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PubMed1994

2.2 A resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains).

BACKGROUND: HIV-1 reverse transcriptase (RT) catalyzes the transformation of single-stranded viral RNA into double-stranded DNA, which is integrated into host cell chromosomes. The molecule is a heterodimer of two subunits, p51 and p66. The amino acid sequence of p51 is identical to the sequence of the amino-terminal subdomains of p66. Earlier crystallographic studies indicate that the RT molecule is flexible, which may explain the difficulty in obtaining high-resolution data for the intact protein. We have therefore determined the structure of a fragment of RT (RT216), which contains only the amino-terminal half of the RT molecule ('finger' and 'palm' subdomains). RESULTS: The crystal structure of RT216 has been refined at 2.2 A resolution to a crystallographic R-value of 20.8%. The structure is very similar to that of the corresponding part of the p66 subunit in the p66/p51 heterodimer, although there is a small difference in the relative orientation of the two subdomains compared with the structure of an RT-DNA-antibody fragment complex. There are a large number of stabilizing contacts (mainly hydrogen bonds and hydrophobic interactions) between the subdomains. The locations of conserved amino acids and the position of some important drug-resistant mutations are described. CONCLUSIONS: The RT216 structure provides detailed three-dimensional information of one important part of HIV-1 RT (including the critical active site residues). We propose a model to explain the inhibitory effect of non-nucleoside inhibitors, which partially accounts for their effect in terms of conformational changes of active site residues.

Binding Sites↗
PubMed1993

Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy.

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.

Adult↗
PubMed2000

Immunogenic properties of reverse transcriptase of HIV type 1 assessed by DNA and protein immunization of rabbits.

Genetic immunization may be one way to prime individuals for a subsequent broad anti-HIV-1 immune response. Reverse transcriptase of HIV-1 (RT) presents a selective target for attempts to arrest replication of HIV-1. Rabbits immunized with a plasmid carrying the gene for reverse transcriptase HIV-1 (RT DNA) developed potent antibody and cellular responses to the gene product. The immunogenic properties of RT DNA and recombinant reverse transcriptase were compared in rabbits. The specific immune responses were similar to those reported previously for HIV-1 infected humans. The array of B and T cell epitopes recognized in RT DNA-immunized rabbits was broader than in rabbits immunized with the recombinant RT. We localized seven novel B and T cell epitopes and concordance between B cell and helper T cell epitopes was observed. B cell epitopes of RT induced proliferation of peripheral blood mononuclear cells and were active as helper T cell epitopes. T cell-proliferative responses to the epitopes of RT preceded or paralleled the production of antibodies of the same specificity. Subdomains of reverse transcriptase involved in the enzymatic activity of RT were highly immunogenic. Anti-RT IgG partially inhibited reverse transcription in vitro.

Amino Acid Sequence↗
PubMed2001

A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.

Alkynes↗
PubMed2001

Occurrence of HIV-1 reverse transcriptase gene mutation at codon 215 in HIV-infected infants.

BACKGROUND: The nucleoside reverse transcriptase inhibitor Zidovudine (ZDV) decreases mother to child transmission of HIV infection. Nevertheless, significant proportions of mothers who are treated during pregnancy with ZDV still transmit the virus. Along with other factors, failure of ZDV prophylaxis may be due to maternal infection with ZDV-resistant strains and their consequent vertical transmission. OBJECTIVES: The purpose of the study was to investigate the occurrence of mutations at codon 215 in HIV-1 infected infants and its association with clinical status and virological parameters. STUDY DESIGN: The cohort consisted of 49 HIV-1 infected infants. Mononuclear cell DNA was isolated from whole blood and served as the input DNA for both a qualitative DNA PCR and the codon 215 assay (nested PCR). HIV-1 viral load (RNA PCR) was measured in plasma by the Roche Amplicor Monitor assay. RESULTS: Twelve of the 49 (24.5%) demonstrated viral strains with mutation at codon 215. A significant difference was found in infants born between 1992 and 1994 (6.3%) compared to those born in 1998-1999 (33.3%). Furthermore, in those infants born in 1998-1999, there was a trend toward an increase in the frequency of zidovudine resistant mutations at codon 215 corresponding to an increase in maternal zidovudine treatment. The mixture of wild and mutant HIV-1 strains was detected in two of 14 infants (14.3%) with a low viral load (<750000 c/ml) compared to nine of 19 (47.4%) infants with extremely high levels of HIV-1 RNA concentration (>750000 c/ml). Only two of 33 tested infants were HIV symptomatic and in both, a mixture of wild and mutant HIV-1 strains was detected. In both infants, the viral load was >750000 c/ml. CONCLUSIONS: This study showed that the overall frequency of ZDV resistant strains in infants born in 1998-1999 was significantly higher than that found in infant samples from 1992 to 1994. By 1998, the standard of care for mothers known to be infected with HIV was treatment with either monotherapy using ZDV or combined therapy using a variety of antiretroviral agents including ZDV. Although the exact role of ZDV resistance in limiting the effectiveness of therapies aimed at blocking vertical transmission of HIV remains to be defined, it is clear that drug resistant strains of HIV are occurring more frequently in all types of HIV infection, including infants.

Anti-HIV Agents↗
PubMed1998

Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase.

Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 microM. Their activity against reverse transcriptase ranged from 7 microM to greater than 100 microM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 microM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 microM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit > 10- to > 100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase.

Understanding the molecular mechanisms of HIV-1 reverse transcriptase (RT) action and drug inhibition is essential for designing effective antiretroviral therapies. Although comparisons of the different crystal forms of RT give insights into the flexibility of different domains, a direct computational assessment of the effect of inhibitor binding on the collective dynamics of RT is lacking. A structure-based approach is used here for exploring the dynamics of RT in unliganded and inhibitor-bound forms. Non-nucleoside RT inhibitors (NNRTI) are shown to interfere directly with the global hinge-bending mechanism that controls the cooperative motions of the p66 fingers and thumb subdomains. The net effect of nevirapine binding is to change the direction of domain movements rather than suppress their mobilities. The second generation NNRTI, efavirenz, on the other hand, shows the stronger effect of simultaneously reorienting domain motions and obstructing the p66 thumb fluctuations. A second hinge site controlling the global rotational reorientations of the RNase H domain is identified, which could serve as a target for potential inhibitors of RNase H activity.

Alkynes↗
PubMed2002

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo.

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.

Base Sequence↗
PubMed2003

Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis.

The M184V mutation in HIV reverse transcriptase (RT) is associated with high-level resistance against the nucleoside inhibitor lamivudine as well as diminished viral replication capacity. We have previously demonstrated that HIV variants containing the M184V mutation were relatively unable to successfully undergo compensatory mutagenesis following deletion of an A-rich loop located upstream of the primer binding site (PBS). To understand the mechanisms involved, we synthesized viral RNA templates containing different compensatory mutations that were emergent during the long-term culture of the A-rich loop-deleted viruses. These templates were then used in cell-free reverse transcription initiation assays and in tRNA primer placement assays performed with either recombinant wild-type RT or recombinant RT containing the M184V substitution. The results showed that the RNA template that contained the A-rich loop deletion was impaired in ability to initiate reverse transcription and that the presence of the M184V substitution in RT amplified this effect. Clearance from pausing at position +3 during synthesis of viral DNA was identified as a sensitive step in this reaction that could not be efficiently bypassed with the M184V mutant enzyme. Increased efficiency of initiation was seen with the deleted RNA templates that also contained mutations identified in the revertant viruses, provided that these mutations facilitated formation of a competent binary tRNA/RNA complex. These findings provide biochemical evidence that initiation of tRNA(Lys3)-primed DNA synthesis is an important rate-limiting step in reverse transcription that correlates with viral replication fitness.

DNA Primers↗
PubMed1995

The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Benzodiazepines↗
PubMed2000

RNA motifs mediating in vivo site-specific nonhomologous recombination in (+) RNA virus enforce in vitro nonhomologous crossovers with HIV-1 reverse transcriptase.

There are several lines of evidence that both RNA viruses and retroviruses recombine according to a copy choice mechanism. Using the brome mosaic virus (BMV)-based system, we recognized elements in the RNA structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. The same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to switch templates during DNA synthesis. We demonstrated that a specific combination of the local double-stranded region with short homologous sequences and a hairpin structure allows template switching by HIV-RT. In contrast to BMV replicase, HIV-RT does not mediate the detectable level of recombination using only the heteroduplex structure, though local hybridization between RNA molecules efficiently pauses primer extension. Moreover, the presented data suggest that a proper arrangement of identified structural motifs can ensure site specificity of RNA-RNA recombination. These results indicate that HIV-RT utilizes the same or a very similar mechanism as BMV replicase to change nonhomologous RNA templates in a site-specific manner.

Base Sequence↗
PubMed1994

Fidelity of HIV-1 reverse transcriptase copying a hypervariable region of the HIV-1 env gene.

The unusually high mutation frequency exhibited by the human immunodeficiency virus (HIV) is a major impediment to developing effective vaccines against the virus and to designing analogs that inhibit viral replication. To investigate the molecular basis of HIV hypermutability, we established cell-free assays to measure the fidelity of HIV-1 reverse transcriptase (RT) in copying either DNA or both RNA and DNA templates that contain the hypervariable region 1 of the HIV-1 env gene (V-1). The fidelity of DNA synthesis was measured by repetitively copying the envelope gene (V-1) DNA by HIV-1 RT, followed by cloning and sequencing these newly synthesized DNA products. We found that the error rate of HIV RT copying either RNA or DNA of the env V-1 region is about one misincorporation per 5 kb polymerized. This rate is similar to that found with the M13mp2 forward mutation assay using the lacZ alpha gene as a template. This similarity suggests that the HIV env hypervariable sequence is not inherently hypermutable. The high error rate of HIV RT suggests that misincorporation by this enzyme is a major source of mutations throughout the viral genome and a determinant for rapid viral evolution. The spectrum of mutations produced by HIV RT in vitro partially correlates with the spectrum of HIV mutations observed in AIDS patients. The differences between these spectra highlight the contribution of phenotypic selection during HIV-1 infection. The overall uniformity of misincorporation of HIV-1 RT further suggests an alternative anti-HIV strategy based on increasing viral mutagenesis by nucleotide analogs.

Base Sequence↗
PubMed1994

Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis.

Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides.

Base Sequence↗
PubMed2004

Artificial neural networks: non-linear QSAR studies of HEPT derivatives as HIV-1 reverse transcriptase inhibitors.

Structure-anti HIV activity relationships were established for a sample of 80 1-[2-hydroxyethoxy-methyl]-6-(phenylthio)thymine (HEPT) using a three-layer neural network (NN). Eight structural descriptors and physicochemical variables were used to characterize the HEPT derivatives under study. The network's architecture and parameters were optimized in order to obtain good results. All the NN architectures were able to establish a satisfactory relationship between the molecular descriptors and the anti-HIV activity. NN proved to give better results than other models in the literature. NN have been shown to be particularly successful in their ability to identify non-linear relationships.

Anti-HIV Agents↗
PubMed2000

Comparison of a point mutation assay with a line probe assay for the detection of the major mutations in the HIV-1 reverse transcriptase gene associated with reduced susceptibility to nucleoside analogues.

This study compares the performance of a line probe assay (LiPA) for the detection of the major mutations associated with reduced sensitivity to nucleoside analogues with a well characterised point mutation assay (PMA). Plasma samples obtained from patients in a trial of four reverse transcriptase inhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutations in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from plasma by the Boom method and amplified by RT-PCR prior to being tested by LiPA or PMA. Assay discrepancies were further investigated by sequencing of the RT gene. Of 275 samples available from 98 trial subjects, 246 samples were successfully amplified by PCR and analysed by LiPA and PMA for six mutations. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a result for 32 codons from 22 samples and PMA failed with 58 codons from 38 samples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequencing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PMA result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are genotypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases.

Acetamides↗
PubMed1993

Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV-1 reverse transcriptase.

Deoxyribonucleotide insertion efficiencies were measured opposite site-directed abasic template lesions using human immunodeficiency virus 1 reverse transcriptase (HIV-1RT), and the efficiencies to continue primer synthesis beyond the lesion, by addition of the "next correct" deoxynucleotide, were measured as a function of sequence context. Insertion of purines was favored over pyrimidines, A > G > T approximately C. Primer extension past the lesion occurred by two distinct mechanisms, either by direct or by misalignment extension. An "A-rule" appeared to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In misalignment extension, the primer terminus is realigned from a site directly opposite the lesion to a new position opposite a neighboring template base 5' to the lesion. Direct extension efficiencies were measured in 16 different configurations, by varying 4 bases at the primer 3'-termini and 4 at the 5'-side (downstream) of the lesion. The predominant order of direct extension was A > G > T approximately C, similar to that observed for insertion. Reduced primer extension rates were not caused by a reduction in HIV-1 RT-DNA binding. Primers terminating in C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment extension efficiencies were 27:1, 2.5:1, and 1:25 for A, G, and C opposite the lesion, respectively. For the case of primers terminating in T, misalignment extension was not observed. A striking result was that while primers were extended past an abasic lesion by HIV-1 RT in both direct and misalignment modes, avian myeloblastosis virus RT failed to catalyze significant extension by either mode.

Animals↗