Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m403306200
ISSN1083-351X
AutoresChandravanu Dash, Hye-Young Yi-Brunozzi, Stuart F.J. Le Grice,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoUnusual 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 dA·rU tract immediately 5′ of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT·rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC·rG and neighboring dT·rA 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. 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 dA·rU tract immediately 5′ of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT·rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC·rG and neighboring dT·rA 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 (-)-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) 1The abbreviations used are: RNase H, ribonuclease H; HIV-1, human immunodeficiency virus type 1; LNA, locked nucleic acid; MGBT, minor groove binding track; nt, nucleotide(s); PPT, polypurine tract; RHPG, RNase H primer grip; RT, reverse transcriptase.1The abbreviations used are: RNase H, ribonuclease H; HIV-1, human immunodeficiency virus type 1; LNA, locked nucleic acid; MGBT, minor groove binding track; nt, nucleotide(s); PPT, polypurine tract; RHPG, RNase H primer grip; RT, reverse transcriptase. catalytic centers of the multifunctional reverse transcriptase (RT), respectively (1Telesnitsky A. Goff S.P. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 121-161Google Scholar). 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The spatial separation of KMnO4-sensitive sites approximates the distance between the MGBT of the p66 thumb and the RNase H catalytic center. Thus, interaction of the MGBT (or other elements of the thumb) with the unzipped portion of the PPT might directly affect positioning the RNase H catalytic center over the PPT/U3 junction.Although plausible, our hypothesis remains speculative, requiring additional evidence that locally altering PPT architecture, while retaining sequence context, affects RT contacts and, ultimately, cleavage at the PPT/U3 junction. Recently, we introduced non-hydrogen-bonding pyrimidine isosteres (40Kool E.T. Biopolymers. 1998; 48: 3-17Crossref PubMed Scopus (113) Google Scholar, 41Kool E.T. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 1-22Crossref PubMed Scopus (406) Google Scholar, 42Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (333) Google Scholar, 43Kool E.T. Morales J.C. Guckian K.M. Angew. Chem. Int. Ed. Engl. 2000; 39: 990-1009Crossref PubMed Scopus (327) Google Scholar) into the HIV-1 PPT (-) DNA template (44Rausch J.W. Qu J. Yi-Brunozzi H.Y. Kool E.T. Le Grice S.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11279-11284Crossref PubMed Scopus (27) Google Scholar), noting that cleavage was particularly sensitive to substitution at the (dC):(rG/dT·rA junction. More recently, the fluorescent cytosine analog pyrrolo-dC was exploited to confirm that a cytosine residue of the unzipped portion of the HIV-1 PPT (-) DNA defined crystallographically was also unpaired in the absence of RT (45Dash C.V. Rausch J.W. Le Grice S.F.J. Nuclic Acids Res. 2004; 32: 1539-1547Crossref PubMed Scopus (65) Google Scholar). These studies coupled with a parallel evaluation of the Ty3 PPT substituted with thymine isosteres (46Lener D. Kvaratskhelia M. Le Grice S.F. J. Biol. Chem. 2003; 278: 26526-26532Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) illustrate the value of nucleoside analogs as probes of PPT structure.Assuming that structural deformations in the wild type PPT (4Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar) introduce local flexibility, we wished to examine how stabilizing the same region might affect its recognition by HIV-1 RT. To achieve this, we prepared a series of (-) DNA templates containing locked nucleic acid (LNA) analogs (47Orum H. Wengel J. Curr. Opin. Mol. Ther. 2001; 3: 239-243PubMed Google Scholar, 48Wengel J. Vester B. Lundberg L.B. Douthwaite S. Sorensen M.D. Babu B.R. Gait M.J. Arzumanov A. Petersen M. Nielsen J.T. Nucleosides Nucleotides Nucleic Acids. 2003; 22: 601-604Crossref PubMed Scopus (21) Google Scholar). These 2′-O-4′-C-methylene-linked bicyclic analogs (Fig. 1A) have the property of locking the deoxyribose ring in the C3′-endo configuration. 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 (49Petersen M. Bondensgaard K. Wengel J. Jacobsen J.P. J. Am. Chem. Soc. 2002; 124: 5974-5982Crossref PubMed Scopus (227) Google Scholar). Using a scanning strategy of overlapping LNA doublets and triplets in the PPT (-) DNA template, A-like duplex geometry was introduced locally throughout the PPT-containing RNA/DNA hybrid and immediately adjacent dA·rU tract, alteration of which also affects PPT function (50Ilyinskii P.O. Desrosiers R.C. EMBO J. 1998; 17: 3766-3774Crossref PubMed Scopus (48) Google Scholar, 51Bacharach E. Gonsky J. Lim D. Goff S.P. J. Virol. 2000; 74: 4755-4764Crossref PubMed Scopus (29) Google Scholar). 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. Finally, we demonstrate here two binding modes for HIV-1 RT on PPT-containing duplexes. These modes mediate hydrolysis at the PPT/U3 junction and 5-6 bp downstream in the U3 region, a combination that has been proposed to regulate (+)-strand synthesis in murine leukemia virus (31Schultz S.J. Zhang M. Champoux J.J. J. Virol. 2003; 77: 5275-5285Crossref PubMed Scopus (21) Google Scholar). A model for each of these hydrolytic modes involving "locking" of the p66 thumb at either side of the internal PPT deformation is proposed. This positioning of RT may also in part explain resistance of the PPT to internal hydrolysis.EXPERIMENTAL PROCEDURESOligonucleotide and Enzyme Preparation—LNA-substituted 40-nt oligodeoxynucleotides were synthesized at a 1-μmol scale on a PE Biosystems Expedite 8909 synthesizer by standard phosphoramidite chemistry with the exception that coupling times for locked 5-methyl cytidine were extended to 15 min. Stepwise coupling yields for incorporation of LNA was >98%, determined by trityl cation monitoring. Sequences and sites of substitution are illustrated in Fig. 1B. LNA phosphoramidites of A, G, T, and 5-methyl cytidine were purchased from Proligo Reagents. Deprotection and cleavage of oligonucleotides from the CPG support was carried out by incubation in 30% ammonium hydroxide for 36 h at 25 °C. Oligonucleotides were purified by preparative polyacrylamide gel electrophoresis and quantified spectrophotometrically (260 nm) assuming a molar extinction coefficient equal to the sum of the constituent deoxynucleotides. A complementary 30-nt RNA containing the HIV-1 3′ PPT and flanking sequences at its 5′ and 3′ termini was purchased from Dharmacon Research (Boulder, CO). Hybrids were prepared by heating equimolar amounts of RNA and DNA to 95 °C in 10 mm Tris-HCl, pH 7.8, 25 mm NaCl for 5 min followed by slow cooling to 4 °C. Samples were stored at -20 °C. p66/p51 HIV-1 RT was purified according to Le Grice et al. (52Le Grice S.F. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-144Crossref PubMed Scopus (120) Google Scholar).PPT Selection—PPT selection was evaluated as previously described (45Dash C.V. Rausch J.W. Le Grice S.F.J. Nuclic Acids Res. 2004; 32: 1539-1547Crossref PubMed Scopus (65) Google Scholar) using p66/p51 HIV-1 RT and 30-nt RNA/40-nt DNA hybrids. 5′-end-labeling of the PPT-containing RNA was performed with T4 polynucleotide kinase and [γ-32P]ATP, whereas 3′-end-labeling was achieved with T4 RNA ligase and [α-32P]CTP. Hybrids were generated by annealing the radiolabeled PPT RNA to each of the LNA-substituted (-) DNA templates as well as to an unsubstituted control oligodeoxynucleotide. Hydrolysis was initiated by adding RT to RNA/DNA hybrids in 10 mm Tris-HCl, pH 8.0, 80 mm NaCl, 5 mm dithiothreitol, and 6 mm MgCl2 at 37 °C with enzyme and RNA/DNA hybrid present at final concentrations of 50 and 200 nm, respectively. Hydrolysis was terminated after 10 min by adding an equal volume of 95% (v/v) formamide containing 0.1% (w/v) bromphenol blue and xylene cyanol, and the products were fractionated by high voltage electrophoresis through 15% (w/v) polyacrylamide, 7 m urea gels, visualized by autoradiography and/or phosphorimaging, and quantified using Quantity One software (Bio-Rad).Circular Dichroism Spectroscopy and Thermal Melting Profiles— Equimolar amounts of 30-nt RNA primer and LNA-substituted 40-nt (-) DNA templates were annealed by heating to 90 °C and slow cooling in degassed 10 mm Na2HPO4/NaH2PO4, pH 7.0, 80 mm NaCl. Nondenaturing gel electrophoresis was used to determine that complete hybridization had been achieved. Circular dichroism spectra were recorded at 25 °C with an AVIV 202 spectrophotometer using a 1-mm path length cuvette. Correction for each spectrum was against the buffer-only spectrum. Nucleic acid duplexes were scanned from 190 to 300 nm. For measurement of melting temperatures (Tm), 10 μg/ml solutions of the same substrates were analyzed in a Beckman DU 640 spectrophotometer. E260 was measured at 0.2 °C intervals from 30 to 95 °C. The Tm of each hybrid was calculated by the "first derivative" method described by the manufacturer.Sensitivity of Template Thymines to KMnO4Oxidation—RNA/DNA hybrids containing dual LNA substitutions between template nucleotides -9 and -15 (defining position -1 as the first PPT base 5′ to the PPT/U3 junction) were subjected to KMnO4 oxidation as previously described (39Kvaratskhelia M. Budihas S.R. Le Grice S.F. J. Biol. Chem. 2002; 277: 16689-16696Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Cleavage products were fractionated by high resolution, denaturing polyacrylamide gel electrophoresis, and quantitated by phosphorimaging.RESULTSExperimental Strategy—The structures of the individual LNA-inducing nucleobases and strategy for their pairwise insertion into the PPT (-) DNA template are illustrated in Figs. 1, A and B, respectively. Because all four nucleobases were commercially available, this allowed us to evaluate the homopolymeric dA·rU tract immediately adjacent to the PPT, a region whose alteration affects PPT usage in HIV-1 (53McWilliams M.J. Julias J.G. Sarafianos S.G. Alvord W.G. Arnold E. Hughes S.H. J. Virol. 2003; 77: 11150-11157Crossref PubMed Scopus (21) Google Scholar), Moloney murine leukemia virus (51Bacharach E. Gonsky J. Lim D. Goff S.P. J. Virol. 2000; 74: 4755-4764Crossref PubMed Scopus (29) Google Scholar), and simian immunodeficiency virus (50Ilyinskii P.O. Desrosiers R.C. EMBO J. 1998; 17: 3766-3774Crossref PubMed Scopus (48) Google Scholar). Although dC was commercially available as the 5-methyl derivative, x-ray crystallography (4Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar) suggests that relatively few contacts are made to nucleobases of the RNA/DNA hybrid, suggesting steric interference would be minimal. The overlapping LNA strategy also allowed us to substitute bases on either side of the junctions between homopolymeric blocks comprising the PPT.As a first step in characterizing LNA-containing RNA/DNA hybrids, CD spectra were compared with that of an unsubstituted substrate. Representative spectra are presented in Fig. 2A for each extremity of the PPT (LNA 15/14 and LNA 2/1) as well as the portion of the PPT where unusual base-pairing leads to unstacking of template base -11C (LNA 11/10) (4Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar, 45Dash C.V. Rausch J.W. Le Grice S.F.J. Nuclic Acids Res. 2004; 32: 1539-1547Crossref PubMed Scopus (65) Google Scholar). As might be expected for an RNA/DNA hybrid (7Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 54Ratmeyer L. Vinayak R. Zhong Y.Y. Zon G. Wilson W.D. Biochemistry. 1994; 33: 5298-5304Crossref PubMed Scopus (166) Google Scholar, 55Hung S.H. Yu Q. Gray D.M. Ratliff R.L. Nucleic Acids Res. 1994; 22: 4326-4334Crossref PubMed Scopus (74) Google Scholar), the spectrum of an unmodified duplex displays a positive band at 267 nm, with minima at 245 and 210 nm. Introducing adjacent LNA analogs at positions -15/-14, -11/-10, or -2/-1 induces a minor change in the 210-nm minimum, suggesting local A-like geometry. Spectra for additional LNA-substituted RNA/DNA hybrids were similar to those in Fig. 2A (data not shown). Thus, introducing adjacent A-form LNA analogs into the PPT (-) DNA template appeared to minimally perturb the structure of the RNA/DNA hybrid.Fig. 2A, representative CD spectra for -2/-1 (left), -11/-10 (center), and -15/-14 LNA-substituted PPT-containing RNA/DNA hybrids. Filled and dashed lines represent wild type and mutant substrates, respectively. B,Tm values for doubly LNA-substituted PPT RNA/DNA hybrids. The shaded portion of the figure represents the unzipped portion of the RT-RNA/DNA co-crystal (4Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar)View Large Image Figure ViewerDownload (PPT)Melting temperatures for doubly substituted RNA/DNA hybrids are presented in Fig. 2B. As expected, substitutions in the dC·rG tract had the most significant impact, raising the Tm as much as 11 °C relative to the unsubstituted control. Interestingly, LNA substitutions of the dT·rA tracts displayed differential effects depending on their position relative to the unzipped portion of the PPT (4Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar). As an example, a -14/-13 substitution raised the Tm to 58.9 °C, whereas that of a -9/-8 substituted duplex was 62.9 °C. In both cases these substitutions are internal to a 5′-TTTT-3′ sequence, minimizing the sequence context. Finally, wild type and LNA-substituted RNA/DNA hybrids migrated similarly on non-denaturing polyacrylamide gels, indicating that major structural distortions such as G-quartets were not induced. (data not shown).Insertion of LNA-inducing Doublets into PPT (-) DNA—The consequences of introducing LNA-inducing doublets on HIV-1 PPT selection is presented in Fig. 3. In Fig. 3A, the PPT primer was radiolabeled at its 5′ terminus. In the absence of modification (lane (-)), we observed cleavage at the PPT/U3 junction and minor cleavage at U3 positions +1 and +2. A similar pattern was observed for template substitutions -20/-19 and -19/-18 (lanes a and b, respectively), which define the extremity of the upstream dA·rU tract. A subtle change in the hydrolysis pattern is induced by LNA substitutions at positions -18/-17 and -17/-16 (lanes c and d, respectively), where cleavage at U3 positions +2 and +3 are induced in addition to the PPT/U3 junction.Fig. 3HIV-1 PPT cleavage after LNA doublet insertions into the (-) DNA template. A, 5′-end labeled PPT primer; B, 3′-labeled PPT primer. The position of the 5′ LNA analog of a doublet is given at the side of each panel for orientation, Lanes a, -20/-19; lanes b, -19/-18; lanes c, -18/-17; lanes d, -17/-16; lanes e, -16/-15; lanes f, -15/-14; lanes g, -14/-13; lanes h, -13/-12; lanes i, -12/-11; lanes j, -11/-10; lanes k, -10/-9; lanes l, -9/-8; lanes m, -8/-7; lanes n, -7/-6; lanes o, -6/-5; lanes p, -5/-4; lanes q, -4/-3; lanes r, -3/-2; lanes s, -2/-1; lanes t, -1/+1; lanes (-), unsubstituted PPT (-) DNA template. The schematic between panels A and B is included for orientation of the homopolymeric tracts of the PPT and upstream region.View Large Image Figure ViewerDownload (PPT)In contrast, altering the geometry of the distal PPT dT·rA tract between positions -16 and -12 has dramatic effects on selection. After -16/-15 and -15/-14 substitution, hydrolysis at U3 positions +1, +2, and +3 are favored over the PPT/U3 junction (lanes e and f, respectively). Cleavage at these novel sites and at the PPT/U3 junction is eliminated when template nucleotides -14/-13 and -13/-12 are substituted (lanes g and h, respectively). PPT/U3 cleavage is restored, albeit to a reduced level, after LNA substitution at positions -12/-11 (lane i). Thus, the combined data of lanes e-i in Fig. 3A suggest that PPT hydrolysis is sensitive to alterations to the geometry of the upstream dT·rA tract. This appears to be less critical for the central dT·rA tract, since substitutions between positions -11 (lane j) and -7 (lane m) do not significantly affect hydrolysis at the PPT/U3 junction.Lanes n-t of Fig. 3A address alterations to the geometry of the dC·rG tract of the PPT. Surprisingly, dramatic inhibition of hydrolysis accompanies substitution of template nucleotides -7/-6 (lane n), although a neighboring -8/-7 substitution is well tolerated (lane m). The effect of a -7/-6 substitution appears to be local, since LNA analogs between positions -6 and -4 (lanes o and p) permit hydrolysis at the PPT/U3 junction. Finally, because A-like geometry is induced in the vicinity of the PPT/U3 junction, hydrolysis is inhibited (lanes q-t). Taken together, the data of Fig. 3A suggest that either end of the PPT-containing RNA/DNA hybrid is significantly less tolerant to alterations in nucleic acid geometry than the central dT·rA tract. In addition, the junction between the dC·rG and neighboring dT·rA tract appears to be particularly susceptible to alteration.Two Modes of PPT Cleavage Are Affected by LNA
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