Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Wisniewski, Michele; Balakrishnan, Mini; Palaniappan, Chockalingam; Fay, Philip J.; Bambara, Robert A.

doi:10.1073/pnas.210392297

Cited by 71 publications

(80 citation statements)

References 29 publications

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“…5b). This orientation clearly cannot support primer extension activity but directly explains the primary RNase-H-cleavage mode observed previously on similar substrates, in which the cleavage site is 18 nt from the 59 terminus of the RNA 17 . The two opposite binding orientations on DNA and RNA primers were also observed on primers encoding an alternative sequence ( Supplementary Fig.…”

mentioning

confidence: 63%

“…The mechanism by which RT discriminates between these substrates and executes the appropriate catalytic function is, however, poorly understood. Although RNase H cleavage analysis suggests the presence of different interaction modes of RT with substrates 16,17 , crystal structures have so far revealed only one enzyme-binding orientation 4,[18][19][20][21][22] .…”

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confidence: 99%

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Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

171

216

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The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

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confidence: 63%

mentioning

confidence: 99%

Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

171

216

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“…This initial fragment is further processed into 8 -10-nt-long smaller fragments, which can dissociate from the template. We refer to the 18 -19-nt and 8 -10-nt cuts as the primary and the secondary cuts, respectively, based on their rate of formation (55). On blunt-ended hybrid substrates, where the RNA 5Ј-end is flush with the DNA 3Ј-end, secondary cleavages are less efficient and stimulated by NC protein (43).…”

Section: Resultsmentioning

confidence: 99%

“…For use in RNase H assays, RNA templates were first dephosphorylated by calf intestine phosphatase and then 5Ј-end-labeled using [␥-32 P]ATP (6000 Ci (222 TBq)/mmol) and polynucleotide kinase. For polymerase-independent RNase H assays, the labeled donor RNA was annealed to the DNA template at a ratio of 1:3 as previously described (55).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Chen¹,

Balakrishnan²,

Roques

et al. 2003

Journal of Biological Chemistry

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Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5 -end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5 -end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5 terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5 terminus. Transfer was unaffected when donor 5 -end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5 -end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication (see Ref. 1 and reference therein). This process is catalyzed by the virus-encoded enzyme reverse transcriptase (RT). 1 RT has two enzymatic activities, the DNA polymerase and the RNase H activities. Reverse transcription is initiated by the partial annealing between the cellular tRNA Lys3 primer and the viral primer binding site, which is located near the 5Ј-end of the viral RNA. Synthesis proceeds to the 5Ј-end of the genomic RNA, creating the minus strand strong stop DNA (ϪsssDNA), which comprises all of the U5 and R sequences from the 5Ј-untranslated region. During minus strand synthesis, RT simultaneously cleaves the copied RNA using its RNase H activity. This frees the ϪsssDNA, enabling its transfer to the homologous R sequence in the 3Ј-untranslated region and continuation of minus strand synthesis. This step is called minus strand strong stop transfer (minus strand transfer), and it is obligatory for reverse transcription and viral replication. Extensive studies have identified the RT-RNase H activity, R sequence homology, and viral nucleocapsid (NC) protein as important components for minus strand transfer (see Ref. 2 and references therein).Viruses with defective RNase H fail to complete reverse transcription, particularly the minus strand transfer step, indicating that RNase H is required for the transfer event (3-8). HIV-1 RT has been shown to partially cleave the template RNA during polymerization, leaving RNA fragments annealed to the cDNA (9, 10). Since there a...

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“…Several efforts have been made in this sense during the last decade, and various mechanisms have been proposed, based either on the infection of cells in culture (ex vivo systems) (7,8) or on the reconstitution of the process of reverse transcription with purified proteins and nucleic acids (in vitro systems) (9 -12). Some hard facts have been jointly established by these two approaches, including the enhancement of template switching observed by decreasing the rate of DNA synthesis (13,14) and the importance of a temporal coupling of RT-encoded polymerase and RNaseH activities (8,15). However, detailed mechanistic models, mostly proposed solely on the basis of in vitro studies, still await an evaluation of their physiological relevance.…”

mentioning

confidence: 99%

The Structure of HIV-1 Genomic RNA in the gp120 Gene Determines a Recombination Hot Spot in Vivo

Galetto¹,

Moumen²,

Giacomoni³

et al. 2004

Journal of Biological Chemistry

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By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

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Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Cited by 71 publications

References 29 publications

Dynamic binding orientations direct activity of HIV reverse transcriptase

Dynamic binding orientations direct activity of HIV reverse transcriptase

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

The Structure of HIV-1 Genomic RNA in the gp120 Gene Determines a Recombination Hot Spot in Vivo

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