Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Smith, Anthony J.; Meyer, Peter R.; Asthana, Deshratn; Ashman, Margarita; Scott, Walter A.

doi:10.1128/aac.49.5.1761-1769.2005

Cited by 30 publications

(46 citation statements)

References 54 publications

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“…However, these altered interactions also decrease the overall interaction with a normal dNTP, an effect that was most evident in the experiments done at low dNTP concentrations. In actively dividing cells, this effect may not be as important; however, HIV-1 RT is known to infect nondividing cells, where the concentration of the four dNTPs can be much lower (3,7,10,33,34). Thus, with the Q151M complex (RT1), high levels of drug resistance may come at the price of decreasing the ability of the virus to replicate in nondividing cells.…”

Section: Figmentioning

confidence: 99%

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

Boyer

Clark

Hughes

2012

J Virol

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bAs anti-HIV therapy becomes more widely available in developing nations, it is clear that drug resistance will continue to be a major problem. The related viruses HIV-1 and HIV-2 share many of the same resistance pathways to nucleoside reverse transcriptase inhibitors (NRTIs). However, clinical data suggest that while HIV-1 reverse transcriptase (RT) usually uses an ATP-dependent excision pathway to develop resistance to the nucleoside analog zidovudine (AZT), HIV-2 RT does not appear to use this pathway. We previously described data that suggested that wild-type (WT) HIV-2 RT has a much lower ability to excise AZT monophosphate (AZTMP) than does WT HIV-1 RT and suggested that this is the reason that HIV-2 RT more readily adopts an exclusion pathway against AZT triphosphate (AZTTP), while HIV-1 RT is better able to exploit the ATP-dependent pyrophosphorolysis mechanism. However, we have now done additional experiments, which show that while HIV-1 RT can adopt either an exclusion-or excision-based resistance mechanism against AZT, HIV-2 RT can use only the exclusion mechanism. All of our attempts to make HIV-2 RT excision competent did not produce an AZT-resistant RT but instead yielded RTs that were less able to polymerize than the WT. This suggests that the exclusion pathway is the only pathway available to HIV-2. HIV-1 infection has been the target of various multidrug therapies, but to date, the effectiveness of all anti-HIV drugs has been blunted by drug-resistant mutations that arise in the genome of the virus. Reverse transcriptase (RT) is an enzyme that contains two enzymatic activities, a DNA polymerase that can copy either an RNA or DNA template, and an RNase H, which degrades RNA if the RNA is part of an RNA/DNA duplex. RT uses these two enzymatic activities to convert the single-stranded RNA genome of the virus into a double-stranded DNA that can be integrated into the genome of the host cell. The synthesis of a DNA copy of the viral genome is a crucial step in the life cycle of the virus, and RT has, for that reason, been the target of a number of different anti-HIV drugs (for reviews, e.g., see references 2, 12, 17, 19, 21, 23, 31, and 32). The earliest anti-HIV therapies involved nucleoside RT inhibitors (NRTIs). These analogs enter the cell and are converted to the triphosphate form (nucleoside RT inhibitor triphosphates [NRTI-TPs]) by host cell kinases. Because the NRTI-TPs are analogs of the normal deoxynucleoside triphosphates (dNTPs), NRTI-TPs are incorporated into the primer strand by RT. However, because the NRTI-TPs do not have a 3=-OH group on the sugar or pseudosugar moiety, an NRTI monophosphate (NRTI-MP) that has been incorporated into viral DNA cannot support continued DNA synthesis and the primer chain is terminated. Decreased susceptibility to NRTIs means that the mutant RT has an enhanced ability to select normal dNTPs over the NRTI-TPs. Two primary mechanisms have been identified by which HIV-1 RT becomes less susceptible to the NRTITPs. One mechanism is exclusion, in which the m...

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Section: Figmentioning

confidence: 99%

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

Boyer

Clark

Hughes

2012

J Virol

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“…The products of the transfer reaction are a dinucleoside tetraphosphate of the form Np 4 ddN and an extendable primer terminus. In vitro, any NTP, deoxynucleoside triphosphate (dNTP), or dideoxyribonucleoside triphosphate (ddNTP) can serve as acceptor, whereas in vivo the acceptor is most likely ATP, resulting in the synthesis of Ap 4 ddN (31). The mechanism by which TAMs give rise to primer unblocking has not been determined, but it has been proposed on the basis of crystal structure and substrate specificity data that the residues affected by TAMs play a role in interaction with the ATP substrate or with the adenosine portion of an intermediate formed during the excision reaction (3,5,22,24).…”

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confidence: 99%

Chain-Terminating Dinucleoside Tetraphosphates Are Substrates for DNA Polymerization by Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Increased Activity against Thymidine Analogue-Resistant Mutants

Meyer¹,

Smith

Matsuura³

et al. 2006

Antimicrob Agents Chemother

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Nucleoside reverse transcriptase inhibitors are an important class of drugs for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Resistance to these drugs is often the result of mutations that increase the transfer of chain-terminating nucleotides from blocked DNA termini to a nucleoside triphosphate acceptor, resulting in the generation of an unblocked DNA chain and synthesis of a dinucleoside polyphosphate containing the chain-terminating deoxynucleoside triphosphate analogue. We have synthesized and purified several dinucleoside tetraphosphates (ddAp 4 ddA, ddCp 4 ddC, ddGp 4 ddG, ddTp 4 ddT, Ap 4 ddG, 2(3)-O-(Nmethylanthraniloyl)-Ap 4 ddG, and AppNHppddG) and show that these compounds can serve as substrates for DNA chain elongation and termination resulting in inhibition of DNA synthesis. Thymidine analogue-resistant mutants of reverse transcriptase are up to 120-fold more sensitive to inhibition by these compounds than is wild-type enzyme. Drugs based on the dinucleoside tetraphosphate structure could delay or prevent the emergence of mutants with enhanced primer unblocking activity. In addition, such drugs could suppress the resistance phenotype of mutant HIV-1 that is present in individuals infected with resistant virus.

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“…This suggests that the levels of ATP that the RT encounters in some infected cells may not be as high as expected (3.0 m⌴). An obvious possibility is that there is a significant amount of HIV replication taking place in nondividing T cells or in other cells where the ATP concentration is Ͻ3 mM (15,18,23,25). If so, the presence of the L210W mutation could allow more efficient binding of ATP at lower concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…The initial excision reactions were performed by using 3.0 mM ATP. This concentration has served as the benchmark by many of the groups who have performed AZTMP excision assays because it is considered to be the physiological concentration of ATP in dividing cells (15,18,23,25). However, this might not be the concentration that the RT sees during polymerization in some infected cells.…”

Section: Discussionmentioning

confidence: 99%

“…We also point out that due to microscopic reversibility, mutations in RT cannot preferentially speed up the excision of AZTMP from the end of the viral DNA using pyrophosphate as an excision substrate without also causing an equivalent increase in the rate at which AZTTP is incorporated. This means that an excision-based mechanism that uses PP i as the excision substrate cannot lead to resistance (17,18).…”

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confidence: 99%

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Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

Boyer

Das

Arnold

et al. 2015

Antimicrob Agents Chemother

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bAlthough anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/ F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3= OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3= end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration. Generally speaking, most mutations in HIV-1 reverse transcriptase (RT) have a negative impact on the enzyme; the primary mutations that cause zidovudine (AZT) resistance are not exceptions. In some cases, secondary mutations are selected in viruses that are replicating in patients because these mutations help to compensate for the deleterious effects of primary resistance mutations. In other cases, the most important effect of a secondary mutation is to cause a further decrease in the susceptibility of the virus to the inhibitor. In this report, we have analyzed the effects of the secondary mutations M41L and L210W, in the presence of the primary mutation T215Y, on both ATP-dependent AZTMP excision and polymerase activities of HIV-1 RT to determine whether these secondary mutations enhance the excision of AZTMP by RT and/or act as compensatory mutations that partially restore the reduced polymerase activity of the T215Y mutant.During the replication of HIV-1, the single-stranded RNA genome is converted into double-stranded DNA by HIV-1 RT. This conversion requires both enzymatic activities of RT: a DNA polymerase that can copy either an RNA or a DNA template and an RNase H that cleaves RNA if, and only if, it is part of an RNA/DNA duplex. Like many DNA polymerases, RT requires both a template and a primer; nucleotides are added sequentially to the 3= end of the primer strand by the polymerase activity of...

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Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Cited by 30 publications

References 54 publications

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

Chain-Terminating Dinucleoside Tetraphosphates Are Substrates for DNA Polymerization by Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Increased Activity against Thymidine Analogue-Resistant Mutants

Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

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