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PubMed1992

Interaction of tRNALys with the p66/p66 form of HIV-1 reverse transcriptase stimulates DNA polymerase and ribonuclease H activities.

The precursor homodimeric p66/p66 form of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) possesses the DNA polymerase and RNase H activities involved in the synthesis of the double-stranded provirus DNA. Reverse transcription is initiated from tRNALys in the case of HIV-1. The present study confirmed that interactions between HIV-1 RT and tRNALys induce protein conformational changes and demonstrated that these interactions stimulate the enzymatic activities associated with the p66 subunit. Thus, the p66/p66 form of the enzyme is strongly stimulated in both DNA polymerase and RNase H activities. Preincubation of the enzyme with tRNA is an obligatory step to obtain the stimulatory effect. The affinity of template, primer, or substrate for RT p66/p66 did not change when the enzyme was preincubated with tRNALys at stimulatory concentrations; the interaction of tRNA with p66/p66 has an effect only on the maximal rate of polymerization. It is further shown that the RNase H domain of RT is much more accessible to protease attack than the DNA polymerase active site.

Chymotrypsin↗

PubMed1992

Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T).

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.

Antiviral Agents↗

PubMed1995

Mechanistic evaluation of new plant-derived compounds that inhibit HIV-1 reverse transcriptase.

Swertifrancheside [1], a new flavonone-xanthone glucoside isolated from Swertia franchetiana, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2], a triterpene isolated from the roots of Maprounea africana, and protolichesterinic acid [3], an aliphatic alpha-methylene-gamma-lactone isolated from the lichen Cetraria islandica, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT), with 50% inhibitory doses (IC50 values) of 43, 3.7, and 24 microM, respectively. They were not cytotoxic with cultured mammalian cells. The kinetic mechanisms by which compounds 1-3 inhibited HIV-1 RT were studied as was their potential to inhibit other nucleic acid polymerases. Swertifrancheside [1] bound to DNA and was shown to be a competitive inhibitor with respect to template-primer, but a mixed-type competitive inhibitor with respect to TTP. On the other hand, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] were mixed-type competitive inhibitors with respect to template-primer and noncompetitive inhibitors with respect to TTP. Therefore, the mechanism of action of 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] as HIV-1 RT inhibitors involves nonspecific binding to the enzyme at nonsubstrate binding sites, whereas swertifrancheside [1] inhibits enzyme activity by binding to the template-primer.

4-Butyrolactone↗

PubMed2000

Effects of beta-L-3'-azido-3'-deoxythymidine 5'-triphosphate on host and viral DNA polymerases.

We have previously reported that several beta-L-thymidine analogues including beta-L-3'-azido-3'-deoxythymidine (beta-L-AZT), beta-L-3'-fluoro-2',3'-dideoxythymidine (beta-L-FLT) and beta-L-2', 3'-didehydro-2',3'-dideoxythymidine (beta-L-D4T) did not inhibit HIV replication in human peripheral blood mononuclear (PBM) cells whereas their corresponding beta-D-counterparts are known as potent and selective anti-HIV agents [Faraj et al., 1997. Nucleosides and Nucleotides 16, 1287-1290]. In order to gain insight on the lack of antiviral activities of these beta-L-derivatives, in vitro enzymatic steady state studies were conducted in the present study with beta-L-AZT. beta-L-AZT 5'-triphosphate (L-AZTTP) was chemically synthesized and found to moderately inhibit wild-type HIV reverse transcriptase (HIV-1 RT) with a K(i) value of 2 microM; while lacking any inhibitory effect towards human DNA polymerase alpha, beta or gamma. However, the inhibitory effect of L-AZTTP towards HIV-1 RT was very modest (266-fold less potent) when compared to its isomer beta-D-AZT 5'-triphosphate (D-AZTTP) which exhibits a K(i) value of 0.0075 microM and this finding was further confirmed by DNA chain termination assay. These data suggest that the absence of antiviral activity of the parent beta-L-AZT may in part be explained by the poor inhibition of the targeted viral enzyme by L-AZTTP, the active metabolite. Finally, L-AZTTP was found to lack affinity for the mutant RT at position 184 (M184V) demonstrating that this mutation confers resistance not only to beta-L-2',3'-dideoxycytidine analogs as previously reported by our group [Faraj et al., 1994. Antimicrob. Agents Chemother. 38, 2300-2305] but as well as to beta-L-2',3'-dideoxythymidine analogs.

Anti-HIV Agents↗

PubMed1997

SIV/HIV-1 hybrid virus expressing the reverse transcriptase gene of HIV-1 remains sensitive to HIV-1-specific reverse transcriptase inhibitors after passage in rhesus macaques.

We have previously described an animal model for the therapy of human immunodeficiency virus type 1 (HIV-1) infection with HIV-1-specific reverse transcriptase (RT) inhibitors based on a simian immunodeficiency virus (SIV), in which the RT gene of SIV was replaced by the RT gene of HIV-1. In vitro, replication of the hybrid virus, RT-SHIV, was delayed compared with parental SIV. RT-SHIV could induce AIDS-like symptoms and pathologic alterations in rhesus macaques. Characterization of re-isolates recovered from RT-SHIV-infected macaques one-half year after infection revealed that the re-isolates replicated with kinetics similar to those of SIV. Inefficient processing of the Gag-Pol precursor of RT-SHIV may be one reason for the retarded growth of RT-SHIV, because the protease cleavage site between the protease gene and the RT gene was frequently mutated in the RT-SHIV re-isolates. Adaptation of RT-SHIV to the growth in macaques did not result in a relevant loss of sensitivity to nonnucleoside RT inhibitors (NNRTIs). However, because a minor sub-population of the RT-SHIV re-isolates contained a mutation conferring low-level resistance to ddI and ddC, the RT-SHIV/macaque model may underestimate the efficacy of these drugs. Nevertheless, this report further supports the suitability, reliability, and usefulness of the RT-SHIV/macaque model to investigate the antiviral properties of most RT inhibitors in an in vivo setting.

Animals↗

PubMed1997

The inhibitory action of fatty acids on DNA polymerase beta.

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.

Animals↗

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↗

PubMed2005

Newer aminopyrimidinimino isatin analogues as non-nucleoside HIV-1 reverse transcriptase inhibitors for HIV and other opportunistic infections of AIDS: design, synthesis and biological evaluation.

Human immuno deficiency virus (HIV) weakens the immune system so that many opportunistic infections (OIs) like tuberculosis, hepatitis, bacterial infections etc can develop. In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-related OIs. Compound 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[3'-(4'-amino-5'-trimethoxybenzyl pyrimidin-2'-yl)imino-1'-(5-methylisatinyl)]methyl]-N1-piperazinyl]-3-quinoline carboxylic acid (10) emerged as the most potent broad-spectrum chemotherapeutic agent active against HIV, HCV, Mycobacterium tuberculosis and various pathogenic bacteria.

AIDS-Related Opportunistic Infections↗

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↗

PubMed2004

Prevalence of drug resistance and newly recognised treatment-related substitutions in the HIV-1 reverse transcriptase and protease genes from HIV-positive patients naïve for anti-retrovirals.

The aim of this study was to assess the prevalence of genetic changes in either the HIV reverse transcriptase (RT) or protease (Pro) genes in a cohort of patients naïve for anti-retroviral therapy. Of 61 patients, 43 (70.5%) were infected with HIV strains harbouring at least one resistance-related mutation, with 41 (67.2%) harbouring newly recognised treatment-related mutations. Among the 61 patients, the prevalence of specific mutations in the RT gene was as follows: 39A, 1.6%; 43E, 1.6%; and 228H, 1.6%. The prevalence of specific mutations in the Pro gene was as follows: 11I, 1.6%; 13V, 26.2%; 35D, 19.6%; 45R, 1.6%; 58E, 1.6%; 62V, 31%; 72V, 11.4%; 72M, 6.5%; 72T, 3.2%; 75I, 1.6%; and 89M, 13%. A higher prevalence of newly recognised mutations was found in strains from patients infected through sexual practices (30/36 = 83.4% vs. 11/25 = 44%; p 0.0023; OR 10.91; 95% CI 3.14-40.39). These findings support the use of resistance testing in patients naïve for anti-retroviral therapy, and suggest that the possible impact of newly recognised treatment-related mutations on clinical outcome requires further investigation.

Adult↗

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↗