HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 29, 27721-27730, July 20, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/29/27721    most recent M100513200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, J. T. Articles by Le Grice, S. F. J. Search for Related Content PubMed PubMed Citation Articles by Miller, J. T. Articles by Le Grice, S. F. J. Social Bookmarking                      What's this?

A Novel Interaction of tRNA^Lys,3 with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis^*

Jennifer T. Miller, Bernard Ehresmann^§, Ulrich Hübscher^¶, and Stuart F. J. Le Grice

From the  HIV Drug Resistance Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, the ^§ Institut de Biologie Moléculaire et Cellulaire du CNRS, rue Rene Decartes, 67084 Strasbourg Cedex, France, and the ^¶ Institute of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich

Received for publication, January 18, 2001, and in revised form, May 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Complementarity between nucleotides at the 5' terminus of tRNA^Lys,3 and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA^Lys,3 hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA^Lys,3 primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)₄ sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cis-acting sequences throughout the RNA genome are important mediators of several events during replication of retroviruses and retrotransposons, including transcription (1, 2), translation (3), nuclear transport (4, 5), and genome packaging (6, 7). With respect to DNA synthesis, these are represented by the primer-binding site (PBS)¹ and polypurine tract, the initiation sites of ()- and (+)-strand DNA synthesis, respectively (3). While the PBS was originally regarded as the sole determinant of primer tRNA binding and correct initiation of ()-strand DNA synthesis, a considerable body of evidence implicates additional intermolecular contacts between the cognate tRNA primer and viral genome in this event. First documented with avian viruses as an interaction between the TC loop of tRNA^Trp and nucleotides of the U5-IR stem (8-12), this notion has been extended to HIV-1, where extensive base pairing between tRNA^Lys,3 and the viral genome controls initiation and the transition to productive elongation (13-19). In the case of the mal isolate of HIV-1, intermolecular pairing between the U-rich tRNA anticodon loop and the A-rich U5-IR loop upstream of the PBS has been demonstrated to play an important role in this transition (15, 20). Intermolecular interactions of this nature are not confined to retroviruses, exemplified by the contribution of D-loop nucleotides of tRNA^iMet to efficient initiation of ()-strand strong-stop DNA synthesis in the Saccharomyces cerevisiae retrotransposons Ty1 and Ty3 (21-24). While many of these studies have been derived through evaluation of recombinant enzymes and chemically or enzymatically synthesized substrates, their significance is supported by a wealth of in vivo data (25-33). Collectively, these findings show that additional base pairing with the viral U5-IR stem-loop can occur at positions throughout the entire tRNA replication primer.

Although feline immunodeficiency virus (FIV) RT shares a heterodimeric organization of its p66 and p51 subunits with the HIV-1 enzyme and likewise exploits tRNA^Lys,3 as its replication primer, the absence of an A-rich U5-IR loop upstream of the PBS suggests base pairing with the tRNA anticodon loop is unlikely. Significant differences between the HIV and FIV initiation complexes is also predicted by our observations that the p66/p66 and p66/p51 forms of FIV RT fail to productively extend tRNA^Lys,3 hybridized to the PBS of the HIV-1 genome while catalyzing the equivalent event on the FIV genome (34). However, we have noted significant pausing and arrest of DNA synthesis following polymerization of ~14 nucleotides of FIV ()-strand strong-stop DNA (34). This feature was observed with both HIV-1 and FIV RT suggesting it was mediated by a structural feature of the tRNA-viral RNA duplex rather than a deficiency in the retroviral polymerase. Further inspection of FIV U5-IR loop sequences indicated extensive homology with nucleotides at the extreme 5'-end of tRNA^Lys,3. Although these tRNA nucleotides would make up in part the acceptor stem in free tRNA^Lys,3, they might be available for pairing upon its hybridization to the FIV PBS, an event mediated through the 18 3' terminal tRNA nucleotides. If correct, this scenario would contrast sharply with the situation in HIV-1, where both chemical and enzymatic mapping indicate that hybridization of tRNA^Lys,3 to PBS-containing RNA is accompanied by extensive rearrangement and intramolecular pairing of its 5' terminus with nucleotides of the TC arm (16).

In the present article, we have evaluated the initiation program of FIV by a variety of approaches, directed at both the tRNA primer and viral RNA genome. The first of these involved nuclease mapping of the 5' end of the FIV genome to which full-length tRNA^Lys,3 and a variant lacking nucleotides constituting part of the D stem-loop and the entire 5' acceptor stem terminus was hybridized. This approach provides preliminary information on the availability of the FIV U5-IR loop for an interaction with the tRNA 5' terminus. Subsequently, we employed tRNA variants containing increasing lengths of ()-DNA at their 3' terminus to determine the stage in ()-strand synthesis at which arrest at position +14 is overcome. Finally, variants of the FIV genome containing a modified U5-IR loop were constructed to determine whether (a) relief of the proposed base pairing is achieved by introducing an unrelated sequence and (b) introduction of an A-rich U5-IR loop induced an initiation program similar to that reported for HIV-1, i.e. extensive pausing between template positions +3 and +5. Data presented herein supports the notion that FIV exploits a novel intermolecular base pairing interaction to control initiation that is independent of hypermodified bases of the tRNA primer. Moreover, when the FIV genome is mutated to introduce an A-rich U5-IR loop, this does not induce an HIV-like initiation program, but rather allows high level DNA synthesis with minimal pausing. Initiation of reverse transcription in many retroviruses and retrotransposons can thus be viewed as a complex, multistep process where productive elongation arises through "escape" synthesis from an abortive initiation complex, in many respects analogous to the initiation program exhibited by prokaryotic RNA polymerase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Restriction enzymes, DNA/RNA modifying enzymes, dNTPs, rNTPs, and glycogen were purchased from Roche Molecular Biochemicals. ³²P-Labeled nucleotides were the products of Amersham Pharmacia Biotech. T7 RNA polymerase was acquired from Promega, Madison, WI. RNase T1 and nuclease S1 were from Life Technologies, Gaithersburg, MD. Synthetic oligonucleotides were obtained from Integrated DNA Technologies, Coralville, IA. All other reagents were of the highest purity and purchased from Fisher, Sigma, and Bio-Rad.

Plasmids-- p34TF10, a plasmid containing the full FIV Petaluma genome, was a gift from Dr. T. North, University of California, Davis, CA (35). For expression of the FIV RT p66 subunit, pFIV RT was created by ligation of an insert generated by PCR and cleaved with BglII/SalI into plasmid pRT (36), cleaved with BamHI and SalI. The template for PCR was pMA132 (37). For expression of the FIV RT His₆-p51 subunit, p6H FIV RT51 was created by ligation of an insert generated by PCR and cleaved with BglII/HindIII into plasmid p6H RT51 (36), cleaved with BamHI and HindIII. The template for PCR was plasmid pMA131 (37). After expression, each subunit represents the authentic sequence with the exception of a Met-Arg-Gly sequence substituted for the amino-terminal isoleucine, and an additional 6 histidine residues at the NH₂ terminus of p51. Each clone was verified by sequencing.

Enzymes-- Recombinant p66/p51 FIV RT was prepared by in vitro reconstitution from strains M15::pDMI.1::pFIVRT (p66) and M15::pDMI.1::p6HFIVRT51 (His-p51) following induction with isopropyl-1-thio--D-galactopyranoside. Enzyme was purified by a combination of metal chelate and ion exchange chromatography (38), and exhibited a 1:1 stoichiometry between subunits when examined by SDS-polyacrylamide gel electrophoresis.

RNA Templates-- Wild type and mutant viral RNAs were prepared by in vitro transcription utilizing a T7 promoter-containing PCR product. Viral RNA templates were produced in two sizes, namely a 416-nt sequence beginning at the start of the R region and a 123-nt species initiating at the start of the U5 region. Primer extension reactions comparing products in these assays with either the short 123-nt or long 416-nt template were indistinguishable. 5'-³²P-Labeled templates were prepared by phosphorylation of RNA lacking a 5' phosphate (see below) utilizing T4 polynucleotide kinase and [-³²P]ATP (3000 Ci/mmol) following standard protocols. RNA templates with a 5'-terminal guanosine residue were prepared by in vitro transcription using T7 RNA polymerase and a 5-fold excess of guanosine included over the other 4 rNTPs.

tRNA Primers-- Synthetic tRNA^Lys,3 was prepared by in vitro transcription utilizing a T7 promoter-containing PCR product, with the addition of [-³²P]UTP (3000 Ci/mmol) if the primer was to be internally labeled. Synthetic tRNA lacking 17 nucleotides at the 5'-end was prepared by in vitro transcription with T7 RNA polymerase of FokI cleaved pLYSF119 containing the gene for 3'-59-mer tRNA^Lys,3 (39). Natural (fully modified) tRNA^Lys,3 was prepared from bovine liver as described previously (40), and labeled at the 3'-end according to the procedure described below. Natural tRNA^Lys,3 was annealed to a PBS-containing DNA oligonucleotide (flanked 5' and 3' by 7 and 10 nt of FIV sequence, respectively) and extended by one deoxyadenosine residue with the large fragment of Escherichia coli DNA polymerase as described (34, 41). This primer was purified by high voltage denaturing acrylamide gel electrophoresis and annealed to the template as detailed below. Extended tRNA/DNA chimeras were also prepared by this method, with the following modifications: ³²P-internally labeled synthetic tRNA^Lys,3 was annealed to one of three different PBS-containing DNA oligonucleotides, each flanked 3' by 10 nt, and at the 5'-end extending 7, 11, and 32 nt 5' to the PBS, respectively. The species extended by 7, 11, and 32 deoxynucleotides were created by adding all four dNTPs to the primer extension reaction and allowing run-off synthesis. To create the species extended by three deoxynucleotides, a mixture of dATP, dGTP, and dTTP was used.

RNA-dependent DNA Synthesis Reactions-- Annealing was facilitated by mixing a 2-fold molar excess of template with primer in 100 mM NaCl and heating to 90 °C for 2 min. This denaturation step was quenched on ice for 2 min, after which the reaction was incubated at 70 °C for 20 min, and stored on ice. The efficiency of annealing was verified by nondenaturing polyacrylamide gel electrophoresis analysis and was always 90-100%. Annealed template/primer was added at a final concentration of 40 nM to a reaction buffer containing 42.9 mM Tris-HCl, pH 7.8, 6 mM MgCl₂, 100 mM KCl, 1 mM dithiothreitol. Enzyme was added to a final concentration of 80 nM and complex formation was allowed to proceed for 1 min at 37 °C. dNTPs were added to a final concentration of 200 µM and aliquots were taken between 10 s and 10 min, extracted with an equal volume of phenol/CHCl₃/isoamyl alcohol (25:24:1), and precipitated with glycogen (final 0.4 mg/ml), sodium acetate, pH 5.2, and ethanol. After precipitation, pellets were washed with 70% EtOH, dried, resuspended in 89 mM Tris borate, pH 8.3, 2 mM EDTA, 7 M urea, 0.1% xylene cyanole, 0.1% bromphenol blue and subjected to high voltage denaturing polyacrylamide gel electrophoresis. Dried gels were evaluated by either phosphorimaging analysis (Bio-Rad screen and Quantity One software) or autoradiography (Kodak BioMax screens and film).

Single-round RNA-dependent DNA Synthesis Reactions-- Annealing reactions were performed as above. Annealed template/primer was added at a final concentration of 40 nM to a reaction buffer containing 42.9 mM Tris/HCl, pH 7.8, 6 mM MgCl₂, 100 mM KCl, 1 mM dithiothreitol. Enzyme was added to a final concentration of 80 nM and complex formation was allowed to proceed for 1 min at 37 °C. A mixture of heparin and dNTPs were added to a final concentration of 0.5 mg/ml and 200 µM, respectively. Aliquots were taken between 10 s and 10 min, extracted with an equal volume of phenol/CHCl₃/isoamyl alcohol (25:24:1) and precipitated with glycogen (final 0.4 mg/ml), sodium acetate, pH 5.2, and ethanol. After precipitation, pellets were washed with 70% EtOH, dried, resuspended in 89 mM Tris borate, pH 8.3, 2 mM EDTA, 7 M urea, 0.1% xylene cyanole, 0.1% bromphenol blue, and subjected to high voltage denaturing polyacrylamide gel electrophoresis. Dried gels were evaluated as above. As a control for trap efficiency, RT and heparin were preincubated with buffer; the reaction was initiated with template/primer and dNTPs and allowed to proceed 10 min.

Enzymatic Cleavage of RNA-- The annealing reaction was composed of 5'-³²P-labeled template RNA, 75 ng/µl, specific activity ~500 cpm/ng, combined with (when appropriate) a 2-fold molar excess of primer in 50 mM Tris-HCl, pH 7.8, 100 mM KCl. This mixture was heated to 90 °C for 2 min. The sample was quenched on ice for 2 min, after which the reaction was incubated at 70 °C for 20 min. Subsequently, MgCl₂ was added to a final concentration of 5 mM and the reaction incubated at room temperature for 15 min. All cleavage reactions were performed in a rack previously cooled to 4 °C and placed on ice prior to assembly of the reaction. S1 nuclease cleavage was performed in a buffer of 30 mM sodium acetate, pH 4.6, 100 mM NaCl, 1 mM ZnCl₂, 5% glycerol, 25 ng/µl yeast tRNA, 7.5 ng/µl template/primer, and 0.7 units/µl nuclease S1. Prior to addition of enzyme, samples were incubated in the cooled rack for 2 min to ensure uniform temperature. Cleavage was initiated by addition of S1 nuclease, freshly diluted in 20 mM Tris, pH 7.5, 0.1 mM zinc acetate, 50 mM NaCl, and 5% glycerol, aliquots were removed at 1, 5, and 10 min, immediately extracted with an equal volume of phenol/CHCl₃/isoamyl alcohol (25:24:1) and precipitated with 0.4 mg/ml glycogen, sodium acetate, pH 5.2, and ethanol. RNase T1 cleavage was performed in a buffer of 35 mM Tris, pH 7.8, 100 mM KCl, 5 mM MgCl₂, 25 ng/µl yeast tRNA, 7.5 ng/µl template/primer, and 0.12 unit/µl RNase T1. Prior to addition of enzyme, reaction mixtures were incubated in the cooled rack for 2 min as above. Cleavage was initiated by addition of RNase T1, freshly diluted in 10 mM sodium phosphate, pH 6.7, 50% glycerol, aliquots were removed at 5 and 10 min, immediately extracted with an equal volume of phenol/CHCl₃/isoamyl alcohol (25:24:1) and precipitated as above. After precipitation, the pellets were washed with 70% EtOH, dried, resuspended in 89 mM Tris borate, pH 8.3, 2 mM EDTA, 7 M urea, 0.1% xylene cyanole, 0.1% bromphenol blue and subjected to high voltage denaturing polyacrylamide gel electrophoresis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Initiation of FIV ()-Strand Strong-stop DNA Synthesis-- Fig. 1A provides a time course of ()-strand strong-stop DNA synthesis catalyzed by heterodimeric (p66/p51) FIV RT on its wild type genome containing a radiolabeled oligodeoxynucleotide hybridized to the PBS. In the immediate vicinity of the initiation site, pausing occurs early in the time course, although the products gradually diminish as they are replaced by the full-length ()-strand strong-stop DNA. A similar DNA synthesis profile was obtained with an oligoribonucleotide hybridized to the PBS (data not shown). In contrast, the pausing profile was significantly altered when the oligonucleotide primers were replaced with the 76-nt natural tRNA^Lys,3 (Fig. 1B). Two pause sites limited the amount of full-length cDNA formed. The first of these occurs at position T + 7 (designating T as the length of the tRNA primer), and rapidly diminishes with extended incubation. Given the length of the nascent DNA, the T + 7 product is unlikely to correspond to the early initiation products observed with HIV-1 RT on its homologous RNA genome. Rather, we believe this corresponds to the gradual replacement of duplex RNA in the nucleic acid-binding cleft with an RNA-DNA hybrid and the transition of this junction over the p66 thumb subdomain as was recently demonstrated for HIV (17-19). However, a T + 14 product accumulates throughout the time course, suggesting a feature of the tRNA-viral RNA complex was transiently halting the FIV replication machinery. In order to determine if the enzyme stalled at these pause sites remains bound or dissociates, primer extension was evaluated in the presence of a heparin trap (Fig. 1C), which restricts the enzyme to a single binding event. RT was pre-bound to the template/primer and the reaction initiated with dNTPs and heparin. As early as 10 s, T + 7 and T + 14 extended primers accumulated. This pattern remained essentially unchanged with prolonged incubation, with 64% termination at T + 7. Of the remaining 36% that reached T + 14, 85% termination efficiency was observed, and only 0.25% of those proceeding through both pause sites reached full-length ()-strand strong-stop. These data indicate that the majority of the enzyme dissociates during the transition from RNA/DNA to RNA/RNA in the nucleic acid-binding cleft. In addition, those that remain associated are likely to encounter a second barrier and dissociate at T + 14. Unextended tRNA primer remains at late time points, even though the reaction is performed in 2-fold enzyme excess. This is likely caused by the fact that the enzyme preparation itself may only be partially active. Previous studies of HIV-1 or EIAV RT isolated in an analogous fashion report values of ~50% active enzyme in those preparations (42-45).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Oligonucleotide and tRNA-primed ()-strand strong-stop DNA synthesis on the FIV genome. A, oligonucleotide-primed synthesis, using an 18-nucleotide DNA primer hybridized to the PBS. Migration positions of the primer and ()-strong-stop (ss) product have been indicated. RNA-dependent DNA synthesis was evaluated after 10 s, 30 s, 1 min, 2 min, 3 min, and 5 min (panels a-f, respectively). The concentration of enzyme was 80 nM and template/primer 40 nM. Lane C, unextended, radiolabeled DNA primer. P indicates the migration position of this DNA primer. B, natural tRNALys,3-primed ()-strand strong-stop synthesis. In addition to the full-length ()-strong-stop product, major pause points at positions T + 7 and T + 14 are indicated. RNA-dependent DNA synthesis was evaluated after 10 s, 30 s, 1 min, 2 min, and 3 min (panels a-e, respectively). The concentration of enzyme was 80 nM and template/primer 40 nM. Lane C, unextended tRNA primer. T indicates the migration position of the tRNA primer. C, tRNALys,3-primed ()-strand strong-stop synthesis in the presence of heparin trap. Natural tRNA was used as primer in this experiment. Migration positions of the ()-strand strong-stop product and major pause points are indicated. Time points, enzyme, and primer template concentrations are as in B. Lane C, unextended tRNA primer. T indicates the migration position of the tRNA primer. D, structure of tRNALys,3 indicating nucleotides of its 5' terminus complementary to the FIV U5-IR loop (black bar). In the tRNA structure, base pairing between the D and TC loops has been indicated by the dashed lines in addition to the base paired D, TC, anticodon, and acceptor stems. Nucleotides absent from the 5'-truncated tRNALys,3 are indicated in bold and italicized (5').

Because the U5-IR loop of the FIV genome is not A-rich (34), it appeared unlikely that T + 14 pausing was mediated through an interaction with the tRNA anticodon loop previously reported for the HIV-1 (mal isolate) system. However, inspection of the sequence of tRNA^Lys,3 indicated that base pairing might occur between U5-IR loop nucleotides and those at the extreme 5' terminus of the tRNA primer (Fig. 1D). Fig. 2B indicates this potential intermolecular duplex would start 16 nucleotides upstream from the initiation site, which would be 2 nucleotides removed from the T + 14 pause site (Fig. 1B). The extreme 5' terminus of tRNA^Lys,3 would be available for this interaction following hybridization of the 18 3' nucleotides to the PBS. Thus, while the U5-IR loop of another lentivirus is again implicated in control of ()-strand strong-stop DNA synthesis, involvement of 5' nucleotides of the tRNA primer represents a mechanism unlike those that have been documented previously (9, 21, 24, 40).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   A, determination of the structure of FIV PBS-containing genomic RNA in the absence (panel i) and presence of tRNALys,3 derivatives (panels ii and iii). RNA structure was evaluated by susceptibility to the nucleases S1 and T1. For each panel, nuclease digestion was performed for 5 or 10 min (lanes a and b, respectively). For S1 digestion, enzyme was at a concentration of 0.7 unit/µl and template primer was present at 7.5 ng/µl. T1 digestion was performed at the same template/primer concentration but with 0.12 unit/µl RNase T1. The migration positions of the 5'-labeled products following digestion within the PBS, U5-IR loop, U-rich loop, and PBS complementary (PBS-C) have been indicated. 5'-End-labeled substrate did not show any truncated products when analyzed without nucleases (data not shown). B, summary of nuclease mapping data in the absence and presence of full-length tRNALys,3. Closed and open squares represent nuclease-sensitive regions prior to tRNA hybridization, while closed and open circles indicate those which are affected following hybridization of the replication primer. The notation +1 represents the site at which strong-stop DNA synthesis initiates (arrow). Structure prediction was performed according to Zuker et al. (56).

Interaction of FIV U5-IR Loop Nucleotides with the 5' Terminus of tRNA^Lys,3-- Prior to a more extensive evaluation of tRNA-primed initiation in FIV, it was important to establish that U5-IR loop nucleotides of the viral genome were available for hybridization with the 5' terminus of tRNA^Lys,3 and subsequently involved in intermolecular base pairing following its association with the PBS. The folding diagram of Fig. 2B predicts an extensively paired U5-IR stem with an 8-nucleotide loop at its apex. The same figure also predicts that the majority of PBS nucleotides are paired in the absence of the tRNA primer, and in the immediate vicinity of a hairpin structure whose loop is U-rich. 5'-End-labeled viral RNA was therefore subjected to mild digestion with the nucleases S1 and T1, which recognize single-stranded regions of nucleic acid and unpaired G residues in RNA, respectively. Subsequently, nuclease mapping was performed in the presence of full-length tRNA^Lys,3 and a synthetic variant from which bases between the D-loop and 5' terminus were deleted. The results of our mapping experiments are presented in Fig. 2A.

In the absence of full-length tRNA^Lys,3, two regions around the PBS are susceptible to S1 digestion (Fig. 2A, panel i). The first of these is the U5-IR loop, and the second is the U-rich loop of the stem-loop adjacent to the PBS. At the same time, we observe little to no digestion of nucleotides constituting the PBS, confirming this is largely paired in the absence of the replication primer. T1 cleavage reveals two unpaired G residues in the U5-IR loop and a single-stranded G adjacent to the U-rich stem-loop (Fig. 2A, panel i). As illustrated in panel ii of Fig. 2A, this pattern changes dramatically in the presence of full-length tRNA^Lys,3. In both cases, nucleotides representing the U5-IR loop are rendered resistant to both nucleases. At the same time, S1 sensitivity of the nucleotides complementary to the PBS dramatically increases, as would be predicted from the hybridization of the PBS with the 18 nucleotides at the tRNA 3' terminus. This effect is mirrored, albeit to a lesser extent, in the case of T1 digestion.

While the enzymatic mapping data in panel i of Fig. 2A indicates extensive rearrangement around the PBS and base pairing of the U5-IR loop, it does not provide direct evidence of involvement of the 5' terminus of tRNA^Lys,3 with this region. Proof of this interaction was provided by hybridizing a tRNA variant lacking sequences at its 5' terminus (panel iii of Fig. 2A). Hybridization of this truncated tRNA was predicted to leave bases of the U5-IR loop nuclease susceptible, but at the same retain the susceptibility of bases previously paired to the PBS, a notion which was borne out experimentally. We also note that retention of S1 sensitivity within the entire U5-IR loop with truncated tRNA^Lys,3 ruled out the possibility of base pairing between the adjacent A residues and U residues of the tRNA anticodon loop. The combined data of Fig. 2 thus illustrates that hybridization of tRNA^Lys,3 to the FIV PBS is accompanied by an additional intermolecular interaction of its free 5' terminus with the U5-IR loop.

Pausing in the Vicinity of the FIV PBS Is Independent of tRNA Modification-- The most extensive base modifications in tRNA^Lys,3 are within the anticodon loop (Fig. 3A), and are known to stabilize the interaction of the HIV-1 (13-19, 40). In contrast, studies with Moloney murine leukemia virus indicate that post-transcriptional base modifications of its cognate replication primer tRNA^Pro inhibit additional base pairing interactions between the viral genome (46). Therefore, we determined whether modifications in tRNA^Lys,3 interrupted FIV DNA synthesis in the vicinity of the PBS. A time course of ()-strand strong-stop synthesis was performed using both natural and synthetic tRNA^Lys,3 hybridized to the FIV genomic RNA template. The results are presented in Fig. 3, B and C, and summarized graphically in Fig. 3D.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Pausing during ()-strand synthesis on the FIV genome is independent of base modifications on the tRNA primer. A, modified bases of the tRNALys,3 contributing to stability of the HIV-1 initiation complex. The upper portion of the figure illustrates the tRNA anticodon stem-loop, while the lower provides the structure of the hypermodified bases S and R. B and C, DNA synthesis profiles primed by natural and synthetic tRNALys,3, respectively. In each panel, lane C, unextended tRNA primer, while lanes a-f are samples evaluated 10 s, 30 s, 1 min, 2 min, 3 min, and 5 min after initiation of DNA synthesis. The concentration of enzyme was 80 nM and template/primer 40 nM. Migration positions of the tRNA primer (T), the major pause product (T + 14) and full-length ()-strand strong-stop (ss) DNA have been indicated. D, upper, amount of total primer extension was quantified for each reaction and plotted versus time. , natural tRNALys,3; , synthetic tRNALys,3. Lower, amount of T + 14 product as a proportion of total extended product. Symbols are as in the upper panel.

When natural tRNA^Lys,3 is hybridized to the viral RNA template, the amount of primer extended is ~3-fold greater (Fig. 3D, upper panel). This is in agreement with studies in HIV-1 where primers containing post-transcriptional modifications were used with greater efficiencies, with regard to initiation of both ()-strand synthesis and (+)-strand transfer (14, 47, 48). Another difference between the two primers was accumulation of the T + 7 product during early stages of ()-strand strong-stop synthesis supported by natural tRNA. This species rapidly disappears and is replaced with the T + 14 reverse transcript. Although less pronounced, a similar pause site is observed with synthetic tRNA. These data suggest that modified nucleotides of tRNA^Lys,3 enhance the efficiency with which it is utilized as a primer for initiation of ()-strand strong-stop synthesis. In addition, modifications present in the primer may facilitate the transition from duplex RNA to an RNA-DNA hybrid in the nucleic acid-binding cleft. Regardless of the tRNA primer employed, significant pausing is evident at template nucleotide +14, suggesting that modified bases do not influence the interaction of the 5'-end of the primer with the viral RNA U5-IR loop. This is not unexpected, since the only modification at the 5' end of tRNA^Lys,3 is a methylated G at position 10, a position not directly involved in the proposed base pairing interaction.

Hybridization of a Truncated tRNA^Lys,3 to the PBS Relieves Pausing-- Experiments reported in Fig. 2 exploited a truncated derivative of tRNA^Lys,3 lacking sequences from the D-loop through the 5' terminus. In order to determine whether pausing on the RNA genome reflected participation of the tRNA 5' terminus in intermolecular base pairing, the ability of this truncated tRNA to support ()-strand strong-stop synthesis was compared with an intact tRNA primer. Based on the data of Fig. 3, tRNA modifications do not appear to influence pausing, thus allowing the use of the in vitro transcribed counterpart. The results of this analysis are presented in Fig. 4. Although some premature termination occurs in the immediate vicinity of the PBS, our data clearly indicates that eliminating nucleotides from the 5' terminus of the tRNA primer relieves pausing at template nucleotide +14. Again, only a small proportion (8-10%) of the primer is extended at late time points, reflecting a combination of the reduced amount of active enzyme in the preparation and the lack of modifications. Together with the nuclease mapping data, the observations of Figs. 2 and 4 provide a strong argument that, following or concomitant with hybridization to the PBS, the 5' terminus of tRNA^Lys,3 forms a stable structure with the U5-IR loop to present a barrier to the FIV replication machinery.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Deleting nucleotides constituting the 5' terminus of tRNALys,3 relieves pausing during initiation. For both the full-length (A) and truncated tRNA-primed event (B), ()-strand strong-stop (ss) DNA synthesis was evaluated after 10 s, 30 s, 1 min, 2 min, 3 min, 5 min, and 10 min (lanes a-g, respectively). Concentrations of enzyme and template/primer were 80 and 40 nM, respectively. Differences in migration positions of the two ()-strong-stop species reflect the use of the truncated primer. Lanes C, unextended tRNA primer; lanes M, X174 HinfI DNA molecular weight marker.

Initiation of ()-Strand DNA Synthesis by tRNA-DNA Chimeras-- In order to better understand structural features of the FIV tRNA-viral RNA complex controlling initiation of ()-strand synthesis, a series of tRNA-DNA chimeras were substituted for tRNA^Lys,3. These chimeras contained increasing amounts of ()-strand DNA at the 3' terminus of the tRNA primer, effectively extending complementarity to the viral RNA genome beyond the PBS. Annealing of these chimeras would be predicted to disrupt intermolecular interactions between the tRNA primer and viral RNA genome. This strategy was successfully employed to study HIV-1 RT mutants defective in initiation (34, 41), as well as provide a detailed kinetic analysis of its initiation process (18). For the present studies, tRNA primers containing ()-strand extensions of 3, 7, 11, and 32 deoxynucleotides were prepared (T/D3, T/D7, T/D11, and T/D32, respectively). This approach is outlined experimentally in Fig. 5A, while Fig. 5B illustrates the efficiency with which each primer was used.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Evaluation of FIV ()-strand strong-stop (ss) DNA synthesis with tRNALys,3 ()-DNA chimeras. A, schematic representation of the system. The 3' terminus of the tRNA/DNA chimeric primers containing 3, 7, 11, and 32 nt of DNA (T/D3, T/D7, T/D11 and T/D32, respectively) is indicated by the tip of bold arrow, while the circled base represents the authentic initiation site. B, ()-strand strong-stop DNA synthesis profiles. In B, i-iii, DNA synthesis was evaluated after 10 s, 30 s, 1 min, 2 min, 3 min, and 5 min (lanes a-f, respectively), while in panel iv, lanes a-e, represent 10 s, 1 min, 2 min, 3 min and 5 min, respectively. The concentration of enzyme was 80 nM and template/primer 40 nM. An asterisk indicates the major pause site.

Hybridization of the T/D3 tRNA^Lys,3 variant had little effect on both pausing, which now occurs at position +11, and the total amount of ()-strand strong-stop DNA was 7%, similar to that obtained in Fig. 4A (Fig. 5B, panel i). Enzymatic footprinting of HIV-1 RT (49, 50) and a kinetic evaluation of DNA synthesis as a function of template length (51) suggest that the translocating enzyme shields 5-7 single-stranded template nucleotides ahead of the polymerase active center. Such data would imply that hybridization of the T/D3 chimera positions the replication complex upstream of the tRNA-viral RNA base pairing interaction, thus pausing would still be realized. The first indication of a tRNA-DNA chimera influencing the base pairing interaction is evident following hybridization of the T/D7 primer (Fig. 5B, panel ii). In this case, although the predicted +7 pause product is evident, the amount of ()-strand strong-stop DNA increases to 20%, i.e. almost 3-fold. The use of the T/D7 primer would place the replication complex in the immediate vicinity of the U5-IR loop, where it may potentially weaken base pairing. If this were the case, the chimeric T/D11 primer might be predicted to resolve the tRNA-viral RNA loop, alleviate pausing completely and result in a further increase in ()-strand synthesis. This notion is borne out by the data of Fig. 5B, panel iii, where almost 88% of the primer is converted to ()-strand strong-stop DNA. As a final control, hybridization of the T/D32 primer, predicted to completely resolve the base paired complex, lead to high level ()-strand strong-stop DNA synthesis, as illustrated in Fig. 5B, panel iv. The use of such chimeric tRNA-DNA primers thus substantiates the existence of this novel tRNA-viral RNA interaction and moreover that its resolution is accompanied by high level ()-strand strong-stop DNA synthesis.

Altering the Nature of the FIV U5-IR Loop Influences the Initiation Program-- In addition to altering the tRNA primer through deletion of 5' nucleotides or addition of a limited number of deoxynucleotides, we elected to evaluate FIV ()-strand strong-stop synthesis on an RNA genome whose U5-IR stem-loop had been altered. The first of these destroyed complementarity with the tRNA 5' terminus while preserving the overall structure of the U5-IR loop. A second viral template variant introduced the A-rich U5-IR loop of the HIV-1 genome which interacts with bases of the tRNA anticodon loop. These template variants examine whether the FIV initiation program is eliminated or adjusted to resemble that observed with HIV-1, respectively. The results of our analyses are presented in Figs. 6 and 7.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Replacement of the FIV U5-IR loop with an unrelated sequence eliminates pausing. The modified U5-IR loop sequence is indicated in A, and the ()-strand strong-stop (ss) DNA synthesis profiles in B. Lanes a-e represent 10-s, 30-s, 1-min, 2-min, 3-min, and 5-min time points, respectively. The concentration of enzyme was 80 nM and template/primer 40 nM. Lane C, unextended tRNA primer. Panel i represents extension from fully modified tRNALys,3, while panel ii is that from its synthetic counterpart.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Replacing the FIV U5-IR loop with the A-rich U5-IR loop of the HIV-1 genome eliminates pausing and fails to impose the initiation program of HIV-1. Alterations to the FIV U5-IR loop are indicated in A. B depicts nuclease sensitivity of this U5-IR loop in the presence of either full-length tRNALys,3 or the derivative lacking sequences at its 5' terminus. S1 digestion was performed for either 5 or 10 min (lanes a and b), while T1 digestion was performed for 1, 5, and 10 min (lanes c-e, respectively). For S1 digestion, enzyme was at a concentration of 0.7 unit/µl and template/primer was present at 7.5 ng/µl. T1 digestion was performed at the same template/primer concentration but with 0.12 unit/µl RNase T1. Positions of the products reflecting digestion within the U5-IR loop, PB, U-rich loop, and PBS complementary (PBS-C) have been indicated. C, DNA synthesis profiles. Lanes a-f represent 10-s, 30-s, 1-min, 2-min, 3-min, and 5-min time points, respectively. The concentration of enzyme was 80 nM and template/primer was 40 nM. ()-ss DNA denotes the position of ()-strand strong-stop DNA product.

Introducing a U5-IR sequence unable to base pair with tRNA^Lys,3 allows DNA synthesis to proceed relatively unimpaired, yielding substantial levels of ()-strand strong-stop DNA, regardless of the nature of the tRNA primer (Fig. 6, B and C). Minor pausing in the vicinity of the PBS is most likely related to the gradual replacement of duplex RNA in the nucleic acid-binding site with an RNA-DNA hybrid at the onset of RNA-dependent DNA synthesis. A similar result was obtained when the HIV-1 U5-IR loop was introduced (Figs. 7C, panels i and ii). The latter observation is of particular importance since such a tRNA-viral RNA interaction has been proposed as a critical determinant of the HIV-1 initiation program (13-16, 40). Enzymatic mapping of tRNA^Lys,3 with this variant of the FIV genome indicates that nucleotides of the heterologous U5-IR loop remain susceptible to S1 digestion, indicating an inability to form a stable structure with the tRNA anticodon loop (Fig. 7B, panels i-iii). Conceivably, this might result from the increased length of the FIV U5-IR stem, i.e. the distance between the potentially interacting partners is simply too great. Alternatively, the HIV-1 U5-IR loop-tRNA anticodon loop interaction is stabilized by a substantial number of additional intermolecular contacts, most of which are absent on the FIV RNA/tRNA^Lys,3 duplex. Recent data from our laboratory shows that p66/p51 FIV RT fails to productively extend tRNA^Lys,3 hybridized to the HIV-1 genome, which suggests these additional interactions are more critical than the tRNA anticodon loop-vRNA U5-IR loop interaction.² Although these notions are presently speculative, the data presented here indicate major differences in the initiation complexes of HIV-1 and FIV despite the use of a common tRNA primer and similar subunit organization of these lentiviral polymerases.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Initiation of retroviral ()- and (+)-strand DNA synthesis is intimately linked with integration of the double-stranded proviral DNA product into the chromosome of the infected cell. Since sequences at the long terminal repeat termini critical to integration are the immediate product of PBS- and polypurine tract-primed synthesis (3), it is reasonable to assume that a higher degree of efficiency and accuracy might be exerted during initiation from each of these primers. In support of this notion, intermolecular interactions beyond homology of the 3' terminal nucleotides with the PBS have now been shown to mediate efficient tRNA-primed initiation. Examples of this vary from the system in avian viruses, comprising a long-range interaction consisting only of tRNA^Trp TC nucleotides with the viral U5-IR stem, to the scenario in HIV-1 where extensive rearrangement of the viral genome accompanies hybridization of tRNA^Lys,3 to the PBS (13, 52). Central to the latter system is an interaction between the U-rich tRNA anticodon loop and the A-rich viral U5-IR loop, stabilized via hypermodified bases in the former (14, 16, 40). tRNA^Lys,3 primers containing these hypermodified bases have been shown to enhance the efficiency of both ()-strand synthesis and (+)-strand transfer in vitro (14, 47, 48). The present study sought to evaluate initiation of ()-strand DNA synthesis in a related lentiviral system whose RT was structurally analogous to the HIV-1 enzyme and likewise exploited a tRNA^Lys,3 primer, but differed in that its U5-IR loop contained an unrelated sequence. Moreover, reconstituting an FIV initiation system might shed light on the inability of the feline enzyme to initiate ()-strand DNA synthesis on the HIV-1 genome despite sharing a common tRNA primer (34).

Although limited enzymatic probing was undertaken here, evaluation of the wild type FIV genome and variants with alterations to the U5-IR loop suggests the major consequence of tRNA binding to the PBS is the interaction of the tRNA^Lys,3 5' terminus with the U5-IR loop. Thus, the FIV initiation program might be considered analogous to its ASLV counterpart, which implicates only the interaction of TC loop nucleotides with the U5-IR stem (9-11) in addition to that with the PBS. Interestingly, heavily modified nucleotides are absent from both the TC loop of tRNA^Trp and the 5' end of tRNA^Lys,3, suggesting that the additional interactions with their respective genomes may be inherently more stable than that between the HIV U5-IR loop and tRNA anticodon loop, which requires both base modifications and multiple regions of base pairing for efficient initiation. For FIV, this most likely resides in the observation that (a) homology between the 5' terminus of tRNA^Lys,3 and the U5-IR loop extends over 8 base pairs, and (b) the intermolecular complex is stabilized by six G:C base pairs. Data of Fig. 7 also indicate that the efficiency of FIV ()-strand strong-stop synthesis is not significantly affected when its U5-IR loop is altered to optimize a potential interaction with the tRNA anticodon loop, i.e. accumulation of the equivalent HIV initiation products is not observed. Collectively, these findings indicate that stabilization of the HIV initiation complex requires additional tRNA/viral RNA interactions. This would account for our earlier observations that it presents a barrier to tRNA-primed initiation on the HIV-1 genome by FIV RT, although the enzyme will productively utilize an oligoribo- or oligodeoxyribonucleotide primer hybridized to the PBS on the same genome.

While our data provides a plausible explanation for the efficiency with which FIV RT uses homologous and heterologous viral RNA templates, it does not provide insights to the mechanism through which the FIV replication machinery is stalled, albeit transiently, shortly after initiation of ()-strand DNA synthesis on its homologous genome. The simple explanation that this reflects an inability to catalyze strand-displacement synthesis can be ruled out, since pausing occurs after synthesis through the majority of the duplex U5-IR stem. Moreover, artificially destabilizing the U5-IR stem through the use of chimeric tRNA/DNA chimeric primers does not eliminate pausing at T + 14 until the DNA component is ~11 nucleotides long. Interestingly, the apex of the U5-IR stem comprises three G:C base pairs, after which the U5-IR loop sequence -G-G-G-C-C- is paired to its complement at the tRNA 5' terminus. Thus, the replication machinery is required to disrupt a 3-base pair intramolecular G:C duplex and immediately thereafter a 5-base pair intermolecular G:C duplex. Disruption of these structures would therefore appear to be the rate-limiting step during initiation. An additional feature of the initiation complex which could contribute to transient pausing is the nature of the duplex in the nucleic acid-binding site, which gradually changes from duplex RNA to an RNA/DNA hybrid as the PBS is cleared. Our recent work with HIV-1 has indicated that polymerization is significantly affected by the nature of the nucleic acid duplex at the base of the p66 thumb subdomain and COOH-terminal RNase H domain (17-19). In the T + 14 paused complex, duplex RNA will still be in the vicinity of the RNase H catalytic center, which may contribute toward the transition from an initiation to elongation complex. Only once the DNA polymerase catalytic center reaches the U5-IR loop is the nucleic acid-binding site occupied over its entire length with an RNA/DNA hybrid, which might induce a transition to the elongation complex. A comparison of the efficiency with which T + 7 and T + 11 tRNA/DNA chimera is used as primer (Fig. 5) lends credence to this model.

Regions of the tRNA replication primer implicated in controlling initiation of reverse transcription have now expanded to include the 3' acceptor stem (the PBS, in all retroviruses and retrotransposons), the TC loop (avian retroviruses) (9-11), anticodon loop (HIV-1 and HIV-2) (14, 16, 53), D-loop (yeast retrotransposons Ty1 and Ty3) (21-24), and 5' acceptor stem (FIV, this work). Moreover, Brule et al. (54) have demonstrated that sequences in the anticodon stem of tRNA^Lys,3 may mediate efficient ()-strand transfer in HIV. Together, this demonstrates "cross-talk" between the tRNA primer and viral RNA genome and its importance in ensuring that DNA synthesis commences with the appropriate efficiency and accuracy. Interestingly, the U5-IR loop of the equine infectious anemia virus genome, a related lentivirus that uses tRNA^Lys,3 as primer, is neither A-rich nor complementary to nucleotides in the 5' acceptor stem. Despite this, we have noted that the DNA synthesis profile during initiation closely resembles that of HIV-1, i.e. accumulation of T + 1  T + 5 products (34). Efforts are currently underway to determine the nature of the intermolecular interactions active in this system. Finally, while accurate initiation from the PBS defines the terminus of the retroviral 3' long terminal repeat, the terminus of its 5' counterpart is established by sequences immediately adjacent to the (+)-strand polypurine tract primer. In this respect, Götte et al. (55), have recently presented a model for temporal coordination between (+)-strand initiation and removal of the polypurine tract primer. Although these studies are restricted to HIV-1, recombinant enzymes from several retroviruses and retrotransposons whose polypurine tracts vary considerably in sequence are now available, which should allow a detailed study of a step critical to synthesis of an infectious provirus.

	ACKNOWLEDGEMENTS

We thank K. Musier-Forsyth and T. Stello for the gift of pLYSF119 containing the gene for 3'-59-mer tRNA^Lys,3. In addition we are grateful to members of our laboratory and K. Musier-Forsyth for critical review of this manuscript and many helpful discussions. We acknowledge G. Bec and G. Keith for providing natural tRNA^Lys,3.

	FOOTNOTES

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

To whom correspondence should be addressed. Tel.: 301-846-5626; Fax: 301-846-6013; E-mail: slegrice@mail.ncifcrf.gov.

Published, JBC Papers in Press, May 15, 2001, DOI 10.1074/jbc.M100513200

² J. T. Miller, M. Amacker, U. Hübscher, B. Ehresmann, and S. F. J. Le Grice, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PBS, primer-binding site; FIV, feline immunodeficiency virus; RT, reverse transcriptase; PCR, polymerase chain reaction; nt, nucleotide(s); HIV, human immunodeficiency virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. T. Miller, A. Khvorova, S. A. Scaringe, and S. F. J. Le Grice Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes Nucleic Acids Res., September 1, 2004; 32(15): 4687 - 4695. [Abstract] [Full Text] [PDF]

				V. Goldschmidt, J.-C. Paillart, M. Rigourd, B. Ehresmann, A.-M. Aubertin, C. Ehresmann, and R. Marquet Structural Variability of the Initiation Complex of HIV-1 Reverse Transcription J. Biol. Chem., August 20, 2004; 279(34): 35923 - 35931. [Abstract] [Full Text] [PDF]

				M. Rigourd, V. Goldschmidt, F. Brule, C. D. Morrow, B. Ehresmann, C. Ehresmann, and R. Marquet Structure-function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNAHis as primer Nucleic Acids Res., October 1, 2003; 31(19): 5764 - 5775. [Abstract] [Full Text] [PDF]

				Y. Iwatani, A. E. Rosen, J. Guo, K. Musier-Forsyth, and J. G. Levin Efficient Initiation of HIV-1 Reverse Transcription in Vitro. REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS J. Biol. Chem., April 11, 2003; 278(16): 14185 - 14195. [Abstract] [Full Text] [PDF]

				V. Goldschmidt, C. Ehresmann, B. Ehresmann, and R. Marquet Does the HIV-1 primer activation signal interact with tRNA3Lys during the initiation of reverse transcription? Nucleic Acids Res., February 1, 2003; 31(3): 850 - 859. [Abstract] [Full Text] [PDF]

				M. Kvaratskhelia, J. T. Miller, S. R. Budihas, L. K. Pannell, and S. F. J. Le Grice Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent PNAS, December 10, 2002; 99(25): 15988 - 15993. [Abstract] [Full Text] [PDF]

				V. Goldschmidt, M. Rigourd, C. Ehresmann, S. F. J. Le Grice, B. Ehresmann, and R. Marquet Direct and Indirect Contributions of RNA Secondary Structure Elements to the Initiation of HIV-1 Reverse Transcription J. Biol. Chem., November 1, 2002; 277(45): 43233 - 43242. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/29/27721    most recent M100513200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, J. T. Articles by Le Grice, S. F. J. Search for Related Content PubMed PubMed Citation Articles by Miller, J. T. Articles by Le Grice, S. F. J. Social Bookmarking                      What's this?