A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of 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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Synergistic Inhibition of HIV-1 Reverse Transcriptase DNA Polymerase Activity and Virus Replication in Vitro by Combinations of Carboxanilide Nonnucleoside Compounds

Biochemistry ◽

10.1021/bi00032a002 ◽

1995 ◽

Vol 34 (32) ◽

pp. 10106-10112 ◽

Cited By ~ 17

Author(s):

Ronald S. Fletcher ◽

Dominique Arion ◽

Gadi Borkow ◽

Mark A. Wainberg ◽

Gary I. Dmitrienko ◽

...

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Virus Replication ◽

Polymerase Activity ◽

Synergistic Inhibition ◽

Hiv 1

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Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

Journal of Virology ◽

10.1128/jvi.02203-17 ◽

2018 ◽

Vol 92 (13) ◽

Cited By ~ 15

Author(s):

Paul L. Boyer ◽

Steven J. Smith ◽

Xue Zhi Zhao ◽

Kalyan Das ◽

Kevin Gruber ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Nucleoside Analogs ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv Replication ◽

Content Type ◽

Rnase Hi ◽

Hiv 1

ABSTRACT We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

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The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis

Journal of Virology ◽

10.1128/jvi.01370-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10906-10910 ◽

Cited By ~ 10

Author(s):

Amnon Hizi

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Leukemia Virus ◽

Polymerase Activity ◽

Rnase H ◽

The Self ◽

Dna Strand ◽

Negative Sense ◽

Ltr Retroelements

ABSTRACT The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

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RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

Journal of Virology ◽

10.1128/jvi.73.8.6573-6581.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6573-6581 ◽

Cited By ~ 24

Author(s):

Christine M. Smith ◽

Jeffrey S. Smith ◽

Monica J. Roth

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Transfer Reaction ◽

Rnase H ◽

Strand Transfer ◽

Transfer Event ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3 Lys.

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Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423900112 ◽

2015 ◽

Vol 112 (22) ◽

pp. 6979-6984 ◽

Cited By ~ 13

Author(s):

Jennifer La ◽

Catherine F. Latham ◽

Ricky N. Tinetti ◽

Adam Johnson ◽

David Tyssen ◽

...

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Competitive Inhibitor ◽

Polymerase Activity ◽

Rnase H ◽

Screening Methods ◽

Klenow Fragment ◽

Resistance Mutations ◽

Rt Inhibitors ◽

Hiv 1

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.

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Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity

Biological Chemistry ◽

10.1515/bc.2010.067 ◽

2010 ◽

Vol 391 (6) ◽

Cited By ~ 3

Author(s):

Sascha N. Stumpp ◽

Bianca Heyn ◽

Susanne Brakmann

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Error Rate ◽

Antiviral Treatment ◽

Rnase H ◽

Single Amino Acid ◽

Selection Scheme ◽

Single Amino Acid Residue ◽

Hiv 1

AbstractHIV-1 reverse transcriptase (HIV-1 RT) copies the RNA genome of HIV-1 into DNA, thereby committing errors at an exceptionally high frequency. Viral offspring evolve rapidly and consequently are capable of evading the immune response as well as antiviral treatment. However, error-prone viral replication could drive HIV close to extinction owing to an intolerable load of deleterious mutations. We applied a genetic selection scheme to identify variants of HIV-1 RT with a further increased error rate to study the relationship between error rate and viral replication. Using this approach, we identified 16 mutator candidates, two of which were purified and further studiedin vitro. One of these variant enzymes showed a generally increased mutation frequency as compared with the reference enzyme. A single amino acid residue, R448, is probably responsible for the observed effect. Mutation of this residue, which is located within the RNase H domain of HIV-1 RT, seems to perturb the interaction with template RNA and consequently affects polymerase activity and fidelity.

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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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