The Motif D Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Critical for Nucleoside 5′-Triphosphate Selectivity

Canard, Bruno; Chowdhury, Kajal; Sarfati, Robert S.; Doublié, Sylvie; Richardson, Charles C.

doi:10.1074/jbc.274.50.35768

Cited by 24 publications

(35 citation statements)

References 35 publications

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“…3B). Mutations of this highly conserved residue in HIV RT result in a severe reduction in activity and specifically affect the selection of the correct nucleotide in the catalytic site (25). Based on the HIV reverse transcriptase studies, the K902N mutation was predicted to severely decrease enzyme activity of hTERT.…”

Section: Resultsmentioning

confidence: 99%

Haploinsufficiency of t e lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

Armanios

Chen

Chang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

425

417

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Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.telomere ͉ aplastic anemia ͉ hTERT

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Section: Resultsmentioning

confidence: 99%

Haploinsufficiency of t e lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

Armanios

Chen

Chang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

425

417

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“…Plasmids, Genes, and Proviral DNA Constructions-The wild-type RT gene construct p66RT7 has been described (24). This plasmid was further modified and used to construct the mutant RT gene at codon 75 (V75T) and the double mutant V75T/Y146F using synthetic oligonucleotides and a strategy described previously (24).…”

Section: Methodsmentioning

confidence: 99%

“…This plasmid was further modified and used to construct the mutant RT gene at codon 75 (V75T) and the double mutant V75T/Y146F using synthetic oligonucleotides and a strategy described previously (24). The same RT gene mutated at codon 75 was used in conjunction with the recombinant HIV-1 proviral AD8 DNA (HIV-1 AD8 (25)) to construct V75T HIV-1 AD8 .…”

Section: Methodsmentioning

confidence: 99%

“…Standard RT activity was assayed using poly(rA)/oligo(dT) 19 or poly(rC)/oligo(dG) 19 and DE81 ion-exchange paper discs as described (24). To determine the K M for a dNTP, the primer/template was kept at a saturating concentration of 200 nM, and the concentrations of the corresponding dNTP were varied from 2 to 50 M. The RT concentration was 10 nM.…”

Section: Methodsmentioning

confidence: 99%

“…Nucleotide Analogue Inhibition at Steady State-The inhibition of reverse transcription was measured as described previously (11,24). Inhibition was expressed as the concentration of inhibitor producing a 50% inhibition (IC 50 ) using the standard reverse transcription assay described above.…”

Section: Methodsmentioning

confidence: 99%

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The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

Selmi

Boretto

Navarro

et al. 2001

Journal of Biological Chemistry

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The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2,3-didehydro-2,3-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site. Antiretroviral therapy directed against the human immunodeficiency virus (HIV)1 is currently increasing the life expectancy of infected individuals. To replicate in infected cells, HIV relies on reverse transcriptase (RT), an essential RNA-and DNA-dependent DNA polymerase encoded by the viral pol gene (1). RT is a major target for drugs acting as inhibitors of retroviral replication. Because dNTPs are natural substrates for RT, more than 20 nucleoside analogues are currently used in vitro, in clinical trials, and in the clinic to inhibit retroviral replication (2, 3). To act as specific RT inhibitors, nucleoside analogues must be converted into 5Ј-triphosphate nucleosides upon phosphorylation by intracellular nucleoside/nucleotide kinases. Most nucleotide analogues do not possess a 3Ј-OH group required for phosphodiester bond formation during DNA synthesis. Therefore, they act as DNA chain terminators once incorporated into the nascent viral DNA.The efficacy of antiretroviral chemotherapy is limited by at least three factors. First, nucleoside analogues have to cross the cellular membrane to be activated to the 5Ј-triphosphate form by cellular kinases. This series of phosphorylation reactions is generally not efficient enough to produce adequate levels of the active nucleoside 5Ј-triphosphate able to compete efficiently with natural dNTPs. Second, RT incorporates nucleotide analogues with, at best, equal efficiency to that of their natural counterparts, i.e. dTTP for both AZTTP and d4TTP. The consequence of these two points is that retroviral replication is not completely suppressed. Third, mutant viruses esca...

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An Integrated System to Study Multiply Substituted Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Boretto

Longhi

Navarro

et al. 2001

Analytical Biochemistry

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The Motif D Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Critical for Nucleoside 5′-Triphosphate Selectivity

Cited by 24 publications

References 35 publications

Haploinsufficiency of t e lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

Haploinsufficiency of t e lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

An Integrated System to Study Multiply Substituted Human Immunodeficiency Virus Type 1 Reverse Transcriptase

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