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PubMedDate not supplied

Deletions in the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase are observed in HIV-1 isolated from subjects during long-term antiretroviral therapy.

OBJECTIVES: To examine the effect of in-frame deletions in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on plasma viremia and phenotypic resistance to antiretroviral drugs. STUDY DESIGN/METHODS: Plasma HIV-1 RNA was isolated from 168 antiretroviral therapy-experienced subjects for quantification of plasma viremia, RT sequence analysis, and phenotypic resistance assays. RESULTS: Four patients were found to harbor HIV-1 strains possessing in-frame, 3-nucleotide deletions at RT codons 67, 69, and 70. In these subjects, phenotypic resistance and high plasma viremia were observed only in a background of multiple resistance mutations. A recombinant virus engineered with an in-frame deletion of RT codon 67 did not have increased resistance to nucleoside reverse transcriptase inhibitors (NRTIs). CONCLUSIONS: Selection for deletions within the beta3-beta4 hairpin loop of the HIV-1 RT is an uncommon event most likely to occur in subjects with long-term antiretroviral experience. The codon 67 deletion does not appear to cause increased phenotypic resistance or increased viremia in the absence of concomitant RT mutations.

Adult↗

PubMed1993

Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro.

Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro. Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure. Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength. Recombinant and virion-derived HIV-1 RTs showed similar pausing patterns. DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the RNA templates were not broken or otherwise unable to support polymerization. Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template. These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausing may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.

Base Sequence↗

PubMedDate not supplied

Mutation L210W of HIV-1 reverse transcriptase in patients receiving combination therapy. Incidence, association with other mutations, and effects on the structure of mutated reverse transcriptase.

Mutation L210W of HIV-1 reverse transcriptase (RT) is one of the six main mutations that confer in vivo resistance to zidovudine. Surprisingly, this mutation has received scant appraisal and its contribution to the genotypic resistance to nucleoside analogs is not well understood. The aim of this study was: (1) to study the frequency of mutation L210W in a large collection of HIV-1 sequences (2,049 samples, including 395 DNA and 1,654 RNA sequences) from patients receiving combination therapy, and (2) to analyze its association with the other mutations that confer resistance to zidovudine. A mutation at codon 210 (mainly L210W) was found in 647 (32%) of the 2,049 sequences analyzed. Only 43 (<7%) of these 647 genomes were also mutated at codon 70 (p < 10(-5)). In contrast, 98% of these 647 sequences were also mutated at codon 215 (essentially T215Y/F), and 94% at codon 41 (mainly M41L). These data showing a close association between L210W, T215Y/F, and M41L, and a mutual exclusion between K70R and L210W, were confirmed by analyzing the sequences stored in the HIV-1 sequences available through the Stanford HIV RT and Protease Database. Follow-up studies demonstrated that L210W appeared always after T215Y/F. This observation is consistent with crystallographic studies which suggested that the aromatic side chain of Trp 210 could stabilize the interaction of Phe/Tyr215 with the dNTP-binding pocket. This molecular cross-talk between amino acid chains occurs nearby the conserved Asp113 residue. Since the lateral chain of Arg70 may also interact with Asp113, this is likely to create a sterical hindrance around this residue. Thus, the R-->K reversion of codon 70 may represent a compensatory mechanism allowing a functional rearrangement of the dNTP-binding pocket in the mutated RT.

Amino Acid Sequence↗

PubMed2001

Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV.

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G-->A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G-->A) to an adenine-to-guanine (A-->G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.

Anti-HIV Agents↗

PubMed2001

HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Algorithms↗

PubMed1993

Two step binding of HIV-1 reverse transcriptase to nucleic acid substrates.

The interactions of HIV-1 reverse transcriptase (HIV-1 RT) with a synthetic 53/19-mer DNA substrate was investigated. For this template-primer HIV-1 RT displayed a Km value of 20 nM. The 53/19-mer competitively inhibited DNA synthesis performed on poly (rC).oligo(dG) with Ki value of 260 nM. This corresponded well to an equilibrium dissociation constant (Kd) of 300 nM, as determined by analytical ultracentrifugation. Since the Kd value is considerably higher than the corresponding Km value it is concluded that the enzyme--DNA complex is further stabilized by the binding of a cognate deoxynucleoside triphosphate and/or catalytic turnover. The association kinetics of HIV-1 RT with the 53/19-mer was measured by the fluorescence stopped-flow technique. RT bound the 53/19-mer with a rate constant of 2 +/- 1 x 10(8) M-1 s-1. The DNA binding step was succeeded by a concentration-independent step with a rate constant of 1.0 +/- 0.5 s-1 suggesting a conformational change of the enzyme. Template-primer binding of RT was influenced by the concentration of MgCl2, displaying a 17-fold increase in the Kd value when Mg2+ was increased from 1 mM to 30 mM. Since neither the association rate constant nor the conformational change was notably affected by changes of the Mg2+ concentration, it is concluded that the dissociation constant is increased by higher concentrations of Mg2+.

Base Sequence↗

PubMed1993

Amino acid substitutions in HIV-1 reverse transcriptase with corresponding residues from HIV-2. Effect on kinetic constants and inhibition by non-nucleoside analogs.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

Amino Acid Sequence↗

PubMed1998

Structure-based design of N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A novel computer model of the HIV reverse transcriptase (RT) non-nucleoside inhibitor (NNI) binding pocket, which was generated using high resolution crystal structure information from 9 individual RT/NNI complexes, revealed previously unrecognized ligand derivatization sites for phenethylthiazolylthiourea (PETT) derivatives. Spatial gaps surrounding the pyridyl ring of the active PETT derivative trovirdine were discovered during modeling procedures. Docking studies using the computer-generated model of the binding pocket (composite binding pocket) suggested that the replacement of the planar pyridyl ring of trovirdine with a nonplanar piperidinyl or piperazinyl ring, which occupy larger volumes, would better fill the spacious Wing 2 region of the butterfly-shaped NNI binding pocket. The anti-HIV activity of the synthesized heterocyclic compounds N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea was examined in HTLVIIIB-infected peripheral blood mononuclear cells. Both compounds were more potent than trovirdine and abrogated HIV replication at nanomolar concentrations without any evidence of cytotoxicity.

Antiviral Agents↗

PubMed1995

The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.

HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV-1 virions.

Actins↗

PubMed1996

Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.

OBJECTIVE: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. METHODS: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > or = 300 x 10(6)/l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. RESULTS: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 10-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. CONCLUSIONS: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.

Adult↗

PubMed1997

Models which explain the inhibition of reverse transcriptase by HIV-1-specific (thio)carboxanilide derivatives.

The (thio)carboxanilide derivatives are potent and selective inhibitors of HIV-1 reverse transcriptase (RT) and have a favourable antiviral activity spectrum. To understand better their mode of action, and to provide a structural basis for further improvement, models of RT complexed with four (thio)carboxanilide inhibitors (UC781, UC10, UC38 and UC84) have been constructed based on the X-ray structure of RT complexed with 9-chloro-TIBO. In the models, the protein conformation is similar to that of the RT-TIBO complex and the complexes are stabilised by hydrogen bonding between the inhibitors and the main chain oxygen of Lys101. Significant hydrophobic interactions include those with Leu100, Val106, Val179, Tyr188, Phe227, Leu234, and His235. The thiocarboxanilides UC781 and UC10 also make important hydrophobic interactions with Trp229. The models are consistent with the inhibitors' relative antiviral potencies and the observed resistance data. They further predict that mutations to Phe227, Trp229, or Leu234 might confer resistance. Since these are not observed, some constraining structural or functional role for these residues in the active enzyme is suggested.

Anilides↗

PubMed1994

Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase.

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.

Amino Acid Sequence↗

PubMed2000

Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase.

We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.

Binding Sites↗

PubMed1996

Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

DNA Primers↗

PubMed1997

Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.

HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.

Amino Acid Sequence↗

PubMed1990

Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT).

The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted. The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form. Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique. The present crystal quality is still not adequate for high resolution X-ray investigation.

Crystallization↗

PubMed1998

Analysis of HIV-1 reverse transcriptase gene mutations in infected children treated with zidovudine.

Prolonged treatment with antiretroviral agents directed against reverse transcription (RT) in patients with HIV-1 infection results in the emergence of virus variants with reduced sensitivity containing mutations in the HIV-1 RT gene. Development of zidovudine (ZDV)-related mutations was studied in a cohort of 24 vertically infected pediatric patients receiving ZDV therapy. Monthly clinical and immunologic evaluation was accompanied by direct sequencing of the HIV-1 RT gene every 4 months. A correlation was observed between the emergence of mutations and the duration of therapy. Mutation at codon 41 was found only in the presence of mutation at codon 215. The presence of the mutations Met41-->Leu and Thr215-->Tyr/Phe did not appear to be related to disease progression. These findings suggest that the mere presence of mutations in the HIV-1 RT gene alone during ZDV monotherapy is not a reliable prognostic marker in the absence of other clinical and virologic information.

Anti-HIV Agents↗

PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗