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Originally published In Press as doi:10.1074/jbc.M403306200 on June 25, 2004

J. Biol. Chem., Vol. 279, Issue 35, 37095-37102, August 27, 2004
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Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template*

Chandravanu Dash, Hye-Young Yi-Brunozzi, and Stuart F. J. Le Grice{ddagger}

From the Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702

Received for publication, March 24, 2004 , and in revised form, June 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)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 3'OH 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 (-) DNA for use as template for plus (+)-strand, DNA-dependent DNA synthesis.

A critical step between (-)- and (+)-strand DNA synthesis is selection of the (+)-strand RNA primer from within the replication intermediate. For HIV-1, RNase H-resistant polypurine tract (PPT) sequences at the center (22) and 3'-end of the genome (23) provide specific initiation sites and are later removed from nascent (+) DNA with equal precision. With respect to the 3' PPT, this mechanism creates 5'-terminal sequences of the double-stranded DNA for recognition by virus-coded 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, 23-31), 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 (32-38). However, our chemical footprinting studies (39) suggest an alternative hypothesis. In the absence of RT, two regions of the PPT (-) DNA template, separated by 12-13 bp, were susceptible to KMnO4 modification, suggesting a departure from standard Watson-Crick geometry. The first includes the region of the PPT referred to as unzipped in the presence of RT (4), whereas a second KMnO4-sensitive site was at the PPT/U3 junction. 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 (40-43) into the HIV-1 PPT (-) DNA template (44), 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 (45). These studies coupled with a parallel evaluation of the Ty3 PPT substituted with thymine isosteres (46) illustrate the value of nucleoside analogs as probes of PPT structure.

Assuming that structural deformations in the wild type PPT (4) 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 (47, 48). 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 (49). 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 (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. 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 (31). 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.



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  		FIG. 1.
A, sugar pucker of LNA analogs. For comparison, the C2'-endo pucker of DNA sugars and C3'-endo pucker of RNA sugars is presented. B, location of LNA doublet insertions in the HIV-1 PPT (-) DNA template. Notations 20/19, 19/18, etc. represent the position of LNA insertion, defining -1 as the first primer base (G) 5' to the PPT/U3 junction. For the RNA/DNA hybrid shown, open and filled boxes represent standard and weakened base pairing as defined by Sarafianos et al. (4). Note that mispairing at two positions results in an unpaired template and primer nucleotide (open circles). Template nucleotides sensitive to KMnO4 oxidation (39) are shaded.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotide 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. (52).

PPT Selection—PPT selection was evaluated as previously described (45) 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 [{gamma}-32P]ATP, whereas 3'-end-labeling was achieved with T4 RNA ligase and [{alpha}-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 KMnO4 Oxidation—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 (39). Cleavage products were fractionated by high resolution, denaturing polyacrylamide gel electrophoresis, and quantitated by phosphorimaging.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental 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 (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. 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) (4, 45). As might be expected for an RNA/DNA hybrid (7, 54, 55), 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.



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  		FIG. 2.
A, 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 (4)

 
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 (4). 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.



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  		FIG. 3.
HIV-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.

 
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 Substitution—While analyzing RNA/DNA hybrids whose RNA was 5'-end-labeled we noted a technical difficulty in that hybrids containing LNA substitutions of the dC·rG tract were increasingly difficult to denature. This is due primarily to the observation that LNA analogs significantly increase the stability of nucleic acid duplexes. In an extreme case, the melting temperature of LNA-containing duplexes can be increased by almost 10 °C per substitution (56). This was particularly serious for substitutions into the dC·rG tract, where Fig. 2B indicates that the Tm can increase from 55.8 to 66.9 °C. One solution to this problem was to relocate the radiolabel to the PPT 3' terminus, since the shorter, radioactive hydrolysis fragment should more readily dissociate. Thus, to exclude the possibility that 5' labeling did not reveal all hydrolysis fragments, doubly substituted RNA/DNA hybrids whose (+) RNA was 3'-end-labeled were examined (Fig. 3B).

In general, the same trend was observed with respect to the position of LNA insertion, namely that the dT·rA and dC·rG tract at either end of the RNA/DNA hybrid (lanes e-i and q-t, respectively) were sensitive to alterations in nucleic acid geometry, whereas the central dT·rA tract was more tolerant to substitution (lanes j-m). As expected, the junction between the dC·rG and neighboring dT·rA tract was particularly susceptible. However, 3' labeling revealed a novel hydrolysis pattern at U3 positions +5 and +6. In particular, when hydrolysis at the PPT/U3 junction was tolerant to LNA substitution, +5/+6 cleavage was eliminated. Conversely, when hydrolysis at the PPT/U3 junction was not tolerated, +5/+6 cleavage persisted. Although unusual, the ability to cleave at +5/+6 of an RNA/DNA hybrid when PPT/U3 hydrolysis is eliminated indirectly illustrates that LNA substitution does not affect the global conformation of the RNA/DNA hybrid. Rather, the data of Fig. 3B suggests that HIV-1 RT might bind the PPT-containing RNA/DNA hybrid at two distinct positions.

To eliminate the possibility that U3 + 5/+6 cleavage did not reflect unusual binding of HIV-1 RT to the template 5' terminus, we analyzed the hydrolysis patterns of two non-substituted RNA/DNA hybrids differing by 5 nt at their 5' termini (Fig. 4). An identical hydrolysis pattern was obtained for each substrate, indicating that terminal effects were unlikely to induce U3 + 5/+6 cleavage. Although we did not perform a detailed kinetic analysis to determine the temporal mode of cleavage, the data of Fig. 4 support a study of the Moloney murine leukemia virus PPT, where Schultz et al. (31) demonstrate hydrolysis at the PPT/U3 junction and in U3, suggesting this mechanism facilitates initiation of (+)-strand synthesis. We have also observed cleavage in the U3 sequences ahead of the PPT of the Saccharomyces cerevisiae LTR-transposon Ty3.2 Thus, as postulated above, our data suggest alternative locations for HIV-1 RT on the PPT. A model for these locations will be presented later.



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  		FIG. 4.
HIV-1 PPT cleavage as a function of the template 5' extension. A schematic indicating the RNA/DNA hybrids studied is indicated above the gels of panels A and B. DNA and RNA strands are depicted by closed and open bars, respectively. Template and primer sequences are those of Fig. 1B, differing in that the (-) DNA template of substrate (B) is 5 nt longer at its 5' terminus. The RNA primer of each RNA/DNA hybrid was 3'-end-labeled with [{alpha}-32P]CTP. PPT hydrolysis was evaluated after 1 min (lanes 1), 3 min (lanes 2), 5 min (lanes 3), 10 min (lanes 4), and 15 min (lanes 5).

 

LNA Triplet Substitutions—A second set of triply substituted PPT (-) DNA templates was synthesized to determine whether increasing the extent of nucleic acid stabilization had more severe consequences for hydrolysis at the PPT/U3 junction. The results of this experiment are presented in Fig. 5. In this case LNA triplets were substituted between template nucleotides -19 and -1. 5'-End-labeling of the PPT primer (Fig. 5A) indicates that a -19/-17 substitution of the upstream dA·rU tract yielded a hydrolysis profile equivalent to the unsubstituted RNA/DNA hybrid (lane a). Relocating the LNA one nucleotide into this tract (-18/-16, lane b) reduced cleavage at the PPT/U3 junction in favor of U3 positions +2 and +3 (lane c). LNA substitutions -16/-14 and -15/-13 abruptly eliminated hydrolysis (lanes d and e, respectively), and this was equally abruptly restored after substitution at any position between -14 and -8 (lanes f-k). Finally, a triplet substitution immediately upstream of the dC·rG/dT·rA junction (lane l) and extending as far as the PPT/U3 junction (lane q) eliminated hydrolysis.



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  		FIG. 5.
HIV-1 PPT cleavage after LNA triplet insertions into the (-) DNA template. Panel A, 5'-end-labeled PPT primer. Panel B, 3'-end-labeled primer. Lanes a, -19/-17; lanes b, -18/-16; lanes c, -17/-15; lanes d, -16/-14; lanes e, -15/-13; lanes f, -14/-12; lanes g, -13/-11; lanes h, -12/-10; lanes i, -11/-9; lanes j, -10/-8; lanes k, -9/-7; lanes l, -8/-6; lanes m, -7/-5; lanes n, -6/-4; lanes o, -5/-3; lanes p, -4/-2; lanes q, -3/-1; lanes (-), unsubstituted PPT (-) DNA

 
Because the problem of thermal denaturation became more acute as the number of LNA analog insertions increases, 3' labeling of the PPT primer (Fig. 5B) was necessary to both verify cleavage at the PPT/U3 junction as well as reveal the novel pattern at U3 positions +5 and +6. Essentially the same pattern was observed for PPT/U3 cleavage, namely manipulation of either end of the PPT-containing RNA/DNA hybrid inhibited hydrolysis (lanes c-e and l-q), whereas the central portion was more tolerant to substitution. Although less noticeable, the dC·rG/dT·rA junction was again sensitive to alteration (Fig. 5B, lanes k-m). As was noted for LNA doublet insertions, an inverse relationship between the efficiency cleavage at the PPT/U3 junction and U3 + 5/+6 was observed, strengthening our contention that HIV-1 RT occupies two different positions on this PPT substrate.

Hydrolysis of HIV-1 PPT Duplexes Containing A-form RNA—As indicated earlier, 2'-O-4'-C-methylene-linked bicyclic LNA analogs lock the sugar ring in an C3'-endo (or A-like) configuration. Thus, in the case of the PPT RNA/DNA hybrids, LNA doublet or triplet insertions into the DNA template might be equivalent to locally introducing a short A-form RNA duplex within the context of an RNA/DNA hybrid. However, the increased thermal stability of LNA substitutions in the DNA of an RNA/DNA hybrid compared with an unsubstituted hybrid or duplex RNA (56, 57) suggests the stability they introduce involves more than simply A-form geometry. This notion is strengthened by the observation that {alpha}-L LNA analogs, whose sugar retains the C2'-endo configuration, have also been shown to increase the thermal stability of RNA/DNA hybrids (58). To address A-form geometry with respect to selection of the HIV-1 PPT, several template variants were synthesized as RNA-DNA chimeras containing increasing length of RNA at their 3' termini. Hybridizing these RNA/DNA chimeras to the PPT RNA primer, thus, created a substrate where increasing amounts of A-form RNA abutted an RNA/DNA chimera. As indicated in Fig. 6A, chimeric templates were constructed to introduce duplex RNA between positions -14 (template 12R) and -20 (template 5R). The hydrolysis profiles of these chimeric PPT substrates are presented in Fig. 6B.



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  		FIG. 6.
Effect of introducing A-form duplex RNA into the distal end of the HIV-1 PPT on hydrolysis at the PPT/U3 junction. A, schematic of substrates. Notations 5R-12R represent the length of the duplex RNA component at the 5' terminus of the PPT (-) DNA template. B, PPT hydrolysis profiles. For these experiments, the PPT primer was 32P-end-labeled at its 5' terminus. Lane a, hybrid 5R; lane b, hybrid 6R; lane c, hybrid 7R; lane d, hybrid 8R; lane e, hybrid 9R; lane f, hybrid 10R; lane g, hybrid 10R; lane h, hybrid 11R; lane i, hybrid 12R; lane (-), wild type PPT RNA/DNA hybrid, no RT.

 
PPT substrate 5R (lane b) introduces duplex RNA immediately upstream of the homopolymeric dA·rU tract and results in cleavage at the PPT/U3 junction and U3 + 1. As increasing lengths of duplex RNA are introduced into the dA·rU tract, we observe very little difference in PPT/U3 cleavage. This is of particular importance for substrates 9R-12R (lanes f-i), since these approximate the position of LNA triplet insertions -17/-15 and -16/-14 (Fig. 5, A and B, lanes c and d), which eliminate cleavage at the PPT/U3 junction. Likewise, LNA doublet insertions -16/-15 and -15/-14 (Fig. 3, A and B, lanes e and f) favor cleavage on the U3 side of the PPT as opposed to the PPT/U3 junction, although duplex RNA over the same region allows correct cleavage. Furthermore, although we introduced 2-3-bp regions of altered geometry via LNA insertion, the chimeras of Fig. 6A introduce a much larger RNA duplex. The data of Fig. 6, thus, suggest that the localized alteration to nucleic acid geometry and alteration in cleavage specificity induced by LNA substitution is more than the consequence of simply introducing an A-like structure.

Chemical Footprinting of LNA-substituted RNA/DNA Hybrids—Previous data (4, 39) indicate the HIV-1 PPT-containing RNA/DNA hybrid adopts an unusual pattern of base pairing between positions -9 and -15. Although the CD spectra of Fig. 2A provide an approximation of duplex structure, thymine sensitivity to KMnO4 was performed to determine whether LNA-induced alteration in PPT cleavage reflected a local alteration or a further destabilization of the unzipped portion of the PPT (4). Pairwise LNA-substituted RNA/DNA hybrids covering template nucleotides -9 to -15 (Fig. 7A) were subjected to KMnO4 cleavage, which highlights thymines in an unpaired or distorted configuration (39).



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  		FIG. 7.
KMnO4 footprinting of selected LNA-substituted, PPT-containing RNA/DNA hybrids. The sequence of the hybrid between positions -8 and -17 is presented in A, above which the LNA-substituted (-) DNA templates subjected to chemical probing are presented. A base-pairing pattern is as presented in Fig. 1. B, KMnO4 footprint analysis. Lane W, wild type RNA/DNA hybrid; lane a, LNA template -9/-10; lane b, LNA template -10/-11; lane c, LNA template -11/-12; lane d, LNA template -13/-14; lane e, LNA template -14/-15; lane f, LNA template -15/-16. Template thymines -7T to -10T and -12T to -15T in addition to thymine +1T at the PPT/U3 junction have been indicated.

 
KMnO4 sensitivity of template thymines between positions -9 and -12 is indicated in Fig. 7B, lanes a-c, respectively. A general pattern emerged that LNA substitutions inhibited KMnO4 modification at the position of insertion, i.e. a -9/-10 LNA doublet reduces chemical attack at template nucleotides -9T and -10T (Fig. 7B, lane a). Moreover, although KMnO4 reactivity at template nucleotide -12T decreases in a -11/-12-subsituted template, reactivity of nucleotides -7T through -10T resemble the control duplex (cf. Fig. 7B, lanes W and c). LNA substitutions of the (dT)4 sequence between template nucleotides -12 and -15 are also localized to that region (Fig. 7B, lanes d-f), although some differences in reactivity were noted. For example, a -13/-14 LNA substitution resulted in loss of reactivity at -14T and -15T (Fig. 7B, lane d), whereas a -14/-15 LNA substitution altered reactivity of -13T and -14T Fig. 7B, lane e). Despite these minor changes the combined data of Figs. 2 and 7 suggest that no global change in PPT structure accompanies LNA substitution of template nucleotides -9 through -15, where major differences in cleavage at the PPT/U3 junction are observed (Fig. 3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In HIV-1, (-)-strand DNA synthesis produces an RNA/DNA hybrid harboring the (+)-strand PPT primer embedded within a considerably larger nucleic acid duplex. Despite this, the PPT is specifically recognized and cleaved at its 3' terminus to facilitate (+)-strand synthesis. It is, therefore, reasonable to postulate that structural features of the PPT/(-) DNA duplex mediate specific recognition by the polymerizing machinery. This notion is strengthened by our recent detection of anomalies in PPT architecture in the absence of RT (39, 45), corresponding to the unzipped portion of the PPT observed in a co-crystal with HIV-1 RT (4). Flexibility in PPT architecture may, therefore, play an important role in sequestering HIV-1 RT via structural motifs in the DNA polymerase and/or RNase H domain. As a complementary approach to the use of pyrimidine isosteres (44, 46), we examined here the consequences of increasing the stability of this RNA/DNA hybrid via a scanning strategy employing LNA analogs in a pairwise and triplet fashion.

Our data suggest that altering duplex geometry of homopolymeric tracts at either end of the RNA/DNA hybrid significantly impair hydrolysis at the PPT/U3 junction, whereas the intervening region is more tolerant to substitution. To explain these findings, the model of Fig. 8 is derived from Sarafianos et al. (4), but RT contacts on the PPT-containing hybrid are realigned to correspond to the RNase H catalytic center positioned over the PPT/U3 junction. In this model the MGBT of the p66 thumb spans approximately positions -13 through -16. One proposed role for the MGBT is as a sensor of nucleic acid geometry during translocation (19). The MGBT is very close to the region of the PPT where LNA substitution eliminates hydrolysis at the PPT/U3 junction (Fig. 3). Because the region encompassing positions -13 to -16 involves unpaired, weakly paired, and mispaired bases, LNA-induced stabilization of this region (most likely via enhanced base stacking (49)) may be incompatible with an interaction with the p66 thumb. Significantly fewer contacts are made between RT and the nucleic acid duplex between its thumb subdomain and RNase H domain in all RT/DNA and RT-RNA/DNA structures (2-4). LNA doublet insertions in this region, i.e. between positions -14/-13 and -9/-8, have a negligible effect on hydrolysis at the PPT 3' terminus, suggesting that, provided proper contacts are made with the MGBT and RHPG, stabilizing nucleic acid structure of the intervening region has little effect. Finally, LNA substitutions from positions -4 to -1 of the dC·rG tract are incompatible with hydrolysis at the PPT 3' terminus. Two mechanisms might account for this. First, modified nucleic acid is introduced into the RHPG, affecting the trajectory of the RNA strand entering the catalytic center. A second equally plausible hypothesis is that inducing A-like geometry at a position of the substrate normally has a more B-like structure (2-4). Although we cannot distinguish between these possibilities, the data of Fig. 3 suggest PPT/U3 cleavage is particularly sensitive to altering geometry at the dC·rG/dT·rA junction, whereas the immediately adjacent region on either side is more tolerant. Interestingly, a similar observation was made with PPT (-) DNA templates containing pyrimidine isosteres (44). Although limited crystallographic data is available, an NMR study of a related DNA duplex (59) suggests local structural polymorphology in this region. Because this region is sensitive to both stabilization (this work) or destabilization (44), a more detailed analysis with additional nucleoside analogs is warranted.



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  		FIG. 8.
Proposed HIV-1 RT binding modes catalyzing hydrolysis at the PPT/U3 junction and U3 positions +5/+6. DNA and RNA sequences are in blue and red, respectively. KMnO4-sensitive DNA bases (39) are filled. Selected contacts to the p66 thumb and RNase H primer grip are taken from the RT-RNA/DNA co-crystal of Sarafianos et al. (4), and have been repositioned to represent positioning of the RNase H active center over the PPT/U3 junction. The U3 + 5 binding mode assumes these contacts are re-positioned 5 bp into U3. In the lower portion of the figure, notations MGBT and RHPG represent the minor groove binding track (16) and RNase H primer grip (4) of p66 RT, respectively. Pol, polymerase.

 
Although cleavage at U3 positions +5 and +6 is analogous to findings with the PPTs of Moloney murine leukemia virus (31) and Ty3,2 the surprising observation here was the sensitivity at these positions relative to the sites of LNA insertion. Figs. 3B and 5B show these sites are not cleaved when the central portion of the PPT is altered, and, conversely, are accessible to RNase H when alterations are introduced into the dC·rG tract and immediately upstream region. One possible explanation for the LNA effect and the mechanism of +5/+6 cleavage is presented in Fig. 8, where RT contacts have again been repositioned to give the RNase H active access to positions +5/+6. Under these circumstances, the MGBT would be relocated to the central portion of the PPT and the RHPG immediately downstream of the PPT/U3 junction. This model predicts that altering the upstream dA·rU and adjacent dT·rA tracts, where contacts to the MGBT would be relinquished, would not affect +5/+6 cleavage. Conversely, if the MGBT contacts the central portion of the PPT in the +5/+6 cleavage mode, this would be sensitive to LNA substitution, as observed experimentally. The alternative is that HIV-1 RT reverses its orientation on the PPT, with the RNase H domain over U3 positions +5/+6. However, this orientation would lead to loss of contact between the DNA polymerase domain and the nucleic acid duplex, which would seem unlikely. Moreover, the data of Fig. 4, where +5/+6 cleavage is retained when the DNA template is extended by 5 nt at its 5' terminus, also suggests reversal of RT orientation is unlikely. Our data indirectly suggests that the interaction of RT with the PPT-containing duplex is controlled primarily by contacts made through the DNA polymerase domain and most likely the thumb subdomain. Although this might be coincidental, the two RT orientations suggested in Fig. 8 would position the MGBT on either site of the structural anomaly within the PPT defined by both x-ray crystallography (4) and by chemical footprinting (39).

Regardless of the molecular basis of HIV-1 PPT selection, our studies with unsubstituted RNA/DNA hybrids have revealed novel cleavage sites 5-6 nt ahead of the PPT/U3 junction. Our observations are reminiscent of a recent report from Schultz et al. (31) who found that Moloney murine leukemia virus RT also cleaved a PPT-containing RNA/DNA hybrid at several positions ahead of the PPT 3' terminus. In our studies of RT from the LTR-retrotransposon Ty3, we also noted cleavage sites in U3 sequences a short distance ahead of the PPT/U3 junction.2 Creating a short gap ahead of the PPT 3' terminus via cleavage at and ahead of the PPT/U3 junction was proposed to allow RT to move unimpaired into (+)-strand synthesis without having to immediately invoke strand displacement activity to remove the viral (+) RNA genome (31). Duplication of these events in related retroviral and retrotransposon systems may indeed illustrate the need to "prime" regions downstream of the PPT 3' terminus for DNA synthesis. Interestingly, to initiate DNA synthesis, HIV-1 RT will have to reverse its orientation, positioning the DNA polymerase catalytic center over the PPT primer 3' OH. Creating a gap in (+) RNA ahead of the PPT/U3 junction and dissociation of a small RNA fragment would expose single-stranded template nucleotides for recognition by the RT finger subdomain. These combined nucleolytic events may conceivably be a mechanism to convert RT orientation, switching from a degradative to synthetic mode.

Altered minor groove width as a consequence of the anomalous structure adopted by this RNA/DNA hybrid has been suggested as a determinant of resistance to internal hydrolysis (4). However, positioning of RT on the duplex as indicated in Fig. 8 is equally plausible. This model proposes that if recognition of the unzipped portion of the PPT is favored by the MGBT of the DNA polymerase domain, this would have the consequence of locating the RNase H catalytic center over the PPT/U3 junction. Under these circumstances, internal regions of the PPT would not be available for hydrolysis. In support of this possibility, we have observed that recessing the (-) DNA template at its 3' terminus relative to the RNA primer sequesters the DNA polymerase catalytic center, favoring internal PPT cleavage over the PPT/U3 junction.3


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, NCI-Frederick, MD 21702. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice{at}ncifcrf.gov.

1 The 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. Back

2 D. Lener and S. Le Grice, unpublished observations. Back

3 J. Rausch and S. Le Grice, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank S. Tarasov, NCI-Frederick, for assistance in CD measurements and G. Klarmann for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Telesnitsky, A., and Goff, S. P. (1997) in Retroviruses (Coffin, J. M., Hughes, S. H., and Varmus, H. E., eds) pp. 121-161, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  2. Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324[Abstract/Free Full Text]
  3. Huang, H., Chopra, R., Verdine, G. L., and Harrison, S. C. (1998) Science 282, 1669-1675[Abstract/Free Full Text]
  4. Sarafianos, S. G., Das, K., Tantillo, C., Clark, A. D., Jr., Ding, J., Whitcomb, J. M., Boyer, P. L., Hughes, S. H., and Arnold, E. (2001) EMBO J. 20, 1449-1461[CrossRef][Medline] [Order article via Infotrieve]
  5. Jacques, P. S., Wohrl, B. M., Ottmann, M., Darlix, J. L., and Le Grice, S. F. (1994) J. Biol. Chem. 269, 26472-26478[Abstract/Free Full Text]
  6. Ghosh, M., Jacques, P. S., Rodgers, D. W., Ottman, M., Darlix, J. L., and Le Grice, S. F. (1996) Biochemistry 35, 8553-8562[CrossRef][Medline] [Order article via Infotrieve]
  7. Powell, M. D., and Levin, J. G. (1996) J. Virol. 70, 5288-5296[Abstract/Free Full Text]
  8. Ghosh, M., Williams, J., Powell, M. D., Levin, J. G., and Le Grice, S. F. (1997) Biochemistry 36, 5758-5768[CrossRef][Medline] [Order article via Infotrieve]
  9. Palaniappan, C., Wisniewski, M., Jacques, P. S., Le Grice, S. F., Fay, P. J., and Bambara, R. A. (1997) J. Biol. Chem. 272, 11157-11164[Abstract/Free Full Text]
  10. Powell, M. D., Ghosh, M., Jacques, P. S., Howard, K. J., Le Grice, S. F., and Levin, J. G. (1997) J. Biol. Chem. 272, 13262-13269[Abstract/Free Full Text]
  11. Wohrl, B. M., Krebs, R., Thrall, S. H., Le Grice, S. F., Scheidig, A. J., and Goody, R. S. (1997) J. Biol. Chem. 272, 17581-17587[Abstract/Free Full Text]
  12. Yu, Q., Ottmann, M., Pechoux, C., Le Grice, S., and Darlix, J. L. (1998) J. Virol. 72, 7676-7680[Abstract/Free Full Text]
  13. Wisniewski, M., Palaniappan, C., Fu, Z., Le Grice, S. F., Fay, P., and Bambara, R. A. (1999) J. Biol. Chem. 274, 28175-28184[Abstract/Free Full Text]
  14. Beard, W. A., Stahl, S. J., Kim, H. R., Bebenek, K., Kumar, A., Strub, M. P., Becerra, S. P., Kunkel, T. A., and Wilson, S. H. (1994) J. Biol. Chem. 269, 28091-28097[Abstract/Free Full Text]
  15. Bebenek, K., Beard, W. A., Casas-Finet, J. R., Kim, H. R., Darden, T. A., Wilson, S. H., and Kunkel, T. A. (1995) J. Biol. Chem. 270, 19516-19523[Abstract/Free Full Text]
  16. Bebenek, K., Beard, W. A., Darden, T. A., Li, L., Prasad, R., Luton, B. A., Gorenstein, D. G., Wilson, S. H., and Kunkel, T. A. (1997) Nat. Struct. Biol. 4, 194-197[CrossRef][Medline] [Order article via Infotrieve]
  17. Beard, W. A., Bebenek, K., Darden, T. A., Li, L., Prasad, R., Kunkel, T. A., and Wilson, S. H. (1998) J. Biol. Chem. 273, 30435-30442[Abstract/Free Full Text]
  18. Powell, M. D., Beard, W. A., Bebenek, K., Howard, K. J., Le Grice, S. F., Darden, T. A., Kunkel, T. A., Wilson, S. H., and Levin, J. G. (1999) J. Biol. Chem. 274, 19885-19893[Abstract/Free Full Text]
  19. Latham, G. J., Forgacs, E., Beard, W. A., Prasad, R., Bebenek, K., Kunkel, T. A., Wilson, S. H., and Lloyd, R. S. (2000) J. Biol. Chem. 275, 15025-15033[Abstract/Free Full Text]
  20. Rausch, J. W., Lener, D., Miller, J. T., Julias, J. G., Hughes, S. H., and Le Grice, S. F. (2002) Biochemistry 41, 4856-4865[CrossRef][Medline] [Order article via Infotrieve]
  21. Julias, J. G., McWilliams, M. J., Sarafianos, S. G., Alvord, W. G., Arnold, E., and Hughes, S. H. (2003) J. Virol. 77, 8548-8554[Abstract/Free Full Text]
  22. Charneau, P., Mirambeau, G., Roux, P., Paulous, S., Buc, H., and Clavel, F. (1994) J. Mol. Biol. 241, 651-662[CrossRef][Medline] [Order article via Infotrieve]
  23. Huber, H. E., and Richardson, C. C. (1990) J. Biol. Chem. 265, 10565-10573[Abstract/Free Full Text]
  24. Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456[CrossRef][Medline] [Order article via Infotrieve]
  25. Wohrl, B. M., and Moelling, K. (1990) Biochemistry 29, 10141-10147[CrossRef][Medline] [Order article via Infotrieve]
  26. Pullen, K. A., Ishimoto, L. K., and Champoux, J. J. (1992) J. Virol. 66, 367-373[Abstract/Free Full Text]
  27. Pullen, K. A., Rattray, A. J., and Champoux, J. J. (1993) J. Biol. Chem. 268, 6221-6227[Abstract/Free Full Text]
  28. Rausch, J. W., and Le Grice, S. F. (1997) J. Biol. Chem. 272, 8602-8610[Abstract/Free Full Text]
  29. Schultz, S. J., Zhang, M., Kelleher, C. D., and Champoux, J. J. (2000) J. Biol. Chem. 275, 32299-32309[Abstract/Free Full Text]
  30. Lim, D., Orlova, M., and Goff, S. P. (2002) J. Virol. 76, 8360-8373[Abstract/Free Full Text]
  31. Schultz, S. J., Zhang, M., and Champoux, J. J. (2003) J. Virol. 77, 5275-5285[Abstract/Free Full Text]
  32. Szyperski, T., Gotte, M., Billeter, M., Perola, E., Cellai, L., Heumann, H., and Wuthrich, K. (1999) J. Biomol. NMR 13, 343-355[CrossRef][Medline] [Order article via Infotrieve]
  33. Fedoroff, O., Salazar, M., and Reid, B. R. (1993) J. Mol. Biol. 233, 509-523[CrossRef][Medline] [Order article via Infotrieve]
  34. Salazar, M., Fedoroff, O., Zhu, L., and Reid, B. R. (1994) J. Mol. Biol. 241, 440-455[CrossRef][Medline] [Order article via Infotrieve]
  35. Zhu, L., Salazar, M., and Reid, B. R. (1995) Biochemistry 34, 2372-2380[CrossRef][Medline] [Order article via Infotrieve]
  36. Horton, N. C., and Finzel, B. C. (1996) J. Mol. Biol. 264, 521-533[CrossRef][Medline] [Order article via Infotrieve]
  37. Han, G. W., Kopka, M. L., Cascio, D., Grzeskowiak, K., and Dickerson, R. E. (1997) J. Mol. Biol. 269, 811-826[CrossRef][Medline] [Order article via Infotrieve]
  38. Fedoroff, O. Y., Ge, Y., and Reid, B. R. (1997) J. Mol. Biol. 269, 225-239[CrossRef][Medline] [Order article via Infotrieve]
  39. Kvaratskhelia, M., Budihas, S. R., and Le Grice, S. F. (2002) J. Biol. Chem. 277, 16689-16696[Abstract/Free Full Text]
  40. Kool, E. T. (1998) Biopolymers 48, 3-17[CrossRef][Medline] [Order article via Infotrieve]
  41. Kool, E. T. (2001) Annu. Rev. Biophys. Biomol. Struct. 30, 1-22[CrossRef][Medline] [Order article via Infotrieve]
  42. Kool, E. T. (2002) Annu. Rev. Biochem. 71, 191-219[CrossRef][Medline] [Order article via Infotrieve]
  43. Kool, E. T., Morales, J. C., and Guckian, K. M. (2000) Angew. Chem. Int. Ed. Engl. 39, 990-1009[CrossRef][Medline] [Order article via Infotrieve]
  44. Rausch, J. W., Qu, J., Yi-Brunozzi, H. Y., Kool, E. T., and Le Grice, S. F. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 11279-11284[Abstract/Free Full Text]
  45. Dash, C. V., Rausch, J. W., and Le Grice, S. F. J. (2004) Nuclic Acids Res. 32, 1539-1547[Abstract/Free Full Text]
  46. Lener, D., Kvaratskhelia, M., and Le Grice, S. F. (2003) J. Biol. Chem. 278, 26526-26532[Abstract/Free Full Text]
  47. Orum, H., and Wengel, J. (2001) Curr. Opin. Mol. Ther. 3, 239-243[Medline] [Order article via Infotrieve]
  48. Wengel, J., Vester, B., Lundberg, L. B., Douthwaite, S., Sorensen, M. D., Babu, B. R., Gait, M. J., Arzumanov, A., Petersen, M., and Nielsen, J. T. (2003) Nucleosides Nucleotides Nucleic Acids 22, 601-604[Medline] [Order article via Infotrieve]
  49. Petersen, M., Bondensgaard, K., Wengel, J., and Jacobsen, J. P. (2002) J. Am. Chem. Soc. 124, 5974-5982[CrossRef][Medline] [Order article via Infotrieve]
  50. Ilyinskii, P. O., and Desrosiers, R. C. (1998) EMBO J. 17, 3766-3774[CrossRef][Medline] [Order article via Infotrieve]
  51. Bacharach, E., Gonsky, J., Lim, D., and Goff, S. P. (2000) J. Virol. 74, 4755-4764[Abstract/Free Full Text]
  52. Le Grice, S. F., Cameron, C. E., and Benkovic, S. J. (1995) Methods Enzymol. 262, 130-144[Medline] [Order article via Infotrieve]
  53. McWilliams, M. J., Julias, J. G., Sarafianos, S. G., Alvord, W. G., Arnold, E., and Hughes, S. H. (2003) J. Virol. 77, 11150-11157[Abstract/Free Full Text]
  54. Ratmeyer, L., Vinayak, R., Zhong, Y. Y., Zon, G., and Wilson, W. D. (1994) Biochemistry 33, 5298-5304[CrossRef][Medline] [Order article via Infotrieve]
  55. Hung, S. H., Yu, Q., Gray, D. M., and Ratliff, R. L. (1994) Nucleic Acids Res. 22, 4326-4334[Abstract/Free Full Text]
  56. Braasch, D. A., and Corey, D. R. (2001) Chem. Biol. 8, 1-7[CrossRef][Medline] [Order article via Infotrieve]
  57. Petersen, M., and Wengel, J. (2003) Trends Biotechnol. 21, 74-81[CrossRef][Medline] [Order article via Infotrieve]
  58. Nielsen, K. M., Petersen, M., Hakansson, A. E., Wengel, J., and Jacobsen, J. P. (2002) Chemistry 8, 3001-3009[Medline] [Order article via Infotrieve]
  59. Kanagawa, M., Okada, Y., Uesugi, S., Doi, H., and Katahira, M. (1998) Nucleosides Nucleotides 17, 831-841[Medline] [Order article via Infotrieve]

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