Slide into Action: Dynamic Shuttling of HIV Reverse Transcriptase on Nucleic Acid Substrates

Liu, Shixin; Abbondanzieri, Elio A.; Rausch, Jason W.; Grice, Stuart F. J. Le; Zhuang, Xiaowei

doi:10.1126/science.1163108

Cited by 148 publications

(147 citation statements)

References 46 publications

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“…Because NNRTIs potently and preferentially inhibit initiation of HIV-1 (ϩ)-strand DNA synthesis (66), SMS suggests that this might be due in part to their ability to induce enzyme binding in a polymerase-incompetent mode. Consistent with the notion that NNRTIs occupy a hydrophobic pocket at the base of the p66 thumb, thereby "loosening" the grip on nucleic acid, SMS also demonstrated increased "sliding" of HIV-1 RT on the template in the presence of NVP (67). Although an unresolved feature of this and related studies (68) was the ability of HIV-1 RT to adopt alternative orientations (collectively referred to as "flipping") without dissociating from its nucleic acid substrate, our work demonstrated the value of SMS in dissecting the intricate events of reverse transcription.…”

Section: Conformational Dynamics Of Reverse Transcriptionsupporting

confidence: 65%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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Synthesis of integration-competent, double-stranded DNA from the (؉)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA-and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (؊)-and (؉)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. HIV-1 DNA SynthesisThe individual steps of HIV-1 DNA synthesis, catalyzed by the multifunctional reverse transcriptase (RT), 2 are summarized schematically in Fig. 1. (Ϫ)-Strand DNA synthesis, initiated from a cellular tRNA (tRNA 3 Lys ) hybridized to the genomeencoded primer-binding site, continues to the 5Ј terminus, creating (Ϫ)-strong-stop DNA. RNase H-mediated degradation of the resulting RNA/DNA hybrid promotes relocation of nascent (Ϫ)-DNA to the genome 3Ј terminus by a strand transfer event that exploits sequence homology between the 5Ј and 3Ј termini. RNA-templated DNA synthesis continues, accompanied by RNase H-mediated degradation of the RNA genome, the exception to which are two short purine-rich segments (the 3Ј-and central polypurine tracts (PPTs)) from which (ϩ)-strand DNA-dependent DNA synthesis is initiated. Newly synthesized (Ϫ)-strand DNA and 18 nucleotides of the covalently attached tRNA 3 Lys primer provide the template for 3Ј-PPTprimed (ϩ)-strand DNA synthesis until the replication complex stalls at a position corresponding to the first modified tRNA base (A57). As a consequence, the C-terminal RNase H domain is positioned at the (Ϫ)-DNA/tRNA junction, and degradation of the tRNA "template" promotes a second or (ϩ)-strand transfer event supported by homology between (Ϫ)-and (ϩ)-strand DNA primer-binding sites. Although bidirectional DNA synthesis would be sufficient to complete DNA synthesis, HIV utilizes a second, central PPT primer, thereby producing a (ϩ)-strand discontinuity (1). Following (ϩ)-strand transfer, 3Ј-PPT-mediated DNA synthesis continues, displacing ϳ100 nucleotides of central PPT-primed (ϩ)-DNA, but abruptly ceases at the central termination sequence, a prominent feature of which is phased A-tracts that induce minor groove compression (1-3), creating the "central flap" (Fig. 1) (4 -7). Although central flap function remains controversial (8, 9), its mutation or deletion has been shown to impair...

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Section: Conformational Dynamics Of Reverse Transcriptionsupporting

confidence: 65%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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“…Individual RNA molecules exchange rarely between the two conformational states for the wild-type sequence, demonstrating that there exists a high-energy barrier for rearrangement of the RNA conformation. Prior single-molecule FRET studies on RT-RNA dynamics, using FRET between RT and RNA Liu et al 2008Liu et al , 2010, have highlighted this conformational heterogeneity as well. Our results show unambiguously that the HIV initiation complex exists in two conformational states, reconciling conflicting results and models of the initiation complex.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

Coey

Larsen

Puglisi

et al. 2016

RNA

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Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5 ′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNA Lys 3 . Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

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“…NNRTIs appear to promote the latter orientation, which inhibits DNA synthesis and facilitates the primer removal. Fluorescence resonance energy transfer-based singlemolecule studies provide strong support for this notion (39,40). Here, we asked whether NNRTI resistance-conferring mutations have the potential to reverse these effects, which could provide a novel mechanism of resistance to this class of inhibitors.…”

Section: Hiv-1mentioning

confidence: 99%

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

Biondi

Beilhartz

McCormick

et al. 2010

Journal of Biological Chemistry

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Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (؉)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.

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Slide into Action: Dynamic Shuttling of HIV Reverse Transcriptase on Nucleic Acid Substrates

Cited by 148 publications

References 46 publications

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

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