Analysis of Plus-strand Primer Selection, Removal, and Reutilization by Retroviral Reverse Transcriptases

Schultz, Sharon J.; Zhang, Miaohua; Kelleher, Colleen D.; Champoux, James J.

doi:10.1074/jbc.m000021200

Cited by 27 publications

(51 citation statements)

References 77 publications

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“…Despite the ability of reverse transcriptase to carry out DNA displacement synthesis at a nick (21,25,43), the PPT primer is not efficiently reutilized for additional plus-strand synthesis (19,37). A similar situation might arise when RNase H creates the PPT primer if RNase H were to make only a single cleavage at the Ϫ1/ϩ1 site.…”

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confidence: 85%

“…An example is the generation of the PPT primer: RNase H efficiently generates the 3Ј end of the PPT primer by a specific cleavage within the PPT at the plus-strand origin, herein referred to as the Ϫ1/ϩ1 site. Importantly, this RNase H cleavage is sequence specific and can occur internally on an RNA-DNA hybrid without 5Ј end-directed binding (14,37). Sequence recognition directly contributes to RNase H specificity for at least two other sites as well.…”

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confidence: 99%

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Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Schultz

Zhang

Champoux

2003

J Virol

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Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3 terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3 terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3 terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3 end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3 terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3 terminus.Shortly after entrance of the viral cores into the cytoplasm of a cell, the single-stranded plus-sense RNA genome of a retrovirus is converted into a double-stranded DNA molecule that subsequently integrates into the host cell genome (4). This process, termed reverse transcription, requires two distinct RNA primers to synthesize the double-stranded DNA. The first primer is a host cell-derived tRNA that is used for the initiation of minus-strand DNA synthesis. The second is a short RNA derived by RNase H cleavages within a purine-rich sequence in the viral genome called the polypurine tract (PPT). This primer is used to begin plus-strand synthesis and is referred to as the plus-strand primer or the PPT primer (reference 2 and references therein).These RNA primers are extended by the viral-encoded reverse transcriptase, a multifunctional enzyme that carries out DNA polymerization, strand displacement synthesis, and the strand transfer reaction and that possesses an RNase H activity required at several steps during genome replication (4). The DNA polymerase activity of reverse transcriptase resides in the N-terminal two-thirds of the protein and utilizes either RNA or DNA as a template. Although displacement synthesis is not as efficient as nondisplacement synthesis (13,15,25,43), reverse transcriptase can simultaneously extend the 3Ј terminus of a DNA primer and displace a downstream nontemplate RNA or DNA strand, a...

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Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Schultz

Zhang

Champoux

2003

J Virol

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“…With respect to the 3Ј PPT, this mechanism creates 5Ј-terminal sequences of the double-stranded DNA for recognition by viruscoded integrase. Although selection and removal of the PPT primer have been accurately recapitulated in biochemical studies with wild type and mutant RT variants (7,20,(23)(24)(25)(26)(27)(28)(29)(30)(31), the structural basis for resistance to internal hydrolysis and specific cleavage at the PPT-U3 junction remains elusive. Analysis of a co-crystal of HIV-1 RT and a PPT-containing RNA/DNA hybrid (4) suggest that the altered groove width observed might not permit correct positioning of the RNA strand in the RNase H catalytic center, a notion put forward by studies of related RNA/DNA hybrids (32)(33)(34)(35)(36)(37)(38).…”

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Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/ DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (؊) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/ DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA⅐rU tract immediately 5 of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT⅐rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC⅐rG and neighboring dT⅐rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3 -end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (؉5/؉6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 ؉ 5/؉6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.Minus (Ϫ)-strand DNA synthesis in retroviruses is accompanied by degradation of viral RNA of the RNA/DNA replication intermediate. These are events mediated by the N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) 1 catalytic centers of the multifunctional reverse transcriptase (RT), respectively (1). Correct positioning of nucleic acids to facilitate each of these steps clearly requires its specific interaction with structural motifs of the virus-coded polymerase. Crystallographic (2-4) and biochemical analysis (5-13) of human immunodeficiency virus type 1 (HIV-1) RT identified the primer grip of the p66 thumb as a motif critical for positioning the primer 3ЈOH before nucleophilic attack on the incoming dNTP. Immediately adjacent to this motif, the minor groove binding track (MGBT) or translocation track is proposed to mediate translocation of the polymerization machinery and act as a sensor of nucleic acid geometry (14 -19). Recently, it was proposed that the RNase H primer grip (RHPG), involving portions of the p66 connection subdomain and RNase H domain, interacts with the DNA strand of an RNA/DNA hybrid, providing the RNA with a trajectory appropriate for hydrolysis within the RNase H catalytic center (4, 20, 21). These combined activities effectively prepare nascent (Ϫ) DNA for use as template for plus (ϩ)-strand, DNA-dependent DNA synthesis.A critical...

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“…Cleavage site specificity is required for proper generation and removal of the PPT (18,26,38,41,45,47,50,52,53,54,57,58,64,75). Previous studies have demonstrated that certain residues within the PPT and its overall helical structure are important determinants for specific cleavage and extension (21,26,33,41,51,55,60,63). The sites of plus-and minus-strand initiation are important because they ultimately define the ends of the full-length proviral DNA, which are recognized by the viral integrase (5,62; reviewed in reference 11).…”

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Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

Miles

Agresta

Khan

et al. 2005

J Virol

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We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFS⌬nef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5 end appeared to have less of an effect on infectivity than mutation of the 3 end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3 end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.After entry of the human immunodeficiency virus type 1 (HIV-1) virion into the host cell, its single-stranded RNA genome is converted into a double-stranded DNA intermediate through the process of reverse transcription (reviewed in reference 11). This process, catalyzed by a virally encoded reverse transcriptase (RT), requires a primer for each strand of DNA to be synthesized. The primer for minus-strand DNA synthesis is provided by a cellular tRNA (tRNA 3 Lys in the case of HIV-1), which is selectively incorporated into the virion during budding (29, 34, 42; reviewed in references 11 and 43). This primer binds to the viral RNA at a region known as the primer-binding site (PBS) (37, 59, 72) to initiate minus-strand synthesis. The plus-strand DNA primer is provided by a 15-nucleotide (nt) purine-rich viral RNA sequence known as the polypurine tract (PPT; 5Ј-AAAAGAAAAGGGGGG-3Ј) (reviewed in reference 56), which is generated from viral RNA by the RNase H activity of RT (18,41,45,47,54,55,58,64). The PPT is highly conserved in most retroviruses and has been shown to be selectively used as the site of plus-strand initiation (18,21,26,41,45,47,51,54,55,67). Cleavage site specificity is required for proper generation and removal of the PPT (18,26,38,41,45,47,50,52,53,54,57,58,64,75). Previous studies have demonstrated that certain residues within the PPT and its overall helical structure are important determinants for specific cleavage and extension ...

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Analysis of Plus-strand Primer Selection, Removal, and Reutilization by Retroviral Reverse Transcriptases

Cited by 27 publications

References 77 publications

Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

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