reported that particles resembling retroviral particles and possessing an RNA-directed DNA polymerase activity can be prepared from platelets. Furthermore, we and others have shown that these particles are present at higher levels in patients with essential thrombocythemia and polycythemia vera. We show here that these particles package RNA molecules that encode HERV-K–related pol genes. A subset of the RNA molecules that are packaged are likely to encode the RNA directed DNA polymerase activity and, because these RNAs possess long/full-length open reading frames for the reverse transcriptase and RNaseH (also for part of the integrase domains in genomic clones) of HERV-K, we propose that these transcripts are indeed strong candidates for encoding the enzyme activity found in these particles. Moreover, by using a modification of the polymerase chain reaction-based reverse transcriptase assay in which activated DNA is added during cDNA synthesis to suppress DNA polymerase-mediated RNA-directed DNA synthesis, we have found that the particle-associated enzyme behaves like a retroviral reverse transcriptase, further supporting the conclusion that retrovirus-like, perhaps HERV-K sequences, encode this enzyme activity.

Novel C-organosilicon derivatives as leads for reverse-transcriptase-mediated anti-HIV-1 activity

Summary In order to prepare novel antiretroviral lead compounds active against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), a family of C-silylalkylated heterocyclic derivatives were synthesized. One of these derivatives 3 inhibited HIV-1 replication in cell culture (IC 50 = 12 ± 4 μM) without significant cytotoxic effects. This compound did not exhibit antiviral activity against herpes simplex virus type 1 and poliovirus type 1. Biochemical studies showed that 3 inhibited the DNA polymerase activity of a recombinant HIV-1 RT. This derivative was shown to be a non-competitive inhibitor with respect to dNTP substrate incorporation. Different levels of inhibition were obtained depending on the template-primer used. Compound 3 did not inhibit either the RNase H activity of HIV-1 RT, or the RNA-directed DNA polymerase activity of HIV-2 RT.

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor.

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template: primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.

Lack of synergy in the inhibition of HIV-1 reverse transcriptase by combinations of the 5′-triphosphates of various anti-HIV nucleoside analogs

3′-Deoxy-3′-azidothymidine (AZT) has been shown to synergistically inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in cell culture when combined with several other 2′,3′-dideoxynucleoside analogs. In an effort to understand the biochemical mechanism of this synergy, we have examined the effect of combinations of the 5′-triphosphate of AZT (AZT-TP) with either ddCTP, ddATP, or the 5′-triphosphate of the carbocyclic analog of 2′,3′didehydro-2′,3′-dideoxyguanosine (carbovir) on both the RNA-directed and DNA-directed DNA polymerase activity of HIV-1 reverse transcriptase. Kinetic studies, which evaluated the ability of these combinations to competitively inhibit the enzyme, showed that AZT-TP could not bind to the enzyme with either the RNA or DNA template at the same time as either of the other three inhibitors. None of these analogs could affect the incorporation of another analog into the DNA chain by the HIV-1 reverse transcriptase. These results indicated that synergistic inhibition of the HIV-1 reverse transcriptase is not responsible for the synergistic antiviral activity seen in cell culture with combinations of these nucleoside analogs.

Kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-88204E.

The bis(heteroaryl)piperazine U-88204E is a potent inhibitor of HIV-1 reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the RNA- and DNA-dependent DNA polymerases of RT were carried out in order to determine whether the inhibitor interacts directly with the template:primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The experimental results were analyzed using steady-state or Briggs-Haldane kinetics, by assuming that the template:primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The results of the analysis show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template:primer and the dNTP binding sites. The potency of U-88204E on the RNA-directed DNA polymerase activity depends on the base composition of the template:primer. The Ki values for the poly(rC):(dG)10-directed reactions were at least 7 times lower than the ones for reactions directed by poly(rA):(dT)10. The inhibitor did not inhibit the RNase H function of HIV-1 RT nor did it impair the RNA-directed DNA polymerase activity of HIV-2 RT. These data thus demonstrate the unique specificity of U-88204E for HIV-1 RT.

The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase

The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.

DETECTION OF RETROVIRUS IN PATIENTS WITH MYELOPROLIFERATIVE DISEASE

RNA-directed DNA polymerase (reverse transcriptase) activity was detected in platelets from 4/4 patients with primary proliferative polycythaemia (PPP), 7/7 patients with essential thrombocythaemia (ET), 1/4 patients with relative "stress" polycythaemia, 0/2 patients with secondary polycythaemia, and 0/3 normal subjects. The activity appeared to be particle-associated and was detected under conditions not appropriate for any known cellular enzymes. Active particulate fractions from 1 patient with PPP and 1 patient with ET were examined by electron microscopy and revealed objects with the features of retroviruses. Similar retrovirus-like particles were observed in 2/2 further patients with PPP and 2/2 further patients with ET but in 0/3 controls.

RNA-directed DNA polymerase activity in human breast cancer biopsy specimens. Relation to estrogen receptor protein.

The expression of a mouse mammary tumor virus is inducible by hormones, and the virus contains a hormone-responsive element. Viral particles and RNA-directed DNA polymerase (RDDP, EC 2.7.7.7; reverse transcriptase) are both detectable in human breast tumors but the frequency and significance of these findings are unknown. Breast tumor biopsy specimens (from either the primary site or a metastasis), frozen in liquid nitrogen at the time of surgery, were routinely obtained to determine estrogen receptor (ERP) concentration. A sample of the tissue was pulverized, homogenized and centrifuged at low speed to remove nuclei and mitochondria. The supernate was then centrifuged at 225,000 g to obtain the cytosol fraction for estrogen and progestin receptor (PgR) assays. Partially purified membranes for the RDDP assays were prepared from the high-speed pellet by discontinuous sucrose density gradient centrifugation. The RDDP assay involved measuring primer-dependent poly(dT) synthesis in the presence of poly(A) as template and oligo-(dT)12-18 as primer. To date, we have studied biopsy specimens from 46 patients with breast cancer. 27 (59%) had ERP and 23 (50%) were RDDP-positive. There was no significant correlation between ERP concentration and RDDP activity. PgR data were available on 36 of the patients; 17 (47%) were positive. No correlation between RDDP and PgR was apparent. Similarly, there was no correlation between RDDP and clinical stage of the disease.

Characterization of virus-like particles from a psoriatic patient with respect to the possible presence of particle-associated RNA and RNA-directed DNA polymerase.

Subcellular particles resembling retroviruses with respect to their morphology, density, and protein composition, and which are present in the urine of a psoriatic patient have been analysed for the presence of high-molecular-weight polyadenylated RNA and RNA-directed DNA polymerase activity. Neither RNA-directed DNA polymerase activity nor high-molecular-weight polyadenylated RNA was detected in preparations containing the purified virus-like particles. This was the case even when the amount of particles analysed exceeded by a factor of 50 or more the amount of avian myeloblastosis virus in which both polymerase activity and RNA were detected.

Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid.

The synthesis of single-stranded globin cDNA by the RNA-directed DNA polymerase activity of reverse transcriptase in the presence of oligothymidylate primers was investigated in order to determine the limitations to higher yields. The results indicated that the associated ribonuclease H activity, an integral part of reverse transcriptase, plays a large role in the synthesis of the first strand of cDNA and that the interplay of the two enzyme activities for any specific set of conditions determines the yield of single-stranded products. In both the presence and the absence of polymerization, the associated ribonuclease H catalyzed the deadenylation of mRNA, producing molecules that were somewhat shorter, highly homogeneous in size, and fully translatable into globin protein. They were also entirely lacking in the ability to serve as templates for cDNA synthesis. The reaction was completely dependent on oligothymidylate and completely independent of deoxyribonucleoside triphosphates. The initial rate of deadenylation was one-fourth the initial rate of initiation of polymerization when saturating levels of deoxyribonucleoside triphosphates were used in the polymerase reaction. In the presence of ribonuclease H activity, the DNA polymerase catalyzed the synthesis of an array of cDNAs including some that were full length. The initiation of polymerization was rate limiting: once synthesis had begun, it required 1-1.5 min to transcribe globin mRNA. However, most primers that were elongated were aborted prematurely. Maximum synthesis of full-length cDNA required stoichiometric levels of enzyme and high triphosphate levels, but regardless of conditions, the sum of completed cDNA and deadenylated mRNA accounted for only 50% of the input mRNA. The data fit a model in which synthesis of full-length cDNA molecules depends on the arrangement of primers and transcription initiation complexes on the poly(A) "tail" of mRNA.

Appearance of reverse transcriptase activity in human fetal thymus cells stimulated by human B-cell.

We have investigated three human fetal thymuses by various induction procedures in a search for retrovirus. Thymus cells were cultured for 7 days together with mitomycin C-treated B cells and examined for reverse transcriptase activity. The following results were obtained. 1) RNA-directed DNA polymerase (RDDP) activity was detected in the culture medium on day 3 and persisted until day 7. This enzyme, after partial purification on phosphocellulose column, showed template-primer specificity of a typical reverse transcriptase. 2) In unstimulated thymus cells, no reverse transcriptase activity was detected. 3) Stimulation with lymphocyte mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A) exerted no effect on inducing the reverse transcriptase activity. 4) No reverse transcriptase activity was obtained in the culture medium of B cells pretreated with mitomycin C. Taken together these results indicate that in human fetal thymus cells reverse transcriptase activity is induced by human B cell stimulation, pointing to an intriguing possibility of protovirus activation.

Inhibition of murine leukaemia virus reverse transcriptase by 2-halogenated polyadenylic acids.

Several new analogues of polyadenylic acid [(A)n], i.e. poly(2-fluoroadenylic acid) [(fl2A)n], poly(2-chloroadenylic acid [(cl2A)n], poly(2-bromoadenylic acid) [(br2A)n] and poly(2-iodoadenylic acid) [(io2A)n] have been synthesized and evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukaemia virus. All (A)n analogues were found to be potent inhibitors of reverse transcriptase, the order of (decreasing) potency being (fl2A)n greater than (io2A)n greater than (br2A)n greater than (cl2A)n. For all four (A)n analogues the inhibition of reverse transcriptase was competitive with respect to the template-primer. (A)n . oligo(dT). The K1 values were 0.02 microgram/ml for (fl2A)n, 0.1 microgram/ml for (io2A)n, 0.5 microgram/ml for (br2A)n and 8 microgram/ml for (cl2A)n. With a Ki of 0.02 microgram/ml (approx. 0.04 microM), (fl2A)n can be considered as one of the most, if not the most, potent polynucleotide inhibitor of reverse transcriptase that has been described so far.

Intracellular ribonucleoprotein complexes of visna virus are infectious.

Sheep choroid plexus cells infected with visna virus produce intracytoplasmic viral ribonucleoprotein complexes with sedimentation values of 120S to 200S and buoyant densities of 1.29 to 1.32 g/cm3. These ribonucleoprotein complexes display an endogenous RNA-directed DNA polymerase activity and contain all of the species of RNA associated with polysomes. An analysis of the polypeptides present in the ribonucleoproteins allowed us to identify the mature internal virion core proteins and their precursor, Pr55gag, as well as the glycosylated envelope precursor gPr150env and small amounts of mature glycoprotein gp135. Ultracentrifugation-purified ribonucleoproteins could infect sheep choroid plexus cells and led to a normal lytic cycle with virus production. Our results suggest that visna virus can propagate by means of intracellular infectious particles.

Human placentas contain a specific inhibitor of RNA-directed DNA polymerase.

Human placental extracts contain a specific inhibitor of mammalian retroviral RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) activity. This inhibitor copurifies with retrovirus-like particles in human placental extracts. The inhibitor can be removed from these particles by salt extraction, which leads to the recovery of the polymerase activity. Thus, the inhibitor does not irreversibly inactivate the particle-associated RNA-directed DNA polymerase activity. The inhibitory preparation contained no nuclease, protease, or phosphatase activity. Because its inhibitory action can be eliminated by the addition of more virus to the reaction, nonspecific inactivation of enzyme substrate has been ruled out. A partial characterization of the inhibitor indicates that it is (i) insensitive to ether, trypsin, and phospholipase C; (ii) stable to heat and pH 2-12; and (iii) nondialyzable.

Intracisternal A-particles in genetically diabetic mice: Identification in pancreas and induction in cultured beta cells

Sucrose density gradient analysis of purified pancreatic homogenates from glycaemic C57BL/Ks diabetes (db/db) mice and their normoglycaemic controls have revealed the presence in the diabetics of increased Mg++-dependent RNA-directed DNA polymerase activity sedimenting with a density of approximately 1.21 g/cm3. Electron microscopy revealed that this fraction contained typical intracisternal A-particles. Purified cultures of pancreatic islet cells 4--7 day old postnatal "misty diabetic" mice and normal siblings were established and then maintained in Eagle's minimal essential medium without serum. Under these conditions, the presence of intracisternal A-particles in beta cells of both mutant and control genotypes was very rare. No change in numbers of intracisternal A-particles was seen after 2--4 days of incubation in Dulbecco's-modified minimal essential medium containing 5.5 mmol/l glucose. However, when the glucose concentration of Dulbecco's medium was elevated to 16.5 mmol/l, ultrastructural changes specific to the beta cell population occurred that were reminiscent of those alterations observed in situ. Intracisternal A-particles were commonly seen in cultured beta cells showing hypersecretion-stress morphology. Since equal numbers of intracisternal A-particles were present in cultured beta cells from normal and mutant mice, it was concluded that the db gene itself was not required for intracisternal A-particle expression. The cell culture results suggest that elevated intracisternal A-particle activity observed in vivo may be produced directly or indirectly by the ambient high blood glucose levels characteristic of this mutant.

Spontaneous Production of a C‐Type RNA Virus in a Cell Line Derived from Rat Glioma

The spontaneous production of a rat C-type RNA virus (ACV) in a cultured cell line (AC cells) established from a chemically induced rat glioma was studied. The characteristics of ACV were: morphology typical of C-type RNA virus; buoyant density of 1.15 g/ml in a sucrose density gradient; RNA directed DNA polymerase activity; viral core with a density of 1.28 to 1.30 g/ml; 70S RNA with dimer structure; and structural protein composed of mainly four polypeptides. Kinetical analysis of DNA-DNA hybridization revealed that DNA sequences homologous to DNA transcripts of RNA of ACV were present in rat cells. RNA directed DNA polymerase of ACV partially cross-reacted with antiserum to the polymerase of Rauscher murine leukemia virus. These data suggest that ACV is an endogenous C-type RNA virus of rat origin.

Normal human placentas contain RNA-directed DNA polymerase activity like that in viruses.

Extracts from over 100 normal human placentas have been examined for RNA-directed DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) activity. More than 80% of these placentas contained this enzyme activity, which banded at a density of 1.15-1.17 g/ml in sucrose. After heat treatment, this enzyme activity was shifted in density to 1.22-1.24 g/ml. The enzymatic activity was greater with (rC)n.(dG)12-18 than with (dC)n.(dG)12-18 and was not stimulated by (dG)12-18 alone. The product of the endogenous reaction, which was sensitive to RNase, had the characteristics of a small DNA associated with a large RNA by hydrogen bonding. Electron microscopic inspection of the material with a density of 1.15-1.17 g/ml revealed numerous retrovirus-like particles with central electron-dense cores and double-membraned envelopes. The enzyme may be associated with the retrovirus-lik particles noted in the trophoblast layer of some human placentas.

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.

Relationship between Nucleic Acids associated with Intracytoplasmic A Particles and Mouse Mammary Tumour Virus RNA

SUMMARY Intracytoplasmic A particles can be found in the cytoplasm of mammary tumours, certain leukaemias and Leydig cell tumours of the mouse. These intracytoplasmic A particles share antigenic determinants with the mouse mammary tumour virus (MTV) and are considered to be precursors of MTV B-type particles on the basis of morphological comparison. Both DNA and RNA have been reported to be associated with these intracytoplasmic A particles (Tanaka, Tamura & Tsujimura, 1972; Smith, Litwach & Longfellow, 1974). In this study it is shown that (1) the RNA of intracytoplasmic A particles is homologous to the RNA of MTV; (2) the DNA of the intracytoplasmic A particles is not a replicative form of the MTV genome, because no hybridization was observed between this intracytoplasmic A particle DNA and the radioactively labelled RNA or cDNA of MTV; (3) the intracytoplasmic A particles also contain an RNA-directed DNA polymerase activity with a cation preference which is similar to that of the reverse transcriptase of the MTV in a poly(rC).oligo(dG)-directed DNA polymerase assay.

Template specificity of rat mitochondrial DNA polymerase

Mitochondrial DNA polymerase was purified 2300-fold over isolated mitochondria from rat liver. Template-primer specficities of this enzyme were investigated. Activated DNA was satisfactorily used as an active template-primer, but both native and denatured DNAs showed a slight activity. Synthetic polynucleotide, poly(dA) · oligo(dT) 10 was found to have a high efficiency under the same condition for activated DNA. When the closed-circular, nicked and gapped ColE1 DNAs were employed as a template-primer, the enzyme could only utilize the gapped DNA, indicating that the displacement synthesis was not catalyzed by the enzyme itself. The enzyme also copied poly(A) · oligo(dT) 10 in high efficiency at pH 7.5 in the presence of MnCl 2. Such RNA-directed DNA polymerase activity of the enzyme was further characterized. Co-fractionated endonuclease activity was completely separated from the enzyme by glycerol gradient centrifugation.