Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Chung, Shinjae; Miller, Jennifer T.; Lapkouski, Mikalai; Tian, Lei; Yang, Wei; Grice, Stuart F. J. Le

doi:10.1074/jbc.m113.465641

Cited by 17 publications

(15 citation statements)

References 32 publications

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“…To expand on our NMR observations, we used DSF to determine the thermal stability of RNH F440A , RNH E438N , RNH F440A/T477A , RNH E438N/T477A , and WT RNH over a range of different buffer pH values (Table ). The WT exhibited a maximum melting temperature ( T m ) of 55.1 ± 2.4°C, at neutral pH, and slightly lower T m values in alkaline buffer conditions.…”

Section: Resultsmentioning

confidence: 99%

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase

et al. 2015

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The mature form of reverse transcriptase (RT) is a heterodimer comprising the intact 66-kDa subunit (p66) and a smaller 51-kDa subunit (p51) that is generated by removal of most of the RNase H (RNH) domain from a p66 subunit by proteolytic cleavage between residues 440/441. Viral infectivity is eliminated by mutations such as F440A and E438N in the proteolytic cleavage sequence, while normal processing and virus infectivity are restored by a compensatory mutation, T477A, that is located more than 10 Å away from the processing site. The molecular basis for this compensatory effect has remained unclear. We therefore investigated structural characteristics of RNH mutants using computational and experimental approaches. Our Nuclear Magnetic Resonance and Differential Scanning Fluorimetry results show that both F440A and E438N mutations disrupt RNH folding. Addition of the T477A mutation restores correct folding of the RNH domain despite the presence of the F440A or E438N mutations. Molecular dynamics simulations suggest that the T477A mutation affects the processing site by altering relative orientations of secondary structure elements. Predictions of sequence tolerance suggest that phenylalanine and tyrosine are structurally preferred at residues 440 and 441, respectively, which are the P1 and P1’ substrate residues known to require bulky side chains for substrate specificity. Interestingly, our study demonstrates that the processing site residues, which are critical for protease substrate specificity and must be exposed to the solvent for efficient processing, also function to maintain proper RNH folding in the p66/p51 heterodimer.

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Section: Resultsmentioning

confidence: 99%

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase

et al. 2015

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“…This is in agreement with a subunit-specific kinetic study of N348I mutant RT showing that the mutation in the p51 subunit decreases RNase H activity 25 . A recent study hypothesized that the loss of a hydrogen bond between N348 and Y427 in p51 by N348I mutation may be responsible for the decreased RNase H activity by the mutant RT 56 . The effects of the N348I/T369I mutations in both subunits are discussed in “Network analysis” section.…”

Section: Resultsmentioning

confidence: 99%

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

2013

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HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations.

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“…Asymmetrical effects in the HIV-1 RT structure further affirmed that mutations in p51 could stabilize the active site on p66, but not vice versa (Figure 2A). Since rigidity reduces HIV-1 RT activity [94], this opens up p51 as a potential new drug target. Given the estimated allosteric-free energies at the DNA polymerase active site (∆∆g site by averaging all ∆∆g residue of the residues involving in the active site to demonstrate stabilizing (∆∆g site < 0) or destabilizing (∆∆g site > 0) effects), it showed that the active site is destabilized by mutations on the thumb domain of p66 (residues 260-321) and on p51 (residues 33-42, 68-78, and 96-114), highlighted in red spheres and red dash ovals in Figure 2B.…”

Section: Analysis Of Allosteric Communicationmentioning

confidence: 99%

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance

Yeo

Goh

et al. 2020

Viruses

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The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) plays a major role in treatment resistance, from the development of vaccines to therapeutic drugs. In addressing the crux of the issue, various attempts to estimate the mutation rate of HIV-1 resulted in a large range of 10−5–10−3 errors/bp/cycle due to the use of different types of investigation methods. In this review, we discuss the different assay methods, their findings on the mutation rates of HIV-1 and how the locations of mutations can be further analyzed for their allosteric effects to allow for new inhibitor designs. Given that HIV is one of the fastest mutating viruses, it serves as a good model for the comprehensive study of viral mutations that can give rise to a more horizontal understanding towards overall viral drug resistance as well as emerging viral diseases.

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Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Cited by 17 publications

References 32 publications

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance

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