Identification of a sequence element immediately upstream of the polypurine tract that is essential for replication of simian immunodeficiency virus

Ilyinskii, Petr O.; Desrosiers, Ronald C.

doi:10.1093/emboj/17.13.3766

Cited by 51 publications

(51 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1, A and B, respectively. Because all four nucleobases were commercially available, this allowed us to evaluate the homopolymeric dA⅐rU tract immediately adjacent to the PPT, a region whose alteration affects PPT usage in HIV-1 (53), Moloney murine leukemia virus (51), and simian immunodeficiency virus (50). Although dC was commercially available as the 5-methyl derivative, x-ray crystallography (4) suggests that relatively few contacts are made to nucleobases of the RNA/ DNA hybrid, suggesting steric interference would be minimal.…”

Section: Resultsmentioning

confidence: 99%

“…LNA substitutions also increase the local organization of the phosphate backbone and significantly enhance the stability of nucleic acid duplexes, the latter most likely reflecting a greater degree of stacking with neighboring bases (49). Using a scanning strategy of overlapping LNA doublets and triplets in the PPT (Ϫ) DNA template, A-like duplex geometry was introduced locally throughout the PPT-containing RNA/ DNA hybrid and immediately adjacent dA⅐rU tract, alteration of which also affects PPT function (50,51). Our current studies indicate that modifying either extremity of the PPT impairs hydrolysis at the PPT/U3 junction, whereas the intervening sequence can be substituted with minimal consequences for hydrolysis.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

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

Grice

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Together, these observations implicate structural features of the PPT-containing duplex as active participants in its recruitment of, and processing by, HIV-1 RT. Because the PPT comprises three homopolymeric tracts and is preceded by a (rU):(dA) tract whose alteration affects virus replication (24,25), the geometry adopted both within these tracts and at their junctions may be pivotal to the accuracy with which the plusstrand primer is processed. Lastly, a contribution of the p66 RNase H primer grip (5) to the accuracy of PPT selection has been suggested from our recent mutagenesis studies (19).…”

Section: Discussionmentioning

confidence: 99%

Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection

Rausch

Yi-Brunozzi

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA͞DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66͞p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA͞DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (؉) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage. R eplication of HIV requires converting single-stranded viral RNA into a double-stranded DNA copy suitable for integration. This process takes place in multiple steps, each catalyzed by HIV reverse transcriptase (RT). Whereas minus-strand DNA synthesis is primed by a host-derived tRNA primer annealed to the RNA genome at the primer binding site, plus-strand synthesis initiates from the 3Ј and central polypurine tracts (PPTs) derived from cleavage the RNA genome after minus-strand synthesis has occurred. These identical, purine-rich sequences are selected for plus-strand priming in part by precise RNase H-mediated cleavage at their 3Ј termini. Initiation from the 3Ј PPT represents an especially critical stage in virus replication, because incorrect priming would result either in truncation of the 5Ј LTR and deletion of one or more transcriptional control elements, or extension of the preintegrative DNA and impaired integration (1, 2). Accordingly, PPT processing constitutes a potentially fruitful target for antiviral therapy, and the mechanistic basis by which this occurs is the subject of the current study.Although the all-purine nature of the PPT sequence renders it moderately resistant to ribonuclease H (RNase H)-mediated hydrolysis (3, 4), recent data implicate structural features of a PPT͞(Ϫ)DNA hybrid in its selecti...

show abstract

“…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).…”

mentioning

confidence: 99%

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

Kvaratskhelia

Budihas

Grice

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 ...

show abstract

Identification of a sequence element immediately upstream of the polypurine tract that is essential for replication of simian immunodeficiency virus

Cited by 51 publications

References 43 publications

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

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

Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection

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

Contact Info

Product

Resources

About