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

2010 ◽  
Vol 391 (6) ◽  
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.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

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.

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

2014 ◽  
Vol 58 (8) ◽  
pp. 4515-4526 ◽  
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.

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

2007 ◽  
Vol 52 (1) ◽  
pp. 329-332 ◽  
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.

scholarly journals Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

2007 ◽  
Vol 81 (15) ◽  
pp. 7852-7859 ◽  
Author(s):  
Jessica H. Brehm ◽  
Dianna Koontz ◽  
Jeffrey D. Meteer ◽  
Vinay Pathak ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Cross Resistance ◽  
Polymerase Domain ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by Degradation Products of Ceftazidime

1997 ◽  
Vol 8 (4) ◽  
pp. 353-362 ◽  
Author(s):  
SW Baertschi ◽  
AS Cantrell ◽  
MT Kuhfeld ◽  
U Lorenz ◽  
DB Boyd ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Degradation Products ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Hiv 1

Previous work by Hafkemeyer et al. (1991) [ Nucleic Acids Research19: 4059–4065] indicated that a degradation product of ceftazidime, termed HP 0.35, was active against the RNase H activity of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptase (RT) in vitro. Attempting to repeat these results, we isolated HP 0.35 from an aqueous degradation of ceftazidime and, after careful purification, we found HP 0.35 to be essentially inactive against both the polymerase and RNase H domains of HIV-1 RT (IC50 of >100 μg mL−1). During the investigation we discovered that polymeric degradation products of ceftazidime inhibited both the polymerase and, to a greater extent, the RNase H activities of HIV-1 RT in vitro (IC50 approximately 0.1 and 0.01 μg mL−1, respectively). Subjecting HP 0.35 to conditions under which it could polymerize induced inhibitory activity similar to that of the polymeric ceftazidime degradation products. It is proposed that the previously reported activity of HP 0.35 may have resulted from the presence of low levels of polymeric material either from incomplete purification or from polymerization of HP 0.35 during storage or in vitro testing.

scholarly journals The Base Component of 3′-Azido-2′,3′-Dideoxynucleosides Influences Resistance Mutations Selected in HIV-1 Reverse Transcriptase

2011 ◽  
Vol 55 (8) ◽  
pp. 3758-3764 ◽  
Author(s):  
Jeffrey D. Meteer ◽  
Dianna Koontz ◽  
Ghazia Asif ◽  
Hong-wang Zhang ◽  
Mervi Detorio ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nucleoside Analogs ◽  
Rnase H ◽  
Major Determinant ◽  
Site Directed Mutagenesis ◽  
Wild Type Virus ◽  
Wild Type ◽  
Resistance Mutations ◽  
Hiv 1

ABSTRACTWe recently reported that HIV-1 resistant to 3′-azido-3′-deoxythymidine (AZT) is not cross-resistant to 3′-azido-2′,3′-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conductedin vitroselection experiments by serial passage of HIV-1LAIin MT-2 cells in increasing concentrations of 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG), 3′-azido-2′,3′-dideoxycytidine (3′-azido-ddC), or 3′-azido-2′,3′-dideoxyadenosine (3′-azido-ddA). 3′-Azido-ddG selected for virus that was 5.3-fold resistant to 3′-azido-ddG compared to wild-type HIV-1LAIpassaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ∼2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3′-azido-ddG resistance. In contrast to 3′-azido-ddG, 3′-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3′-azido-ddA, even at concentrations of 3′-azido-ddA that yielded high intracellular levels of 3′-azido-ddA-5′-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

scholarly journals The Nucleoside Analog BMS-986001 Shows GreaterIn VitroActivity against HIV-2 than against HIV-1

2015 ◽  
Vol 59 (12) ◽  
pp. 7437-7446 ◽  
Author(s):  
Robert A. Smith ◽  
Dana N. Raugi ◽  
Vincent H. Wu ◽  
Sally S. Leong ◽  
Kate M. Parker ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Treatment Options ◽  
Nucleoside Analog ◽  
Single Amino Acid ◽  
Intrinsic Resistance ◽  
Immunodeficiency Virus ◽  
Treatment Naïve ◽  
Hiv 1

ABSTRACTTreatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated thein vitroactivity of the investigational nucleoside analog BMS-986001 (2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4′-ethynyl stavudine, or 4′-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture.

Cleavage of the HIV-1 p66 reverse transcriptase/RNase H by the p9 protease in vitro generates active p15 RNase H

1991 ◽  
Vol 118 (3-4) ◽  
pp. 179-188 ◽  
Author(s):  
T. Schulze ◽  
M. Nawrath ◽  
Karin Moelling
Keyword(s):  
Reverse Transcriptase ◽  
Rnase H ◽  
Hiv 1

scholarly journals The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity

2010 ◽  
Vol 54 (6) ◽  
pp. 2401-2408 ◽  
Author(s):  
Hong-Tao Xu ◽  
Yudong Quan ◽  
Susan M. Schader ◽  
Maureen Oliveira ◽  
Tamara Bar-Magen ◽  
...  
Keyword(s):  
Viral Replication ◽  
Reverse Transcriptase ◽  
First Generation ◽  
Resistance Mutation ◽  
Transcriptase Inhibitor ◽  
Rnase H ◽  
Replication Capacity ◽  
Dna And Rna ◽  
Enzymatic Function ◽  
Hiv 1

ABSTRACT The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.

scholarly journals HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

2019 ◽  
Vol 2019 ◽  
pp. 1-28 ◽  
Author(s):  
Ekaterina Bayurova ◽  
Juris Jansons ◽  
Dace Skrastina ◽  
Olga Smirnova ◽  
Dzeina Mezale ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Reverse Transcriptase ◽  
Immune Suppression ◽  
Viral Infections ◽  
Kaposi Sarcoma ◽  
Rnase H ◽  
Infected People ◽  
Increased Risk ◽  
Hiv 1

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

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