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PubMed1995

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids.

The binding of HIV reverse transcriptase (RT) to heteroduplexes was examined using a substrate consisting of a 42 nt chimeric nucleic acid composed. (5'-->3') of 23 nt of RNA and 19 of DNA. This chimera was hybridized to an internal region of a relatively long complementary DNA or RNA. When the chimera was bound to DNA and conditions limiting cleavage to a single binding event between the enzyme and substrate were employed initial RNase H-directed cleavages occurred 19-21 nt from the chimera 5'-terminus. A 42 nt strand identical in sequence to the chimera and composed of only RNA was cleaved at the same locations. Reducing the length of the DNA portion of the chimera from 19 to 7 nt did not alter the cleavage positions, suggesting that cleavage was not coordinated by the DNA 3'-terminus. Under the same conditions cleavage was not detected when the chimera was bound to RNA. In contrast, addition of dNTPs to the DNA 3'-terminus of the chimera occurred only when the chimera was bound to RNA. The results support preferable binding of RT to RNA-DNA versus DNA-DNA hybrid regions and a model in which the orientation of binding to heteroduplexes is 5'-->3' (relative to the RNA strand), polymerase to RNase H active site, with sites associated with the DNA and RNA strand respectively.

Base Sequence↗

PubMed1996

Strand transfer is enhanced by mismatched nucleotides at the 3' primer terminus: a possible link between HIV reverse transcriptase fidelity and recombination.

Strand transfer catalyzed by HIV reverse transcriptase (RT) was examined. The system consisted of a 142 nt RNA (donor) to which a 50 nt DNA primer was hybridized. The primer bound such that its 3' terminal nucleotide hybridized to the 12th nt from the 5' end of the donor. The 3' terminal nucleotide on the primer was either a G, A or T residue. Since the corresponding nucleotide of the donor was a C, the G formed a matched terminus and the A or T a mismatched terminus. The efficiency with which DNA bound to the donor transferred to a second RNA, termed acceptor, was monitored. The acceptor was homologous to the donor for all but the last 9 nt at the 5' end of the donor. Therefore, homologous strand transfer could occur at any point prior to the DNA being extended into the nonhomologous region on the donor. Strand transfer occurred approximately twice as efficiently with the mismatched versus matched substrates. The mismatched nucleotide was fixed into transfer products indicating that excision of the mismatch was not required for RT extension or transfer. Results suggest that base misincorporations by RT may promote recombination by enhancing strand transfer.

Base Sequence↗

PubMed1992

A preliminary 3-D model of the tertiary fold of the polymerase domain of HIV-1 reverse transcriptase.

Development of a 3-D model of the reverse transcriptase from type 1 human immunodeficiency virus (HIV-1 RT), a key enzyme in the pathogenesis of the virus, presents a significant challenge. Three-dimensional structural information is not available for any close homolog, the only 3-D structural data being that of the Klenow fragment (KF) of Escherichia coli DNA polymerase I, for which coordinates of only the alpha-carbons are available. A recently published study of the sequences of a large number of polymerases led to the identification of three common sequence patterns, nominally motif A, motif B and motif C, and to the hypothesis that the various DNA and RNA polymerases including E. coli DNA polymerase I and HIV-1 RT share a common structural motif around their respective polymerase active sites. The preliminary results of recent structural studies on two other polymerases also support this hypothesis. Based on the assumption of structural homology in the active site regions of their polymerase domains, the HIV-1 RT and KF sequences were aligned using pattern-based secondary structure predictions as a guide and motifs A, B and C as 'anchor points'. However, as suggested by the results of chemical modification experiments, it was assumed that the order of the motifs in KF, viz. A, B and C, differed from that of the related motifs A, C and B' in HIV-1 RT, a rearrangement that could have been brought about by an exon shuffling type of mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H.

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.

Endoribonucleases↗

PubMed2000

Structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching.

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.

Amino Acid Motifs↗

PubMedDate not supplied

Detection of reverse transcriptase activity in human breast tumors.

The function of reverse transcriptase (RT) in retroviruses is to copy their RNA genomes in DNA to be integrated into the host genome. We report a high purity preparation of this enzyme by adsorption onto poly(U)-sepharose. We analyzed RT activity with poly(rA)--oligo (dT)12-18, poly(dA)--oligo (dT)12-18, poly(rC)--oligo (dG)12-18 and poly(rCm)--oligo (dG)12-18 to obtain a more accurate pattern of RT. 8 out of 16 malignant breast samples were positive for RT activity, more specifically with poly(rC)--oligo (dG)12-18 and poly(rCm)--oligo (dG)12-18. The 5 non-malignant mammary samples tested were negative for RT activity. Assays with poly(rC)--oligo (dG)12-18 and poly(rCm)--oligo (dG)12-18. The 5 non- malignant mammary samples tested were negative for RT activity. Assays with N--nitroso--N--methylurea--induced rat mammary carcinomas were negative for RT activity.

Animals↗

PubMed1997

Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay.

We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1. The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments. The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/microgram total RNA in MOLT-3/TMQ70, MOLT-3/ TMQ800, and MOLT-3/VCR1,000, respectively. The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis. In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detet P-gp. This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Comparison of the sensitivities of two non-isotopic reverse transcriptase (RT) assays for human immunodeficiency virus type 1 RT.

The sensitivities of two reverse transcriptase (RT) assays, an enzyme-linked oligonucleotide sorbent assay (ELOSA)-RT assay and a non-radioisotopic (non-RI) RT assay were compared. For measuring recombinant HIV-1 RT, the ELOSA-RT assay was 8 times less sensitive in dilution endpoint and 16 times less sensitive in measurement of RT from pelleted HIV-1 than the non-RI RT assay. Higher level of interference by an RNA-DNA hybrid observed in the former assay may indicate that the reduction in sensitivity was due to the presence of viral RNA in the sample of pelleted virus. The ELOSA-RT assay was interfered with to a great extent than the non-RI RT assay by fetal bovine serum and thus may be unsuitable for measuring RT from HIV-1 in a culture supernatant.

Enzyme-Linked Immunosorbent Assay↗

PubMed1994

HIV-1 reverse transcriptase inhibitor design using artificial neural networks.

Artificial neural networks were used to analyze and predict the human immunodeficiency virus type 1 reverse transcriptase inhibitors. The training and control sets included 44 molecules (most of them are well-known substances such as AZT, dde, etc.). The activities of the molecules were taken from literature. Topological indices were calculated and used as molecular parameters. The four most informative parameters were chosen and applied to predict activities of both new and control molecules. We used a network pruning algorithm and network ensembles to obtain the final classifier. Increasing of neural network generalization of the new data was observed, when using the aforementioned methods. The prognosis of new molecules revealed one molecule as possibly very active. It was confirmed by further biological tests.

Algorithms↗

PubMed2002

Optimization of reverse transcriptase PCR to detect viable Shiga-toxin-producing Escherichia coli.

The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated. Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of E. coli slt-II mRNA is useful for detection of viable STEC in ground beef.

Animals↗

PubMed2001

Biochemical mechanism of human immunodeficiency virus type 1 reverse transcriptase resistance to stavudine.

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.

Adenosine Triphosphate↗

PubMed1996

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Affinity Labels↗

PubMed1999

Virologic and immunologic response to nucleoside reverse-transcriptase inhibitor therapy among human immunodeficiency virus-infected infants and children.

Plasma human immunodeficiency virus RNA and CD4 lymphocyte response to nucleoside reverse-transcriptase therapy were evaluated in a large, comparative pediatric trial. Both baseline values and changes in the two laboratory markers over time correlated well with clinical outcome and possessed independent predictive value. In comparison of RNA reduction from baseline between the dideoxyinosine (ddI) and zidovudine+ddI therapeutic arms, marginal superiority of the combination arm was not correlated with an observed clinical benefit. Despite the size of this trial and the significantly higher rate of clinical end points in the zidovudine monotherapy group, attempts to establish surrogacy for plasma RNA were difficult. Nevertheless, plasma RNA and CD4 lymphocyte count together possess strong clinical predictive power and are valuable tools for both the clinician and the evaluation of new therapies.

Adolescent↗

PubMed1997

Increased polymerase fidelity of the 3TC-resistant variants of HIV-1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These 3TC-resistant RT variants have a single point mutation that changes the 184Met residue into either Val or Ile. Both codon 184 variants are frequently observed in 3TC-treated patients and can also be selected in cell culture infections. We demonstrated previously that the 184Ile and 184Val RT enzymes exhibit a processivity defect in in vitro assays, with 184Ile being the least processive enzyme [Met(wt) >Val >Ile]. In this study, we measured the polymerase fidelity of the wild-type (184Met) and 3TC-resistant RT enzymes (184Ile and 184Val) on DNA and RNA templates. Both virion- extracted and Escherichia coli -expressed recombinant RT enzymes were used to measure the nucleotide misinsertion and mispair extension efficiencies. The 3TC-resistant enzymes were more accurate than the wild-type RT protein in both type of assays. The order of accuracy observed for the codon 184 variants [Ile >Val >Met(wt)] may suggest an inverse correlation between the fidelity and processivity properties of these enzymes.

DNA↗

PubMed1990

Inhibition of avian myeloblastosis virus reverse transcriptase by aurochloric acid.

We investigated the inhibitory effects of aurochloric acid (AuCl4H) on reverse transcriptase (RT) derived from avian myeloblastosis virus and DNA polymerase alpha (pol. alpha) purified from HeLa S3 cells. The activities of RT, pol. alpha and E. coli DNA polymerase I (pol. I) with dTTP as the substrate were inhibited 50% at AuCl4H concentrations of 18 microM, 43 microM and 230 microM, respectively. AuCl4H inhibited RT activity competitively with respect to the substrate, dTTP, and uncompetitively with the template/primer, (rA)n(dT)12-18. In assays with dGTP as the substrate, 50% inhibitions of RT, pol. alpha and pol. I activities were observed at AuCl4H concentrations of 100 microM, 450 microM and 580 microM, respectively. AuCl4H inhibited RT activity uncompetitively with respect to the substrate, dGTP, and noncompetitively with the template/primer, (rC)n(dG)12-18. AuCl4H at concentrations causing more than 50% inhibition of RT activity had little inhibitory effect on the colony-forming ability of HeLa cells or their syntheses of DNA, RNA and protein.

Avian Myeloblastosis Virus↗

PubMed2002

HIV-1 subtype C reverse transcriptase sequences from drug-naive pregnant women in South Africa.

The HIV-1 reverse transcriptase genes from 37 HIV-1-positive pregnant women attending an antenatal clinical in Soweto, South Africa were sequenced and analyzed for the presence of drug resistance mutations. All women were antiretroviral drug naive, but were being screened as potential participants in clinical trials of antiretroviral drugs aimed at preventing mother-to-child transmission. Sequence analysis revealed that all belonged to HIV-1 subtype C, the predominant subtype among heterosexual populations in South Africa. Twenty-three amino acid loci associated with resistance to zidovudine, lamivudine, didanosine, stavudine, and nevirapine were examined and found not to encode mutations that would confer resistance to these drugs. Polymorphisms at these loci occurred infrequently, with three patients harboring the A98S and V179I polymorphisms. An additional three patients harbored V118I, which can function as an accessory resistance mutation, but in this context is also likely to be a polymorphism. These data show that pregnant women who are candidates for receiving antiretroviral drug therapies do not contain naturally occurring or preexisting drug resistance mutations and that such drug therapies are likely to be highly effective in this setting.

Amino Acid Sequence↗

PubMed2006

Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures.

In an effort to improve reverse transcriptase (RT) fidelity, we measured the error rate of Moloney murine leukemia virus (MMLV) RT in the presence of several autonomous and DNA polymerase-associated 3'-5' exonucleases using a lacZ forward mutation assay. A number of 3'-5' exonucleases were found to lower the error rate of MMLV RT, including p53, Escherichia coli DNA polymerase III epsilon subunit, and the proofreading activities associated with T4, varphi29, and E. coli pol I DNA polymerases. The bacterial epsilon subunit increased RNA-dependent DNA synthesis fidelity by approximately threefold and was the only 3'-5' exonuclease tested that did not deleteriously affect RT-PCR yields. Further testing showed that RT-PCR mutant frequencies were reduced significantly by performing cDNA synthesis in the presence of epsilon subunit, followed by PCR with a high-fidelity proofreading DNA polymerase. DNA sequence analysis was used to show that the combination of MMLV RT/epsilon subunit and PfuUltra DNA polymerase produces approximately eightfold fewer errors compared with the commonly used combination of MMLV RT and a Taq-based high-fidelity blend, consistent with predictions based on experimentally determined polymerase error rates.

Base Sequence↗

PubMed2002

Reverse transcriptase and protease sequence evolution in two HIV-1-infected couples.

We analyzed the reverse transcriptase (RT) and protease sequences of HIV-1 isolates obtained over 7 years from two couples with known transmission histories. Phylogenetic trees constructed from the sequence data reflected the known transmission histories, despite the fact that the drug resistance mutations were most consistent with the drug treatment histories. However, the RT sequences from one couple diverged by 2.9% even before therapy was begun, and three (0.9%) of 339 unrelated individuals had viruses that shared a common ancestor with sequences from the recipient member of the couple but not with sequences from the transmitter. The divergence between the first two isolates from this couple is consistent with a pretransmission interval during which the transmitter developed a heterogeneous virus population. The closeness between the three controls and the recipient's first RT sequence may indicate slower evolution on the branches of the control sequences. Although the RT and protease genes contain phylogenetic information, they are suboptimal for reconstructing transmission history because the genetic distance between RT and protease isolates from unrelated individuals may occasionally approximate the distance between RT and protease isolates from related individuals.

Amino Acid Sequence↗