Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3′-azido-2′,3′-deoxythymidine-resistant HIV isolates

2002 ◽  
Vol 35 (3) ◽  
pp. 155 ◽  
Author(s):  
Xing-Wu Shao ◽  
Sandra Hjalmarsson ◽  
Johan Lennerstrand ◽  
Bo Svennerholm ◽  
Jonas Blomberg ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Chain Termination

Application of a Chain Termination Assay for Characterization of Reverse Transcriptase from AZT-Resistant HIV Isolates

1996 ◽  
Vol 7 (6) ◽  
pp. 313-320 ◽  
Author(s):  
J. Lennerstrand ◽  
A.-S. Rytting ◽  
L. Vrang ◽  
J.S. Gronowitz ◽  
C.F.R. Källander
Keyword(s):  
Cell Culture ◽  
Reverse Transcriptase ◽  
Sequence Data ◽  
Chain Termination ◽  
Assay System ◽  
Resistant Isolate ◽  
Wild Type ◽  
Resistance Mutations ◽  
A Chain

An enzymatic assay based on utilization of one primer/enzyme molecule was specifically designed for evaluation of the chain termination capacity of reverse transcriptase (RT) from HIV-1 isolates. In this assay system (CT assay) there was a 3.2-fold difference between the AZT-triphosphate (AZT-TP) concentrations required to terminate 50% of the primers (TC50) for a highly resistant isolate, carrying the four common mutations at positions 67, 70, 215, and 219; and two wild type isolates. Two of three other isolates with reduced sensitivity to AZT in cell culture exhibited intermediate values in CT assay, while one behaved as the wild type isolates. There was a correlation P = 0.05 ( r = 0.86, n = 6) between the ED50 values found in cell culture and the TC50 values found in CT assay. This relationship was not found in a similar assay system which measured competition between AZT-TP and 1 μM tymidine triphosphate (TTP) at the enzymatic level. The sequence data of the current isolates gave some information concerning which mutations in the RT gene specifically affect the enzymatic properties measured in CT assay. Mutation only at amino acid 70 had no effect, but the TC50 values found increased with accumulation of the other common AZT resistance mutations.

scholarly journals Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains.

1985 ◽  
Vol 260 (14) ◽  
pp. 8243-8249 ◽  
Author(s):  
D Grandgenett ◽  
T Quinn ◽  
P J Hippenmeyer ◽  
S Oroszlan
Keyword(s):  
Reverse Transcriptase ◽  
Structural Characterization

scholarly journals High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Betty Ha ◽  
Kevin P. Larsen ◽  
Jingji Zhang ◽  
Ziao Fu ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Viral Entry ◽  
Reverse Transcription ◽  
Molecular Mechanisms ◽  
Dissociation Kinetics ◽  
Structural Mechanism ◽  
Medium Resolution ◽  
Kinetics Of ◽  
Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

scholarly journals Characterization of Active Reverse Transcriptase and Nucleoprotein Complexes of the Yeast Retrotransposon Ty3in Vitro

1999 ◽  
Vol 274 (51) ◽  
pp. 36643-36648 ◽  
Author(s):  
Gaël Cristofari ◽  
Caroline Gabus ◽  
Damien Ficheux ◽  
Marion Bona ◽  
Stuart F. J. Le Grice ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nucleoprotein Complexes ◽  
Yeast Retrotransposon

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors

1989 ◽  
Vol 23 (suppl A) ◽  
pp. 47-54 ◽  
Author(s):  
M. Tisdale ◽  
B. A. Larder ◽  
D. M. Lowe ◽  
D. K. Stammers ◽  
D. J. M. Purifoy ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Structural Characterization ◽  
Hiv Reverse Transcriptase ◽  
Specific Virus

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

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