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.

Development of Human Immunodeficiency Virus Type 1Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor Resistant-Strains

2021 ◽  
Author(s):  
Maria E. Cilento ◽  
Aaron B. Reeve ◽  
Eleftherios Michailidis ◽  
Tatiana V. Ilina ◽  
Eva Nagy ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Rnase H ◽  
Phase Iii ◽  
Wild Type ◽  
Resistance Mutations ◽  
Primary Determinant ◽  
Hiv 1

4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV-1, in Phase III clinical trials. EFdA resistance is not well characterized. To study EFdA-resistance patterns as it may emerge in naïve or tenofovir- (TFV), emtricitabine/lamivudine- (FTC/3TC), or zidovudine- (AZT) treated patients we performed viral passaging experiments starting with wild-type, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless the starting viral sequence, all selected EFdA-resistant variants included the M184V RT mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than WT HIV-1. These mutants also had significantly lower specific infectivity than WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (k cat /K m ) of WT vs. A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data, suggest that EFdA is an excellent therapeutic candidate for naïve, AZT-, FTC/3TC, and especially tenofovir-treated patients.

scholarly journals 4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication

2011 ◽  
Vol 55 (5) ◽  
pp. 2379-2389 ◽  
Author(s):  
B. Christie Vu ◽  
Paul L. Boyer ◽  
Maqbool A. Siddiqui ◽  
Victor E. Marquez ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Cultured Cells ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Resistance Mutations ◽  
Viral Dna ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTIt is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine.In vitroexperiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

scholarly journals Mutations That Abrogate Human Immunodeficiency Virus Type 1 Reverse Transcriptase Dimerization Affect Maturation of the Reverse Transcriptase Heterodimer

2005 ◽  
Vol 79 (16) ◽  
pp. 10247-10257 ◽  
Author(s):  
Johanna Wapling ◽  
Katie L. Moore ◽  
Secondo Sonza ◽  
Johnson Mak ◽  
Gilda Tachedjian
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.

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 Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

2001 ◽  
Vol 75 (10) ◽  
pp. 4832-4842 ◽  
Author(s):  
Paul L. Boyer ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Nucleoside Analogs ◽  
Azido Group ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5′-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

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 L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness

2013 ◽  
Vol 94 (7) ◽  
pp. 1597-1607 ◽  
Author(s):  
Jiong Wang ◽  
Dongge Li ◽  
Robert A. Bambara ◽  
Hongmei Yang ◽  
Carrie Dykes
Keyword(s):  
Reverse Transcriptase ◽  
Resistance Mutation ◽  
Transcriptase Inhibitor ◽  
Rnase H ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Nnrti Resistance ◽  
Reverse Transcriptase Inhibitor ◽  
Subsequent Addition ◽  
Hiv 1

The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

scholarly journals Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Paul L. Boyer ◽  
Kevin Melody ◽  
Steven J. Smith ◽  
Linda L. Dunn ◽  
Chris Kline ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Hydrophobic Region ◽  
Nnrti Resistance ◽  
Hiv 1

ABSTRACTTwo mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation.IMPORTANCEAlthough antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

scholarly journals Highly potent oxathiin carboxanilide derivatives with efficacy against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus isolates.

1997 ◽  
Vol 41 (4) ◽  
pp. 831-837 ◽  
Author(s):  
R W Buckheit ◽  
M J Snow ◽  
V Fliakas-Boltz ◽  
T L Kinjerski ◽  
J D Russell ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
In Vitro Selection ◽  
Nucleoside Analogs ◽  
Side Chain ◽  
Resistant Virus ◽  
Immunodeficiency Virus ◽  
Virus Isolates ◽  
Hiv 1

The structure-activity relationships of a series of compounds related to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) oxathiin carboxanilide have been described (R. W. Buckheit, Jr., T. L. Kinjerski, V. Fliakas-Boltz, J. D. Russell, T. L. Stup, L. A. Pallansch, W. G. Brouwer, D. C. Dao, W. A. Harrison, R. J. Schultz, J. P. Bader, and S. S. Yang, Antimicrob. Agents Chemother. 39:2718-2727, 1996). From these studies, the furanyl-containing analog UC10 was identified as the most potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication and a promising candidate for further development. Three new UC analogs (UC040, UC82, and UC781) have been determined to inhibit laboratory-derived and low-passage-number, primary virus isolates at low nanomolar concentrations in both established and fresh human cells. Each of the compounds synergistically interacted with the nucleoside analogs zidovudine, dideoxyinosine, dideoxycytosine, and lamivudine to inhibit HIV-1 replication. As a group, the UC compounds were found to be less active against viruses with the L100I, K103N, and Y181C amino acid changes in the RT and, upon in vitro selection, yielded resistant virus with the Y181C mutation in the RT. The most potent of the three new compounds, UC781, contains a furanyl side chain, similar to UC10, but differs in having an extended ether side chain instead of an oxime chain. The broad therapeutic index of UC781 (>62,000) resulted in effective inhibition of NNRTI-resistant virus isolates at high nanomolar concentrations. Furthermore, UC781 and the NNRTI costatolide were able to synergistically inhibit HIV-1 replication when used in combination, suggesting that UC781 may interact with the RT differently than the other UC analogs. The favorable anti-HIV properties of the UC compounds suggest they should be considered for further clinical development.

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