Drug Targeting of HIV-1 RNA·DNA Hybrid Structures:  Thermodynamics of Recognition and Impact on Reverse Transcriptase-Mediated Ribonuclease H Activity and Viral Replication†

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02729-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4515-4526 ◽

Cited By ~ 2

Author(s):

Hong-Tao Xu ◽

Susan P. Colby-Germinario ◽

Maureen Oliveira ◽

Daniel Rajotte ◽

Richard Bethell ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Rnase H ◽

Hiv Reverse Transcriptase ◽

Compound A ◽

Enzymatic Function ◽

Biochemical Assays ◽

Rt Inhibitors ◽

The Impact ◽

Hiv 1

ABSTRACTA W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.

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Human Immunodeficiency Virus Types 1 and 2 Exhibit Comparable Sensitivities to Zidovudine and Other Nucleoside Analog Inhibitors In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01004-07 ◽

2007 ◽

Vol 52 (1) ◽

pp. 329-332 ◽

Cited By ~ 27

Author(s):

Robert A. Smith ◽

Geoffrey S. Gottlieb ◽

Donovan J. Anderson ◽

Crystal L. Pyrak ◽

Bradley D. Preston

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Replication ◽

Reverse Transcriptase ◽

Antiviral Therapy ◽

Nucleoside Analogs ◽

Nucleoside Analog ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Indicator Cell

ABSTRACT Using an indicator cell assay that directly quantifies viral replication, we show that human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) exhibit similar sensitivities to 3′-azido-3′-deoxythymidine (zidovudine) as well as other nucleoside analog inhibitors of reverse transcriptase. These data support the use of nucleoside analogs for antiviral therapy of HIV-2 infection.

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Pyrrolyl Pyrazoles as Non-Diketo Acid Inhibitors of the HIV-1 Ribonuclease H Function of Reverse Transcriptase

ACS Medicinal Chemistry Letters ◽

10.1021/acsmedchemlett.9b00617 ◽

2020 ◽

Vol 11 (5) ◽

pp. 798-805 ◽

Cited By ~ 2

Author(s):

Antonella Messore ◽

Angela Corona ◽

Valentina Noemi Madia ◽

Francesco Saccoliti ◽

Valeria Tudino ◽

...

Keyword(s):

Reverse Transcriptase ◽

Ribonuclease H ◽

Hiv 1

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Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H

Physical Chemistry Chemical Physics ◽

10.1039/c8cp01843j ◽

2018 ◽

Vol 20 (37) ◽

pp. 23873-23884 ◽

Cited By ~ 16

Author(s):

Fengyuan Yang ◽

Guoxun Zheng ◽

Tingting Fu ◽

Xiaofeng Li ◽

Gao Tu ◽

...

Keyword(s):

Drug Resistance ◽

Reverse Transcriptase ◽

Binding Mode ◽

Hiv Treatment ◽

Rnase H ◽

Ribonuclease H ◽

Resistance Profile ◽

Anti Hiv ◽

Hiv 1 ◽

Dual Target

The recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 IN and RNase H is beneficial to counteract the failure of anti-HIV treatment due to drug resistance.

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