Solution structure sf r(gaggacug):d(CAGTCCTC) hybrid: implications for the initiation of HIV-1(+)-strand synthesis

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the ␣H and ␣I helices in the thumb subdomain play in specific RNase H cleavage at the 3-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3-end. Further analysis of ␣H mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.The virus-encoded enzyme reverse transcriptase (RT) 1 of human immunodeficiency virus type 1 (HIV-1) and other retroviruses catalyzes the conversion of genomic RNA to a doublestranded DNA replicative intermediate. As minus-strand DNA is synthesized, the RNA template is degraded by the RNase H activity of RT, which cleaves the RNA strand in an RNA-DNA hybrid (Refs. 1 and 2; for reviews, see . This results in the production of many small RNA fragments, any one of which could potentially serve as a primer to initiate synthesis of plus-strand DNA (6). However, a short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation (Refs. 7-12, 61; for a review, see Ref. 6). Why the PPT sequence is selected from all of the other available primers has been the subject of much speculation.One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and therefore survives as the sole RNA primer available for plus-strand initiation. However, experimental evidence from HIV-1 (13-15) 2 and murine leukemia virus (MuLV) (9, 16, 17) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3Ј-end of the PPT is lost when the polymerase domain is removed (18,19). Finally, Escherichia coli RNase H catalyzes cleavages within the MuLV PPT (9,16,17,19) as well as within the HIV-1 PPT. 3A second possibility to explain selection of the PPT primer is related to its unique helical structure (12,20) and the shape and width of the major groove, which is wider than that of other RNA-DNA hybrids (20, 21). These structural factors could cause bi...

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confidence: 67%

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confidence: 99%

Residues in the αH and αI Helices of the HIV-1 Reverse Transcriptase Thumb Subdomain Required for the Specificity of RNase H-catalyzed Removal of the Polypurine Tract Primer

Powell¹,

Beard²,

Bębenek³

et al. 1999

“…We suggest that distortion in the (rA) 4 :(dT) 4 tract is induced by the unique PPT sequence. The crystal structure of the A-rich RNA/DNA hybrid r(caaagaaaag): d(CTTTTCTTTG) revealed A-like molecule (30), whereas the NMR structure of the G-rich hybrid r(gaggacug):d(CAGTC-CTC) indicated that the dimensions of the major groove are reminiscent of B-type DNA duplexes (11). Combining these diverse PPT-based structures within one molecule may conceivably lead to the distortion we observe.…”

Section: The Rt Positioning On the 3ј Or 5ј Primer Terminus Is Not Crmentioning

confidence: 91%

“…A crucial role of the guanine-rich segment in the specific hydrolysis of the primer was highlighted by biochemical studies (3,4), which showed that mutations in this region result in imprecise cleavage of the PPT. Furthermore, the NMR structure of an 8-bp RNA/DNA oligonucleotide containing the last four 3Ј-terminal residues of the PPT and the first 4 bp of U3 shows that the width and shape of the major groove are unusual (11), with a bend of ϳ13°between the two halves of this hybrid. Taken together, it is clear that structural studies should encompass the entire PPT rather than its separate constituents when attempting to define the specificity of cleavage at the PPT/U3 junction.…”

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confidence: 99%

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

Kvaratskhelia

Budihas

Grice

2002

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 ...

“…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). However, our chemical footprinting studies (39) suggest an alternative hypothesis.…”

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confidence: 83%

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

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...