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PubMed1996

An unusual mechanism of self-primed reverse transcription requires the RNase H domain of reverse transcriptase to cleave an RNA duplex.

The reverse transcription of retroviruses and long terminal repeat-containing retrotransposons requires that tRNA species serve as primers. We recently reported that the long terminal repeat-containing retrotransposon Tf1 is a unique exception in that reverse transcription is independent of tRNA and is instead initiated by a self-priming mechanism. The first 11 bases of the Tf1 transcript fold back and anneal to the primer binding site in a process that results in the priming of minus-strand strong-stop DNA. Data presented here demonstrate that a cleavage occurs between the 11th and 12th bases of the transcript, resulting in the generation of the primer. Mutagenesis experiments presented here indicate that the RNase H domain of the Tf1 reverse transcriptase is required for the cleavage reaction, suggesting that this RNase H may have the novel ability to cleave double-stranded RNA at the end of a duplexed region.

Amino Acid Sequence↗
PubMed2001

A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase.

One of the difficulties that can impede structural work on a molecule of interest is limited solubility. Although functionally similar to the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), the Moloney murine leukemia virus reverse transcriptase (MMLV RT) differs both in architecture and solubility properties. Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have introduced a single amino acid substitution in the connection domain of an N-terminally truncated MMLV RT (L435K) that significantly improves the solubility of the enzyme eliminating the need for nonionic detergents in buffering storage solutions. The substituted enzyme retains near wild-type polymerase activity. An important consequence of the improved solubility of the L435K MMLV RT has been the ability to obtain diffraction quality crystals.

Amino Acid Substitution↗
PubMed1991

Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2.

The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural-functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied.

Catalysis↗
PubMed1983

The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function.

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.

Adenosine Triphosphate↗
PubMed2000

Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity.

OBJECTIVE: This paper reviews the function of the mitochondria and the mechanisms by which nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs) cause mitochondrial toxicity. BACKGROUND: Highly active antiretroviral therapy (HAART) reduces rates of morbidity and mortality due to HIV disease. However, long-term treatment with these drugs may be associated with adverse effects. Nucleoside and nucleotide analogues are potent inhibitors of HIV reverse transcriptase and have become the cornerstone of HAART. Unfortunately, these drugs have also been shown to inhibit cellular polymerases, most notably mitochondrial DNA polymerase gamma. RESULTS: Studies of the NRTIs in enzyme assays and cell cultures demonstrate the following hierarchy of mitochondrial DNA polymerase gamma inhibition: zalcitabine > didanosine > stavudine > lamivudine > zidovudine > abacavir. In vitro investigations have also documented impairment of the mitochondrial enzymes adenylate kinase and the adenosine diphosphate/adenosine triphosphate translocator. Inhibition of DNA polymerase gamma and other mitochondrial enzymes can gradually lead to mitochondrial dysfunction and cellular toxicity. The clinical manifestations of NRTI-induced mitochondrial toxicity resemble those of inherited mitochondrial diseases (ie, hepatic steatosis, lactic acidosis, myopathy, nephrotoxicity, peripheral neuropathy, and pancreatitis). Fat redistribution syndrome, or HIV-associated lipodystrophy, is another side effect attributed in part to NRTI therapy. The morphologic and metabolic complications of this syndrome are similar to those of the mitochondrial disorder known as multiple symmetric lipomatosis: suggesting that this too may be related to mitochondrial toxicity. The pathophysiology of less common adverse effects of nucleoside analogue therapy, such as diabetes, ototoxicity, and retinal lesions, may be related to mitochondrial dysfunction but have not been adequately studied. CONCLUSION: NRTls can block both HIV reverse transcriptase and mitochondrial DNA polymerase gamma. Inhibition of the latter enzyme is the most likely cause of the adverse effects associated with these drugs.

Animals↗
PubMed2001

Requirements for minus-strand transfer catalyzed by Rous sarcoma virus reverse transcriptase.

We have examined the specific minus-strand transfer reactions that occur after the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma virus (RSV) reverse transcriptases. Three different types of reverse transcriptases can be isolated from virions of RSV: heterodimeric alphabeta and homodimeric alpha and beta. The mechanism of minus-strand transfer was examined using a model primer-template substrate corresponding to the 5'- and 3'-terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand transfer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the alpha homodimer lacking the integrase domain can perform strand transfer almost as efficiently as the alphabeta and alphaPol heterodimers. In contrast, the activities of beta and Pol for minus-strand transfer are reduced. Furthermore, a two- to fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.

Avian Sarcoma Viruses↗
PubMed2005

Panel of prototypical infectious molecular HIV-1 clones containing multiple nucleoside reverse transcriptase inhibitor resistance mutations.

We have created a panel of recombinant HIV-1 infectious clones containing common patterns of reverse transcriptase (RT) mutations responsible for resistance to each of the currently available nucleoside reverse transcriptase inhibitors (NRTI), and we have submitted the panel to the National Institutes of Health AIDS Research and Reference Reagent Programme. Testing the activity of new antiretroviral compounds against this panel of drug-resistant clones will determine their relative activity against many clinically relevant NRTI-resistant viruses.

Clone Cells↗
PubMed1999

Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitor in gynaecological cancer cell lines.

Telomerase is a ribonucleoprotein which has a RNA template to bind and extend telomere ends, so prolonging the life of tumour cells. The aim of this study was to determine whether transcriptase function of telomerase could be inhibited by the reverse transcriptase inhibitors (RTI); azydothymidine (AZT), dideoxyinosine (ddI) and AZT-5' triphosphate (AZT-TP). We examined their effects on the proliferation of cancer cells and the antitumour effects of cisplatin in vitro. The three agents did not cause major changes in telomerase activity or telomere length in MCAS cells. However, in HEC-1 cells changes in telomerase activity and telomere length were observed that were dependent on the RTI concentration and duration of exposure. ddI and AZT-TP reduced telomerase activity and shortened the length of the telomere. In the presence of RTI, the antitumour effects of cisplatin were enhanced. This was particularly evident in HEC-1 cells where there was a marked reduction in cell proliferation, appearance of morphological changes and senescent-like cells in the presence of ddI or AZT-TP. In MCAS cells, TP53 expression was increased by ddI and AZT-TP, while p21 expression was unchanged. In HEC-1 cells the expression of both TP53 and P21 was increased by ddI. Continuous administration of RTI enhanced the cell growth inhibition of cisplatin. RTI also inhibited the proliferation of some cells.

Blotting, Southern↗
PubMed1997

Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1.

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).

DNA↗
PubMed2004

Interaction of HIV-1 reverse transcriptase with modified oligonucleotide primers containing 2'-O-beta-D-ribofuranosyladenosine.

Modified synthetic oligodeoxyribonucleotides containing 2'-O-beta-D-ribofuranosyladenosine were used as primers in the RNA-dependent DNA synthesis catalyzed by HIV-1 reverse transcriptase. The degree of elongation of the primers depends on the position of the additional ribose unit, its presence in the specific position of the primer (-4) (and only in it) completely preventing elongation. Computer-modeled binding of the modified primers to the active site of reverse transcriptase demonstrated that steric hindrances arising from the interaction of the additional ribose residue with the reverse transcriptase region 262-270 interacting with the minor groove of the DNA substrate prevents elongation in the above mentioned case.

Adenosine↗
PubMed1991

Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase and human DNA polymerases alpha, beta, and gamma by the 5'-triphosphates of carbovir, 3'-azido-3'-deoxythymidine, 2',3'-dideoxyguanosine and 3'-deoxythymidine. A novel RNA template for the evaluation of antiretroviral drugs.

Carbovir (the carbocyclic analog of 2'-3'-didehydro-2',3'-dideoxyguanosine) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. Assays were developed to assess the mechanism of inhibition by the 5'-triphosphate of carbovir of HIV-1 reverse transcriptase using either RNA or DNA templates that contain all four natural nucleotides. Carbovir-TP was a potent inhibitor of HIV-1 reverse transcriptase using either template with Ki values similar to that observed by AZT-TP, ddGTP, and ddTTP. The kinetic constants for incorporation of these nucleotide analogs into DNA by HIV-1 reverse transcriptase using either template were similar to the values seen for their respective natural nucleotides. In addition, the incorporation of either carbovir-TP or AZT-TP in the presence of dGTP or dTTP, respectively, indicated that the mechanism of inhibition by these two nucleotide analogs was due to their incorporation into the DNA resulting in chain termination. Carbovir-TP was not a potent inhibitor of DNA polymerase alpha, beta, or gamma, or DNA primase. Given the potent activity of carbovir-TP against HIV-1 reverse transcriptase and its lack of activity against human DNA polymerases, we believe that further evaluation of this compound as a potential drug for the treatment of HIV-1 infection is warranted.

Antiviral Agents↗
PubMed1995

K65R mutation of human immunodeficiency virus type 1 reverse transcriptase encodes cross-resistance to 9-(2-phosphonylmethoxyethyl)adenine.

Cloned variants of human immunodeficiency virus type 1 that contain the K65R mutation in reverse transcriptase have previously been shown to display approximately 10- to 30-fold resistance against 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 2',3'-dideoxy-3'-thiacytidine. On the basis of tissue culture studies with both primary T cells and established cell lines, we now report that the K65R mutation confers approximately 12- to 15-fold resistance to 9-(2-phosphonylmethoxyethyl)adenine (PMEA). Likewise, a chain termination system revealed that mutated recombinant K65R reverse transcriptase displays resistance to PMEA diphosphate, the active metabolite of PMEA, in cell-free enzyme assays. Parallel studies have shown that the M184V mutation in reverse transcriptase, associated with high-level resistance against the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine, does not confer resistance to PMEA in tissue culture. Viruses and enzymes that included both the K65R and M184V mutations were resistant to PMEA and PMEa diphosphate, respectively, but only to the extent conferred by the K65R mutation alone.

Adenine↗
PubMed2001

Proteolytic processing of the human T-cell lymphotropic virus 1 reverse transcriptase: identification of the N-terminal cleavage site by mass spectrometry.

Human T-cell lymphotropic virus 1 (HTLV-1) is a type C human retrovirus, which is the causative agent of Adult T-cell Leukemia and other diseases. The reverse transcriptase of HTLV-1 (E.C. 2.7.7.49) is synthesized as part of a Gag--Pro--Pol precursor protein, and the mature Gag, Pro, and Pol proteins, including the reverse transcriptase, are created by proteolytic processing catalyzed by the viral protease. The location of the proteolytic cleavage site, which creates the N-terminus of mature HTLV-1 reverse transcriptase, has not been previously identified. By using sequence comparisons of several retroviral polymerases, as well as information about the location of the ribosomal frameshift, we tentatively identified this N-terminal processing site. PCR amplification was used to construct a clone, which spans a region of the pro--pol junction of HTLV-1, to produce a recombinant Pro--Pol protein spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombinant Pro--Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysis of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cleavage site to be between Leu(147)--Pro(148) of the pro ORF. This is the first physical identification of the authentic amino acid sequence of the reverse transcriptase of HTLV-1. The data reported here provides a basis for further investigation of the function and structural aspects of protein-nucleic interaction.

Amino Acid Sequence↗
PubMed2007

Non-nucleoside reverse transcriptase inhibitors: a review.

Non-nucleoside reverse transcriptase inhibitors form the backbone of antiretroviral treatment for many HIV-infected individuals. The tolerability, pill burden and efficacy associated with this class of agents make them a frequent choice for first-line therapy. Here we review nevirapine and efavirenz in terms of efficacy, resistance and toxicity, focusing particularly on the use of nevirapine to prevent mother-to-child transmission in developing countries.

Antiretroviral Therapy, Highly Active↗
PubMedDate not supplied

[Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids].

The interactions of HIV-I reverse transcriptase with some alkaloids were studied. Among nine compounds tested three--berberine, palmatine and sanguiritrine--inhibited RT. The dependence of the inhibition on the type of template-primer was also demonstrated. The kinetic analysis as well as circular dichroism experiments suggest the complex mechanism of RT inhibition by alkaloids. This mechanism includes both enzyme-alkaloid and alkaloid-template interactions; the latter effect also results in RT inhibition.

Alkaloids↗
PubMed2004

Simple, short peptide derivatives of a sulfonylindolecarboxamide (L-737,126) active in vitro against HIV-1 wild type and variants carrying non-nucleoside reverse transcriptase inhibitor resistance mutations.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) active against NNRTI-resistant mutants were obtained by introducing two methyl groups at positions 3 and 5 of the benzenesulfonyl moiety of L-737,126 (1) and coupling one to three glycinamide/alaninamide units to its carboxyamide function. In cell-based assays, the new derivatives showed activities against HIV-1 wild type and NNRTI-resistant mutants [Y181C, K103N-Y181C, and triple mutant (K103R, V179D, P225H) highly resistant to efavirenz] superior to that of the parent indole derivative 1.

Anti-HIV Agents↗
PubMedDate not supplied

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself.

Acquired Immunodeficiency Syndrome↗