Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

2003

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Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (؉) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (؉)-RNA and failure to release (؉)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (؊)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T 3 F substitution and enhanced ribonuclease H (RNase H) activity ϳ12-13 bp downstream. In the most pronounced case, dual T 3 F substitution at PPT positions ؊1/؊2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (؊)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT ϳ12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T 3 F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5 vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper. Although reverse transcriptase (RT)1 -associated ribonuclease H (RNase H) activity degrades RNA of the RNA/DNA replication intermediate with little sequence specificity, it must precisely remove the tRNA and polypurine tract (PPT) primers of (Ϫ)-strand (1) and (ϩ)-strand DNA synthesis (2), respectively, to generate sequences at the 5Ј and 3Ј termini of the double-stranded DNA recognized by the integration machinery (3-8). Since the PPT is most likely embedded in a considerably larger RNA/DNA hybrid, precise hydrolysis at the PPT-U3 junction observed in vitro (9) suggests unique structural features may participate by correctly positioning this junction in the RNase H catalytic center. Our recent chemical footprinting of HIV-1 PPT-containing RNA/DNA hybrids (10) and a comparison with the crystal structure of HIV-1 RT bound to a related duplex (11) support this notion. Nucleic acid in the RT-RNA/DNA co-crystal is distorted 8 -14 bp upstream of the PPT-U3 junction, comprising weakly paired, unpaired, and mispaired bases (Fig. 1A). Subsequent chemical footprinting studies (10) revealed that template thymines of this region and thymine ϩ1 (i.e. immediately 3Ј to the PPT) deviate from standard Watson-Crick base pairing in the absence of the retroviral enzyme. The finding that these naturally occurring HIV-1 PPT distortions are 10 -14 bp apart was particularly intriguing, sinc...

Section: Ty3 Ppt Processingmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Although Reverse Transcriptase (Rt)mentioning

confidence: 99%

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Nonpolar Thymine Isosteres in the Ty3 Polypurine Tract DNA Template Modulate Processing and Provide a Model for Its Recognition by Ty3 Reverse Transcriptase

Lener

Kvaratskhelia

2003

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“…Because the bulk of the replication intermediate is nonspecifically hydrolyzed, structural features of the PPT (a) render it RNase H-insensitive and (b) control precise cleavage at the junction with adjacent U3 DNA or RNA sequences (1). Although several reports have studied PPT processing relative to alterations in its sequence (2)(3)(4)(5)(6) or that of the cognate retroviral polymerase (7)(8)(9), the structural basis for this remains elusive. The recent structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to a PPTcontaining RNA/DNA duplex has provided important mechanistic insights regarding the PPT resistance to hydrolysis (10).…”

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confidence: 99%

Pre-existing Distortions in Nucleic Acid Structure Aid Polypurine Tract Selection by HIV-1 Reverse Transcriptase

Kvaratskhelia

Budihas

2002

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Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO 4 ) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine ؉1 is detected when the PPT primer is extended by DNA or RNA at its 3 terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the ؉1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA) 4 :(dT) 4 or (rG) 6 :(dC) 6 tracts. Importantly, there is a correlation between the structural deformation at base pair ؉1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO 4 modification also revealed unusually high reactivity for one of two (dT) 4 : (rA) 4 duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.In retroviruses, the accuracy with which the (ϩ) strand, polypurine tract (PPT) 1 primer is selected from the RNA-DNA replication intermediate and excised from nascent (ϩ) strand DNA defines the 5Ј long terminal repeat terminus and is critical for production of an integration-competent provirus (1). Because the bulk of the replication intermediate is nonspecifically hydrolyzed, structural features of the PPT (a) render it RNase H-insensitive and (b) control precise cleavage at the junction with adjacent U3 DNA or RNA sequences (1). Although several reports have studied PPT processing relative to alterations in its sequence (2-6) or that of the cognate retroviral polymerase (7-9), the structural basis for this remains elusive. The recent structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to a PPTcontaining RNA/DNA duplex has provided important mechanistic insights regarding the PPT resistance to hydrolysis (10). These authors identified an unusual distortion within the (rA) 4 :(dT) 4 stretches of the PPT, i.e. misaligned base pairing between the template and primer nucleotides, suggesting that extension of this deformation into the RNase H active site may confer resistance to hydrolysis. The crystal structure also revealed extensive contacts between the RNase H "primer grip" and the RNA/DNA hybrid. However, because a structure of the complete RNA/DNA duplex PPT in the absence of RT is unavailable, it ...

“…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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confidence: 99%

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

Dash

Yi-Brunozzi

2004

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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...