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PubMed1993

Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase.

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.

Base Sequence↗

PubMed2005

Long-term analysis of the resistance development in HIV-1 positive patients treated with protease and reverse transcriptase inhibitors: correlation of the genotype and disease progression.

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.

Amino Acid Substitution↗

PubMed2001

SJ-3366, a unique and highly potent nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) that also inhibits HIV-2.

We have identified and characterized a potent new nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1) that also is active against HIV-2 and which interferes with virus replication by two distinct mechanisms. 1-(3-Cyclopenten-1-yl)methyl-6-(3,5-dimethylbenzoyl)-5-ethyl-2,4-pyrimidinedione (SJ-3366) inhibits HIV-1 replication at concentrations of approximately 1 nM, with a therapeutic index of greater than 4 x 10(6). The efficacy and toxicity of SJ-3366 are consistent when evaluated with established or fresh human cells, and the compound is equipotent against all strains of HIV-1 evaluated, including syncytium-inducing, non-syncytium-inducing, monocyte/macrophage-tropic, and subtype virus strains. Distinct from other members of the pharmacologic class of NNRTIs, SJ-3366 inhibited laboratory and clinical strains of HIV-2 at a concentration of approximately 150 nM, yielding a therapeutic index of approximately 20,000. Like most NNRTIs, the compound was less active when challenged with HIV-1 strains possessing the Y181C, K103N, and Y188C amino acid changes in the RT and selected for a virus with a Y181C amino acid change in the RT after five tissue culture passages in the presence of the compound. In combination anti-HIV assays with nucleoside and nonnucleoside RT and protease inhibitors, additive interactions occurred with all compounds tested with the exception of dideoxyinosine, with which a synergistic interaction was found. Biochemically, SJ-3366 exhibited a K(i) value of 3.2 nM, with a mixed mechanism of inhibition against HIV-1 RT, but it did not inhibit HIV-2 RT. SJ-3366 also inhibited the entry of both HIV-1 and HIV-2 into target cells. On the basis of its therapeutic index and multiple mechanisms of anti-HIV action, SJ-3366 represents an exciting new compound for use in HIV-infected individuals.

Drug Resistance, Microbial↗

PubMed1998

Novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 7. 8-Arylethyldipyridodiazepinones as potent broad-spectrum inhibitors of wild-type and mutant enzymes.

Like other nonnucleoside inhibitors of HIV-1 reverse transcriptase, the dipyridodiazepinone nevirapine (Viramune, 1) selects for drug resistant variants of HIV-1, both in cell culture and in patients. In particular, the mutation of residue 181 from tyrosine to cysteine (Y181C) is associated with resistance to most reported nonnucleoside inhibitors. Introduction of an arylethyl substituent at the 8-position of the tricyclic dipyridodiazepinone skeleton confers enhanced potency against Y181C RT. Several analogues of this series display good broad spectrum potency against a panel of mutant enzymes.

Amino Acid Substitution↗

PubMed2003

Site-specific footprinting reveals differences in the translocation status of HIV-1 reverse transcriptase. Implications for polymerase translocation and drug resistance.

Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre- and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

Amino Acid Substitution↗

PubMed1986

Ribonuclease H(70) from Saccharomyces cerevisiae possesses cryptic reverse transcriptase activity.

Yeast cells contain a protein of molecular size 70 kDa that possesses RNase H activity. A polyclonal antibody against it reacts in addition with proteins of molecular sizes 160 kDa from yeast extracts. All these immunologically related proteins exhibit reverse transcriptase activity and in this respect they resemble the products of retroviral pol genes, relatives of which reside in Ty elements and mitochondrial introns of yeast. Experimental evidence, however, indicates that the protein described here that combines RNase H and reverse transcriptase activity is not coded for by a known element of the retrotransposon family. It may originate from a cellular gene distantly related to retrotransposon sequences.

Endoribonucleases↗

PubMed1994

Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

The fidelity of DNA strand transfer reactions catalyzed by human immunodeficiency virus type 1 reverse transcriptase has been studied in vitro. A model system involving two sequential DNA strand transfers was developed to simulate the process of forced copy-choice recombination. A propensity for nucleotide misincorporation at the junction of the strand transfer, as determined by DNA sequencing of the reaction products, was found consistent with a model involving the addition of nontemplate-directed nucleotides prior to the transfer of nascent DNA onto the accepting RNA template [Peliska, J. A., & Benkovic, S. J. (1992) Science 258, 1112]. The kinetic and mechanistic factors that may dictate which nucleotide bases are incorporated at recombination sites during strand transfer and the possible consequences of recombination-induced mutagenesis in vivo are discussed.

Base Sequence↗

PubMed2004

In vitro evaluation of nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virus microbicides.

The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV.

Anilides↗

PubMed2001

Single-tube single-enzyme reverse transcriptase PCR assay for detection of bovine viral diarrhea virus in pooled bovine serum.

A reverse transcriptase PCR (RT-PCR) was developed for use as a diagnostic screening test for the detection of bovine viral diarrhea virus (BVDV) in pooled bovine serum samples. Individual serum samples from 60 dairy cattle herds located in Pennsylvania were evaluated by the microplate virus isolation method, and pooled sera were analyzed by RT-PCR. RT-PCR was sensitive and specific and detected a single viremic serum sample in up to 100 pooled serum samples. RT-PCR analysis of pooled sera provides a rapid and cost-effective method for the screening of cattle herds for the presence of animals persistently infected with BVDV.

Animals↗

PubMed2002

The M184V mutation in HIV-1 reverse transcriptase reduces the restoration of wild-type replication by attenuated viruses.

OBJECTIVE: To study the ability of HIV constructs containing the M184V substitution in reverse transcriptase (RT), which causes resistance to lamivudine, to evolve mutations that compensate for deletions within the HIV genome. METHODS: Viruses containing deletions in non-coding regions of the viral genome were examined in tissue culture to see whether the additional presence of M184V delays the reestablishment of wild-type replication kinetics. Potential compensatory mutations were identified by sequencing, and site-directed mutagenesis was carried out to confirm the biological relevance of such substitutions. The rate of initiation of reverse transcription was measured using either recombinant wild-type RT or RT containing M184V. RESULTS: M184V-containing viruses were unable to undergo compensatory mutagenesis to reestablish wild-type replication kinetics, whereas viruses that did not contain M184V were able to mutate extensively. This ability was demonstrated most extensively in viruses deleted of an "A-rich loop", located upstream of the primer-binding site, which is involved in initiation of reverse transcription. The rate of such initiation was severely diminished in virus containing the RT enzyme carrying the M184V substitution. This inhibitory effect was significantly enhanced in a biochemical system that included both the M184V mutant enzyme and a viral DNA template that contained the deletion in the A-rich loop. CONCLUSIONS: These findings provide further biological and biochemical evidence that M184V-containing viruses are impaired in replication fitness. Viruses that had the A-rich-loop deleted were able to reestablish replication ability quickly in the wild-type RT, which provides further evidence for the plasticity of the HIV genome.

Cells, Cultured↗

PubMed2003

Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

Hepatotoxicity is an adverse effect of all available classes of antiretrovirals, including nucleoside reverse transcriptase inhibitors (NRTI). A syndrome of hepatic steatosis and lactic acidosis has been recognized as a rare, potentially fatal complication since the advent of NRTI monotherapy in the early 1990s. Today, NRTI remain the backbone of antiretroviral combination regimens, and, with the success of current treatment strategies, exposure to two or more of these agents may occur over a number of years. Hepatic steatosis and lactic acidosis are accordingly being observed more frequently, along with a more recently recognized syndrome of chronic hyperlactatemia. These as well as other adverse effects of NRTI are mediated by inhibition of human DNA polymerase gamma, resulting in mitochondrial dysfunction in the liver and other tissues. Early recognition and intervention are essential to avert serious outcomes.

Anti-HIV Agents↗

PubMed2001

Two telomerase reverse transcriptases (TERTs) expressed in Candida albicans.

The human pathogenic yeast Candida albicans contains two telomerase reverse transcriptase (TERT) genes. CaTERT1 and CaTERT2 appear either to be two alleles of the same gene or two entirely different genes that encode 867-residue proteins that differ by five amino acids. Both TERTs have a calculated pI of 9.5 and a M(r) of 100.9 kDa and are the smallest TERTs identified to date. Both genes appear to be expressed. Based on sequence similarity between CaTERT1 and the Saccharomyces cerevisiae orthologue Est2p, we suggest a revised alignment for motif E of Est2p. The identification of these TERT genes provides the first opportunity to study telomerase in an important human pathogen.

Alleles↗

PubMed1992

The effects of cysteine mutations on the reverse transcriptases of human immunodeficiency virus types 1 and 2.

Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme. HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445). Both of the cysteines in HIV-1 RT are in the polymerase domain. To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations. Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT. Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification. The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant. The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT. Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity. The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue. We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein. We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.

Cysteine↗

PubMed1991

Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase.

Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit.

Chimera↗

PubMed2000

Resistance profile of the human immunodeficiency virus type 1 reverse transcriptase inhibitor abacavir (1592U89) after monotherapy and combination therapy. CNA2001 Investigative Group.

Abacavir (1592U89) is a nucleoside inhibitor of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT). Resistance to abacavir was studied with abacavir alone and with abacavir in combination with other nucleoside analogues in cell culture, in virus isolates from zidovudine/lamivudine clinical trials, and in the first dose-escalating 12-week clinical trial (CNA2001) to evaluate abacavir clinical potency. Abacavir alone in vitro selected for mutations at HIV RT codons K65R, L74V, Y115F, and M184V. However, abacavir combined with zidovudine selected against virus with the M184V mutation. Abacavir therapy in vivo resulted in large decreases in HIV load (>1 log), even in 1 subject who had the M184V mutation at baseline. A total of 51% of subjects showed new mutations at any of codons K65R, L74V, and M184V after abacavir monotherapy, compared with 11% who received zidovudine/abacavir. Small changes (2- to 4-fold) in abacavir susceptibility were detected. On stopping therapy, reselection of the pretherapy sequence occurred within 4 weeks.

Acquired Immunodeficiency Syndrome↗

PubMed1994

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures.

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

Cells, Cultured↗

PubMed2005

Incidence of acute pancreatitis and nucleoside reverse transcriptase inhibitors usage.

Acute pancreatitis (AP) is a well-known adverse effect of nucleoside reverse transcriptase inhibitors (NRTIs). Therefore, we performed a prospective, cohort study to examine the incidence rates (IRs) and rate ratios (RRs) of AP for each NRTI. A total of 116 HIV patients were included in the final analysis comprising 445.6 person-years of follow-up. Twelve cases of AP were recorded. The lowest IR for AP was for didanosine (ddI) (IR=0.03 per 100 person-years, 95% confidence interval [CI] = 0.01-0.05), and the highest for ddI + stavudine (d4T) (IR = 0.08, 95% CI = 0.07-012). Compared with ddI alone, the RR of AP was 2.21 (95% CI = 1.32-9.31) for d4T, and 3.13 (95% CI = 1.43-12.56) for ddI + d4T. Other risk factors for AP were CD4 cell count <200 cells/mm(3) and female sex. Our results suggest that the use of d4T alone or combined with ddI should not be used as first-line therapy, especially in women or patients with CD4-cell count <200 cells/mm(3).

Adult↗

PubMed1998

Increased misincorporation fidelity observed for nucleoside analog resistance mutations M184V and E89G in human immunodeficiency virus type 1 reverse transcriptase does not correlate with the overall error rate measured in vitro.

Nucleoside analog-resistant variants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that displayed higher in vitro polymerase fidelity were previously identified via nucleotide insertion and mispair extension assays. To evaluate the contribution of increased nucleotide insertion and primer extension fidelities on the overall error rate of HIV-1 RT, we have measured the impact of two such mutations, E89G and M184V, on DNA copying fidelity in an M13 phage-based forward mutation assay. Using this assay, we observed mutation frequencies of 8.60 x 10(-3), 6.26 x 10(-3), 5.53 x 10(-3), and 12.30 x 10(-3) for wild-type, E89G, M184V, and double-mutant E89G/M184V HIV-1 RTs, respectively. Therefore, the overall polymerase fidelities of wild-type, E89G, M184V, and E89G/M184V HIV-1 RTs are similar (less than twofold differences) for DNA-dependent DNA synthesis. Thus, rather large increases in fidelity of deoxynucleoside triphosphate insertion and mispair extension observed previously appear not to influence the overall error rate of these mutants. However, a qualitative analysis of the mutations induced revealed significant differences in the mutational spectra between the wild-type and mutant enzymes.

Anti-HIV Agents↗