Effect of RNA Secondary Structure on the Kinetics of DNA Synthesis Catalyzed by HIV-1 Reverse Transcriptase†

Biochemistry ◽  
1997 ◽  
Vol 36 (41) ◽  
pp. 12459-12467 ◽  
Author(s):  
Zucai Suo ◽  
Kenneth A. Johnson
Keyword(s):  
Secondary Structure ◽  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Rna Secondary Structure ◽  
Kinetics Of ◽  
Hiv 1

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 Structures of Complexes Formed by HIV-1 Reverse Transcriptase at a Termination Site of DNA Synthesis

2001 ◽  
Vol 276 (33) ◽  
pp. 31439-31448 ◽  
Author(s):  
Marc Lavigne ◽  
Lucette Polomack ◽  
Henri Buc
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Hiv 1

scholarly journals Mutating a Region of HIV-1 Reverse Transcriptase Implicated in tRNALys-3Binding and the Consequences for (−)-Strand DNA Synthesis

1998 ◽  
Vol 273 (23) ◽  
pp. 14523-14532 ◽  
Author(s):  
Eric J. Arts ◽  
Jennifer T. Miller ◽  
Bernard Ehresmann ◽  
Stuart F. J. Le Grice
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Hiv 1

The double mutation L109M and R448M of HIV-1 reverse transcriptase decreases fidelity of DNA synthesis by promoting mismatch elongation

2015 ◽  
Vol 396 (12) ◽  
pp. 1315-1323
Author(s):  
Bianca Heyn ◽  
Nicole Pogodalla ◽  
Susanne Brakmann
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Error Rate ◽  
Double Mutant ◽  
Dissociation Rate ◽  
Double Mutation ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Simultaneous Substitution

Abstract Changes of Leu109 and Arg448 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have as yet not been associated with altered fitness. However, in a recent study, we described that the simultaneous substitution of L109 and R448 by methionine leads to an error-producing polymerase phenotype that is not observed for the isolated substitutions. The double mutant increased the error rate of DNA-dependent DNA synthesis 3.1-fold as compared to the wildtype enzyme and showed a mutational spectrum with a fraction of 28% frameshift mutations and 48% transitions. We show here that weaker binding of DNA:DNA primer-templates as indicated by an increased dissociation rate constant (koff) could account for the higher frameshift error rate. Furthermore, we were able to explain the prevalence of transition mutations with the finding that HIV-1 RT variant L109M/R448M preferred misincorporation of C opposite A and elongation of C:A mismatches.

scholarly journals Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis

2007 ◽  
Vol 81 (9) ◽  
pp. 4798-4807 ◽  
Author(s):  
David C. Thomas ◽  
Yegor A. Voronin ◽  
Galina N. Nikolenko ◽  
Jianbo Chen ◽  
Wei-Shau Hu ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Ex Vivo ◽  
Dna Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Specific Amplification ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

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