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PubMed2004

The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA.

The primer tRNA for reverse transcription in HIV-1, tRNALys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC) sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.

Animals↗
PubMed1993

The interaction of illimaquinone, a selective inhibitor of the RNase H activity, with the reverse transcriptases of human immunodeficiency and murine leukemia retroviruses.

Illimaquinone, a natural marine product, was shown by us to inhibit preferentially the ribonuclease H (RNase H) activity of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). We have also shown that illimaquinone inhibits the RNase H activity of HIV-2 RT in addition to that of HIV-1 RT, murine leukemia virus RT, and Escherichia coli RNase H. Chemical modifications of HIV-1 RT by sulfhydryl-specific reagents, such as N-ethylmaleimide (NEM) have been demonstrated to specifically inhibit the RNase H activity of the enzyme. Since our previous studies have suggested that cysteine 280 in HIV-1 RT interacts with the sulfhydryl reagents, we have examined the possibility that illimaquinone interacts with the RT molecules via amino acid residues located in the vicinity of cysteine 280 in both HIV-1 and HIV-2 RTs. In the combined effect studies of illimaquinone and NEM, the two structurally unrelated compounds were shown to be mutually exclusive, exhibiting an antagonistic interaction with both HIV-1 and murine leukemia virus-associated RNase H activities. This implicates cysteine 280, in both HIV-1 and HIV-2 RTs, to be in close proximity to the putative binding site of the enzyme to illimaquinone. The above conclusion is further supported by the fact that the RNase H activity of an enzymatically active mutant of HIV-1 RT, in which cysteine 280 was replaced by serine, was substantially more resistant to illimaquinone than the corresponding activity of the wild-type enzyme. The fact that NEM failed to inhibit E. coli RNase H as opposed to illimaquinone highlights a major difference between the retroviral and bacterial RNase H.

Cloning, Molecular↗
PubMed1999

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors.

This article describes a molecular-field-based similarity method for aligning molecules by matching their steric and electrostatic fields and an application of the method to the alignment of three structurally diverse non-nucleoside HIV-1 reverse transcriptase inhibitors. A brief description of the method, as implemented in the program MIMIC, is presented, including a discussion of pairwise and multi-molecule similarity-based matching. The application provides an example that illustrates how relative binding orientations of molecules can be determined in the absence of detailed structural information on their target protein. In the particular system studied here, availability of the X-ray crystal structures of the respective ligand-protein complexes provides a means for constructing an 'experimental model' of the relative binding orientations of the three inhibitors. The experimental model is derived by using MIMIC to align the steric fields of the three protein P66 subunit main chains, producing an overlay with a 1.41 A average rms distance between the corresponding C alpha's in the three chains. The inter-chain residue similarities for the backbone structures show that the main-chain conformations are conserved in the region of the inhibitor-binding site, with the major deviations located primarily in the 'finger' and RNase H regions. The resulting inhibitor structure overlay provides an experimental-based model that can be used to evaluate the quality of the direct a priori inhibitor alignment obtained using MIMIC. It is found that the 'best' pairwise alignments do not always correspond to the experimental model alignments. Therefore, simply combining the best pairwise alignments will not necessarily produce the optimal multi-molecule alignment. However, the best simultaneous three-molecule alignment was found to reproduce the experimental inhibitor alignment model. A pairwise consistency index has been derived which gauges the quality of combining the pairwise alignments and aids in efficiently forming the optimal multi-molecule alignment analysis. Two post-alignment procedures are described that provide information on feature-based and field-based pharmacophoric patterns. The former corresponds to traditional pharmacophore models and is derived from the contribution of individual atoms to the total similarity. The latter is based on molecular regions rather than atoms and is constructed by computing the percent contribution to the similarity of individual points in a regular lattice surrounding the molecules, which when contoured and colored visually depict regions of highly conserved similarity. A discussion of how the information provided by each of the procedures is useful in drug design is also presented.

Crystallography, X-Ray↗
PubMed1993

A nonnucleoside reverse transcriptase inhibitor active on human immunodeficiency virus type 1 isolates resistant to related inhibitors.

Pyridinone derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and HIV-1 replication in cell culture. However, the potential clinical usefulness of these compounds as monotherapeutic agents may be limited by the selection of inhibitor-resistant viral variants. Resistance in cell culture is due primarily to mutational alterations at RT amino acid residues 103 and 181. A recombinant HIV-1 RT containing both of these mutations was used to screen a panel of pyridinone analogs for inhibitory activity. L-696,229 and L-697,661, pyridinones currently undergoing clinical evaluation, were more than 4,000-fold weaker against the mutant enzyme than against the wild-type enzyme. In contrast, one derivative of L-696,229, L-702,019 (3-[2-(4,7-dichlorobenzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyrid in-2(1H)-thione), showed only three-fold different potencies against the two enzymes. L-702,019 was also a potent inhibitor of the replication of mutant HIV-1 containing the individual mutations at amino acid 103 or 181 as well as of clinical isolates resistant to L-697,661 and L-696,229. Isolation and analysis of resistant viral variants in cell culture showed that significant resistance to L-702,019 could be engendered only by multiple amino acid substitutions in RT. Accordingly, these studies demonstrated the potential of identifying second-generation specific HIV-1 RT inhibitors that can overcome the viral resistance selected by the first generation of inhibitors.

Antiviral Agents↗
PubMed2003

Hybrid canonical-correlation neural-network approach applied to nonnucleoside HIV-1 reverse transcriptase inhibitors (HEPT derivatives).

Beneficial antiviral HIV-1 chemotherapy is associated with adverse reactions. To optimize the desired actions and to lower the side effects of nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors (NNRTIs), quantitative structure-activity relationships (QSARs) were studied by using a series of HEPT derivatives of NNRTIs. Hypothesis testing requires that certain assumptions are approximately satisfied in statistically based QSARs, however. A complementary approach is based on artificial neural network analysis. Model building can be made without the manifold assumptions of statistically based QSAR approaches but the problem is that the number of neural weights increase exponentially (danger of overfitting) under certain circumstances. A way to get more reliable results is to reduce the dimensionality of the two subsets (biological and chemical variables). A suitable method is the canonical correlation analysis. The two subsets of canonical variates are used as outputs (biologically derived variates) and inputs (chemically derived variates) of an optimized backpropagation neural network approach. The contribution summarizes the most recent results of this canonical-correlation backpropagation-neural network QSAR approach. It is shown that noncovalent interactions (lipophilic, steric, hydrogen-bonding, and inductive forces of the substituents) are responsible for the antiviral and cytotoxic actions. The outcome of this analysis produces an internally highly self-consistent result (model robustness). The predictive performance is tested. The butterfly-like conformation of the predicted compound is consistent with the butterfly-like model of other NNRTIs. Molecular simulation shows that the complexed drug interacts with the Tyr181 and Tyr188 residues of the RT. The uracil ring of the drug binds directly with Lys101, and the acyclic side chain (with an intact free hydroxyl function) binds with Lys103. The suggested noncovalent interaction forces are equivalent with that found by the QSAR analysis.

Anti-HIV Agents↗
PubMed2005

Drug resistance mutations in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase differentially affect the phosphorolysis-dependent primer unblocking activity in the presence of stavudine and zidovudine and its inhibition by efavirenz.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.

Alkynes↗
PubMed1996

Genotypic and phenotypic changes during culture of a multinucleoside-resistant human immunodeficiency virus type 1 strain in the presence and absence of additional reverse transcriptase inhibitors.

The observation that human immunodeficiency virus type 1 (HIV-1) mutations conferring resistance to one reverse transcriptase (RT) inhibitor may suppress resistance to other RT inhibitors provides a rationale for treating HIV-1 with certain RT inhibitor combinations. We examined phenotypic and genotypic changes during culture of a multinucleoside (zidovudine, didanosine, zalcitibine, and stavudine)-resistant HIV-1 strain with and without additional RT inhibitors (nevirapine and lamivudine). The development of nevirapine or lamivudine resistance by the multinucleoside-resistant strain was not accompanied by a reduction in zidovudine or didanosine resistance.

Antiviral Agents↗
PubMed1983

Reverse transcriptase activity and particles of retroviral density in cultured canine lymphosarcoma supernatants.

Lymphoid tissue from 43 cases of canine lymphosarcoma and from 40 clinically normal dogs have been examined for markers of retrovirus infection. From 69-76% of culture supernatants from lymphosarcomas were shown to contain particles of retroviral density and to possess poly rC-oligo dG templated polymerase (reverse transcriptase) activity compared with 17-24% of culture supernatants from normal canine lymphoid cells. In 6 culture supernatants from cases of lymphosarcoma, high molecular weight 60-70S RNA was detected and shown to be found in association with this particulate reverse transcriptase activity. No such RNA was detected in 6 culture supernatants from normal canine lymphoid cells.

Animals↗
PubMed2001

Type-specific detection of echovirus 30 isolates using degenerate reverse transcriptase PCR primers.

Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.

Amino Acid Sequence↗
PubMed1998

Allosteric inhibitors against HIV-1 reverse transcriptase: design and synthesis of MKC-442 analogues having an omega-functionalized acyclic structure.

Based on X-ray crystallographic analysis of MKC-442/human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) complex, analogues in which the N1-substituent is replaced with omega-functionalized alkyl groups were designed to improve the affinity for the enzyme. Synthesis of these compounds was carried out starting from MKC-442 by a sequence of reactions (N3-protection, removal of N1-ethoxymethyl group, alkylation, and N3-deprotection). The compounds were evaluated for anti-HIV activity. Structure-activity relationships are discussed in terms of the possible interaction with the enzyme.

Allosteric Site↗
PubMed2004

Capravirine, a nonnucleoside reverse-transcriptase inhibitor in patients infected with HIV-1: a phase 1 study.

Capravirine is a nonnucleoside reverse-transcriptase inhibitor (NNRTI) with a unique resistance profile. Although single mutations allow resistance to established NNRTIs, human immunodeficiency virus (HIV)-1 must undergo multiple mutations to achieve resistance to capravirine. In the present phase 1 study, capravirine was administered orally for up to 28 days to 55 HIV-1-infected individuals with CD4+ T lymphocyte counts of 50-500 cells/microL. The most frequent adverse events were diarrhea (5%) and nausea (4%), with no drug-related rashes observed. The day 15 median (mean) HIV-1 load decreased by 1.34 (1.45) log(10) copies/mL in the patients receiving 25 mg/kg/day. Capravirine demonstrated potent antiviral activity, even in antiretroviral-experienced patients.

Administration, Oral↗
PubMed1975

Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions.

Three temperature-sensitive mutants of the Rauscher strain of murine leukemia virus are defective in early post-penetration functions required both for leukemia virus infection and for initiation of transformation of cells by their pseudotypes of murine sarcoma virus. In the present study, the reverse transcriptase of one of these mutants (ts 29) is shown to be thermolabile compared with the enzymes of the wild-type virus and several other temperature-sensitive mutants. These findings provide evidence that the reverse transcriptase is required both for leukemia virus infection and for initition of transformation by the replication-defective murine sarcoma virus genome.

Absorption↗
PubMed2001

Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.

The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K(m) values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.

Alanine↗
PubMed1999

Clinical resistance patterns and responses to two sequential protease inhibitor regimens in saquinavir and reverse transcriptase inhibitor-experienced persons.

The efficacy of sequential protease inhibitor therapy was studied in 16 human immunodeficiency virus (HIV) 1-infected persons in whom saquinavir with multiple nucleoside reverse transcriptase (RT) inhibitors (NRTI) had failed. Nelfinavir plus two NRTIs (new or continued) resulted in minimal (0.59 log RNA copies/mL) and transient (8 weeks) suppression of plasma HIV RNA levels. Rapid failure was surprisingly associated with baseline presence of protease gene mutation L90M (P=.04) in the absence of D30N and with RT mutations D67N (P<.01), K70R/S (P=.02), and K219Q/W/R/E (P<.01). Ten patients were subsequently switched to indinavir plus nevirapine and 2 NRTIs, resulting in a median 1.62 log reduction in plasma HIV RNA, with 3 patients maintaining 400 copies/mL for 24 weeks. These results suggest that nelfinavir may have limited utility after saquinavir failure, particularly without potent concomitant therapy. Combining an NRTI with a new protease inhibitor for rescue may improve response.

Adult↗
PubMed1998

Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.

The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.

Binding Sites↗
PubMed1995

[Recombinant reverse transcriptase from Rous sarcoma virus. Kinetics and inhibition of DNA polymerase activity].

Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E. coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer and dTTP as nucleotide substrate. Kinetic constants for steady state conditions were determined. The substrate initial velocity patterns point to an ordered mechanism which results in the formation of a ternary complex, in which the template-primer is the first to bind to the enzyme. Inhibition of the DNA-polymerase activity of the enzyme by various inhibitors was studied. Analysis of final products of the DNA-polymerase reaction revealed the presence of distribution syntheses of the DNA chain by the alpha alpha-subunit form of the enzyme.

Avian Sarcoma Viruses↗
PubMed1992

Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase.

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.

Chromatography, Gel↗
PubMed2002

TaqMan junction probes and the reverse transcriptase polymerase chain reaction: detection of alveolar rhabdomyosarcoma, synovial sarcoma, and desmoplastic small round cell tumor.

The reverse transcriptase polymerase chain reaction (RT-PCR) provides a technique to diagnose a group of sarcomas and small round cell tumors that contain specific chromosomal translocations and chimeric gene fusion products. We adapted real-time qualitative RT-PCR to utilize dual-labeled, fluorogenic, TaqMan probes, which hybridize to targets that overlap the junction of the chimeric gene fusions in alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT). Assays were confirmed on cell lines and tissue samples; appropriate negative amplification assays were obtained when each tumor-specific probe and primer set was used on different neoplasms and cell lines that were not expected to harbor the specific translocations and chimeric gene fusions. Although our cases are few, we speculate that as more molecular variants of ARMS, SS, and DSRCT are discovered, clinical correlations based on precise molecular features will be required and fusion site specificity will be assured by the use of junction-based TaqMan probes.

Carcinoma, Small Cell↗