A drug-sensitizing RNA interaction of HIV-1 reverse transcriptase.
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “HIV Reverse Transcriptase”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 8. 8-Aryloxymethyl- and 8-arylthiomethyldipyridodiazepinones.
Nevirapine (I) is the first human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor to reach regulatory approval. As a result of a second generation program around the tricyclic core system of nevirapine, 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-(2-(pyridin-4-yl)ethyl)-6H-dipyrido[3, 2-b:2',3'-e][1,4]diazepin-6-one (II)1a and 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-phenylethyl-6H-dipyrido[3,2-b:2', 3'-e][1,4]diazepin-6-one (III)1a were identified as broad spectrum HIV-1 RT inhibitors. A detailed examination of replacing either of the methylenes of the 8-ethyl linker of II or III is presented. It was found that 8-aryloxymethyl and 8-arylthiomethyl are the preferred pattern of substitution for potency against RT. The most potent compounds were further evaluated against a panel of clinically significant mutant RT enzymes (K103N, V106A, G190A, P236L) and in cytotoxicity and in vitro metabolism assays. The most potent compound was 2-chloro-8-phenylthiomethyl analogue 37 which displayed sub-100 nM activity against all HIV-1 RT enzymes tested.
Animals↗
Glutamic residue 438 within the protease-sensitive subdomain of HIV-1 reverse transcriptase is critical for heterodimer processing in viral particles.
The biological form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer consisting of two polypeptides, p66 and p51, which have identical N-termini. The p51 polypeptide is generated by action of viral protease cleaving the p66 polypeptide between residues Phe440 and Tyr441. Dimerization has been mostly studied using bacterially purified RT bearing amino acid changes in either subunit, but not in the context of HIV-1 particles. We introduced changes of conserved amino acid residues 430-438 into the protease-sensitive subdomain of the p66 subunit and analyzed the reverse transcriptase processing and function using purified variants and their corresponding HIV-1 recombinant clones. Our mutational analysis shows that the conserved Glu438 residue is critical for proper heterodimerization and function of virion-associated RT, but not of bacterially expressed RT. In contrast, the conserved Glu430, Glu432, and Pro433 residues are not important for dimerization of virion-associated RT. The network of interactions made by the Glu438 carboxyl group with neighboring residues is critical to protect the Phe440-Tyr441 from cleavage in the context of the p66/p51 heterodimer and may explain why the p66/p51 is not processed further to p51/p51.
Amino Acid Sequence↗
Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem.
During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult. At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds. These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related. Mechanisms producing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. Several general trends emerged from the study. First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T. Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end. A and C as template bases in the template-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.
Base Sequence↗
Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.
The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.
Anilino Naphthalenesulfonates↗
Pre-existing distortions in nucleic acid structure aid polypurine tract selection by HIV-1 reverse transcriptase.
Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO(4)) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine +1 is detected when the PPT primer is extended by DNA or RNA at its 3' terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the +1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA)(4):(dT)(4) or (rG)(6):(dC)(6) tracts. Importantly, there is a correlation between the structural deformation at base pair +1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO(4) modification also revealed unusually high reactivity for one of two (dT)(4):(rA)(4) duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.
Base Pair Mismatch↗
Crystallography and the design of anti-AIDS drugs: conformational flexibility and positional adaptability are important in the design of non-nucleoside HIV-1 reverse transcriptase inhibitors.
Drug resistance is a key cause of failure for treatment of HIV infection. The efficacy of non-nucleoside reverse transcriptase inhibiting (NNRTI) drugs is impaired by rapid emergence of drug-resistance mutations. A multidisciplinary effort led to the discovery of the potent NNRTIs dapivirine and etravirine, both of which are diarylpyrimidine (DAPY) derivatives. Systematic structural and molecular modeling studies of HIV-1 reverse transcriptase (RT)/NNRTI complexes revealed different modes of inhibitor binding, and some of the DAPY inhibitors can bind to RT in different conformations. The torsional flexibility ("wiggling") of the inhibitors can generate numerous conformational variants and the compactness of the inhibitors permits significant repositioning and reorientation (translation and rotation) within the pocket ("jiggling"). Such adaptations appear to be critical for the ability of the diarylpyrimidine NNRTIs to retain their potency against a wide range of drug-resistant HIV-1 RTs. Exploitation of inhibitor conformational flexibility (such as torsional flexibility about strategically located chemical bonds) can be a powerful element of drug design, especially for the design of drugs that will be effective against rapidly mutating targets (which is a collection of related targets).
Acquired Immunodeficiency Syndrome↗
Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.
In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.
Amino Acid Sequence↗
Substrate requirements for secondary cleavage by HIV-1 reverse transcriptase RNase H.
During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5' end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the blunt-ended substrate, suggesting that NC compensates for the unfavorable substrate structure.
Base Sequence↗
The emergence of different resistance mechanisms toward nucleoside inhibitors is explained by the properties of the wild type HIV-1 reverse transcriptase.
Nucleoside reverse transcriptase inhibitors (NRTIs) represent one of the main drug families used against AIDS. Once incorporated in DNA, they act as chain terminators, due to the lack of a 3'-hydroxyl group. As for the other anti-human immunodeficiency virus type 1 drugs, their efficiency is limited by the emergence of resistant viral strains. Unexpectedly, previous studies indicated that resistance toward NRTIs is achieved via two distinct and generally exclusive mechanisms. Resistance mutations either decrease the efficiency of NRTIs incorporation or increase their excision from the extended primer. To understand the emergence of different resistance mechanisms toward a single inhibitor class, we compared the incorporation and the pyrophosphorolysis of several NRTIs using wild type reverse transcriptase (WT RT). We found that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway. Indeed, our results suggest that the pathway by which RT become resistant toward a given NRTI can be predicted by studying the inhibition of WT RT, because the resistance mutations do not confer new properties to the mutant enzyme, but rather exacerbate pre-existing properties of the WT enzyme. They also help to understand the low cross-resistance toward d4T observed with the 3'-azido-3'-deoxythymidine (AZT or zidovudine)-resistant RT.
Acquired Immunodeficiency Syndrome↗
A mutation in the primer grip region of HIV-1 reverse transcriptase that confers reduced fidelity of DNA synthesis.
A compensatory mutation (M230I) in the primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) restores the replication capacity of virus having a Y115W mutation in their RT coding region. The Y115W substitution impairs DNA polymerase activity and produces an enzyme with a lower fidelity of DNA synthesis. Gel-based fidelity assays with the double mutant Y115W/M230I revealed that the M230I substitution increased the accuracy of mutant Y115W. Y115W/M230I showed wild-type misinsertion fidelity in assays performed with DNA/DNA templates. However, when present alone, M230I conferred reduced fidelity as determined in misinsertion and mispair extension fidelity assays, as well as in primer extension assays carried out with three dNTPs. The mutant M230I showed a 3.3-16-fold increase in misinsertion efficiency for G, C and T opposite T, compared with the wild-type enzyme. Its fidelity was not influenced by nucleotide substitutions in the template/primer around the incorporation site. However, its accuracy was apparently affected by the structure of the 5'-overhang of the template strand. Unlike wild-type HIV-1 RT, nucleotide selectivity of mutant M230I at dT:dG, dT:dC and dT:dT mispairs was almost exclusively dependent on the K(m) values for correct and incorrect dNTPs, a characteristic that has not been described for other low fidelity mutants of HIV-1 RT.
DNA Primers↗
Molecular modeling of HIV-1 reverse transcriptase drug-resistant mutant strains: implications for the mechanism of polymerase action.
A computer model of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) either alone, or complexed with a non-nucleoside inhibitor (NNI), was constructed using crystal coordinate data from a subset of the protein surrounding the binding pocket region. Molecular mechanics calculations were carried out on solvated wild-type RT and RT that contained modifications corresponding to resistance-engendering mutations. Results from the calculations revealed that the r.m.s. difference between 12 modified proteins and that of wild-type RT could be qualitatively correlated with the measured polymerase activity of the enzyme in the presence of these mutations. In addition, the level of activity was related to the measured distance between the primer grip and dNTP binding regions of the protein. These data suggest a direct correlation between RT structure and function. Complexes of RT-8-C1 TIBO and RT-alpha-APA were also minimized in models containing modifications corresponding to key drug-resistant mutants. The variant complexes all showed weaker binding than wild-type RT, while giving rise to similar, but critical changes in the protein. Therefore, the design of new inhibitors should center on obtaining stronger binding drugs to key drug-resistant RT variants.
Anti-HIV Agents↗
HIV-1 reverse transcriptase sequence in plasma and cerebrospinal fluid of patients with AIDS dementia complex treated with Abacavir.
OBJECTIVE: To assess HIV-1 RNA levels and the relationship between HIV-1 reverse transcriptase (RT) genotype from plasma and cerebrospinal fluid (CSF) during treatment with abacavir (Ziagen, ABC) or placebo in combination with stable background therapy (SBG) in subjects with AIDS dementia complex (ADC) (study CNA3001). DESIGN: One-hundred and five HIV-1 infected adults with ADC were randomized to receive either ABC (600 mg twice daily) or ABC-matched placebo (twice daily) in addition to SBG for 12 weeks. METHODS: Plasma and CSF were collected for population sequencing at baseline and week 12 (CSF optional). Sequences were analyzed for mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTI). RESULTS: Sixty out of sixty-seven subjects with baseline plasma HIV-RT sequence data harbored virus with > or = 1 NRTI-associated mutations; 50 out of 67 had the M184V mutation. At week 12, more subjects in the ABC group had plasma HIV-1 RNA < or = 400 copies/ml than the SBG group (46% versus 13%, P = 0.002). Non-response to ABC was associated with multiple baseline zidovudine (ZDV)/stavudine (d4T)-associated mutations. Baseline RT mutation patterns differed in 14 out of 21 (67%) paired samples from plasma and CSF. Four subjects experienced > 1 log10 copies/ml reductions in CSF HIV-1 RNA, two in the absence of reductions in plasma HIV-1 RNA and two with undetectable plasma HIV-1 RNA at baseline. CONCLUSIONS: Substantial decreases in plasma and CSF HIV-1 RNA following addition of ABC were not precluded by baseline HIV-1 NRTI-associated mutations, including the M184V mutation, but non-responders commonly harbored multiple ZDV/d4T-associated mutations. HIV-1 RNA responses and RT genotype appear to be discordant between CSF and plasma in some subjects.
AIDS Dementia Complex↗
RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III.
RNase D was recently reported as a new enzymatic activity associated with HIV-1 reverse transcriptase (RT), cleaving RNA at two positions within the double-stranded region of the tRNA primer-viral RNA template complex (Ben-Artzi et al., Proc. Natl. Acad. Sci. USA 89 (1992) 927-931). This would make RNase D a fourth distinct activity of HIV-1 RT, in addition to RNA- and DNA-dependent DNA polymerase and RNase H. Using a specific substrate containing tRNA(Lys,3) hybridized to the primer binding site, we were able to detect the reported RNase D activity in our preparations of recombinant HIV-1 RT. This activity was also present in several active-site mutants of RT, suggesting that it is independent of the RNase H and polymerase functionalities of RT. Furthermore, we found that the cleavage specificity of RNase D is the same as that of RNase III isolated from E.coli. A likely explantation of these results--that the observed RNase D activity is attributable to traces of RNase III contamination--was further strengthened by the finding that the recombinant preparations of HIV-1 RT can specifically cleave a phage T7-derived double-stranded RNA processing signal, which has been used as a model substrate for detection of E.coli RNase III. Moreover, RT purified from an RNase III- strain of E.coli displayed no cleavage of the tRNA primer-RNA template complex.
Bacteriophage T7↗
Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization.
"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
Amino Acid Sequence↗
The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors alters the size distribution of RNase H cleavages.
We investigated the effects of the nonnucleoside reverse transcriptase inhibitor-resistant mutant Y181C on RNA 5'-end-directed RNase H cleavage by HIV-1 reverse transcriptase, using an RNA.DNA hybrid in which a radiolabeled RNA 5' end was recessed. Y181C produced a higher ratio of secondary (9 nucleotide long) to primary (18 nucleotide long) products than wild type. When the RNA was 3'-end-labeled, Y181C generated a long product, which results when secondary cleavage precedes the primary. When using an RNA.DNA hybrid in which the labeled RNA 5' end and DNA 3' end were flush, formation of secondary product by both enzymes was inhibited. Under these conditions, Y181C cleaved closer to the RNA 5' end than wild type. Studies with this substrate labeled at the RNA 3' end showed that Y181C is no more likely than wild type to cleave toward the RNA 3' end. Thus, Y181C RT has a strong preference to cleave in the direction of the RNA 5' end even when secondary cleavage is prevented, resulting in a disruption of the normal sequence of primary followed by secondary cleavages.
3' Untranslated Regions↗
An enzymatically active chimeric HIV-1 reverse transcriptase (RT) with the RNase-H domain of murine leukemia virus RT exists as a monomer.
The existence of retroviral reverse transcriptases as monomers or dimers is rather intriguing. A classical example of the former is murine leukemia virus reverse transcriptase (MuLV RT), while human immunodeficiency virus type 1 (HIV-1) RT represents the latter. A careful scrutiny of the amino acid sequence alignment of the two enzymes pinpoints the region tentatively responsible for this phenomenon. We report here the construction of a chimeric enzyme containing the first 425 amino acid residues from the N-terminal domain of HIV-1 RT and 200 amino acid residues from the C-terminal domain of MuLV RT. The chimeric enzyme exists as a monomer with intact DNA polymerase and RNase-H functions.
Amino Acid Sequence↗
Reverse transcriptase fidelity and HIV-1 variation.
Explore the source record for details and available documents.
Anti-HIV Agents↗