AZT-Related Mutation Lys70Arg in Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Confers Decrease in Susceptibility to ddATP inin VitroRT Inhibition Assay

Sharma, P. L.; Chatis, Pamela A.; Dogon, Alex L.; Mayers, Douglas L.; McCutchan, Francine E.; Page, Charlotte; Crumpacker, Clyde S.

doi:10.1006/viro.1996.0620

Cited by 2 publications

(3 citation statements)

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“…HIV-1 RT mutations L74V and M184V have been shown to confer a loss of replication fitness to viruses in human peripheral blood mononuclear cells (2,24,25). Site-directed mutagenesis was performed to create a double mutant proviral clone with L74V and M184V mutations in pNL4-3 background, by using the pALTER mutagenesis system (Promega, Madison, Wis.) (23). Previously described protocols were used for plasmid transfection and viral propagation (23)(24)(25).…”

Section: Methodsmentioning

confidence: 99%

“…Site-directed mutagenesis was performed to create a double mutant proviral clone with L74V and M184V mutations in pNL4-3 background, by using the pALTER mutagenesis system (Promega, Madison, Wis.) (23). Previously described protocols were used for plasmid transfection and viral propagation (23)(24)(25). Briefly, WT pNL4-3 and double-mutant L74V ϩ M184V were transfected in phytohemagglutinin-stimulated human peripheral blood mononuclear cells via electroporation, and the replication of the virus was monitored by measuring RT activity in cell-free supernatants.…”

Section: Methodsmentioning

confidence: 99%

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Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

2003

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The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

2003

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“…Various point mutations were created in the background of proviral clone pNL4-3 (Adachi et al, 1986) by using pALTER −1 mutagenesis system of Promega (Madison, WI) according to manufacturer’s guidelines and our previously described protocols (Sharma et al, 1996; Nurpeisov et al, 2003; Sharma and Crumpacker, 1999; Sharma et al, 2004). To eliminate the possibility of reversion during reverse transcription in mutant K65R+L74V virus, we used primary human embryonic kidney cells (293), R which are non-permissive for HIV-1 infection.…”

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confidence: 99%

Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R+74V

Sharma¹,

Nettles²,

Feldman³

et al. 2009

Antiviral Research

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While HIV-1 reverse transcriptase (RT) mutations of M to V at position 184 are commonly observed in the clinic, the double mutation of 65R+74V is rarely seen. It has been demonstrated that rapid R→K reversion occurs at RT codon 65 during replication of HIV-1 in human peripheral blood mononuclear cells containing 65R+74V mutations and that processivity of the RT is reduced relative to wild type. However, clinical studies show that M184V can be detected after several months of therapy interruption, suggesting more effective processivity. Herein, the in vitro RT processivity of genetically engineered M184V and double mutant 65R+74V was compared. Virion-associated RTs of WT pNL4-3, K65R, L74V, M184V and 65R+74V were used to perform RT processivity assays in the presence of trap, poly(rC)-oligo(dG). Both RTs with 184V and 65R+74V mutations exhibited similar processivity when compared with each other and a significantly decreased processivity as compared to WT RT. Both mutant RTs synthesized shorter cDNA molecules (37–42 nt) relative to WT RT, which made longer (65–70 nt) cDNA molecules. Since these surprising biochemical results cannot explain the clinical phenotype, a hypothesis is presented to explain the discrepancy and suggest new approaches for future testing.

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AZT-Related Mutation Lys70Arg in Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Confers Decrease in Susceptibility to ddATP inin VitroRT Inhibition Assay

Cited by 2 publications

References 0 publications

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R+74V

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