Role of Post-transcriptional Modifications of Primer tRNALys,3 in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription*

Sylvie AuxilienDagger , Gérard Keith§, Stuart F. J. Le Grice, and Jean-Luc DarlixDagger parallel

From Dagger  LaboRetro ENS, INSERM U412, 46 allée d'Italie, 69364 Lyon cedex 07, France, § Institut de Biologie Moleculaire et Cellulaire, CNRS UPR 9002, 15 rue Descartes, 67084 Strasbourg cedex, France, and the  Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
Top
Abstract
Introduction
References

During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.

    INTRODUCTION
Top
Abstract
Introduction
References

The distinctive characteristic of retroviruses is the conversion of their RNA genome into double-stranded DNA, which is subsequently integrated into the cellular genome. Reverse transcription is a complex process composed of multiple steps catalyzed by the viral enzyme reverse transcriptase (RT)1 (reviewed in Ref. 1). The general model of reverse transcription includes two obligatory DNA transfer reactions (Fig. 1A). RT initiates minus strand DNA synthesis by elongation of a specific cellular tRNA primer annealed to the primer binding site (PBS) and continues as far as the 5' end of the RNA genome, generating the so-called strong stop cDNA (ss-cDNA). The concomitant degradation of the RNA template by the RNase H activity of RT makes the ss-cDNA single stranded. ss-cDNA is transferred by NC protein (2, 3) to the 3' end of either the template RNA or the other RNA molecule present in the virus particle to complete (-) strand DNA synthesis. (+) strand synthesis is primed by an RNase H-resistant sequence, the polypurine tract (PPT), and continues as far as a double-stranded RNA region corresponding to the 18-nt tRNA-viral PBS RNA complex. RT needs to unwind this tRNA-PBS RNA to reverse-transcribe the 3' end of primer tRNA. The stop signal for this (+) strand synthesis has been proposed to be the methylated adenosine at position 58 (m1A-58) of primer tRNA (4, 5) but it is not the absolute termination site of RT in the case of HIV (6, 7). Termination at m1A-58 results in the (+) strong stop DNA species carrying a complete (+) PBS sequence at its 3' terminus. Before (+) strand DNA transfer, the tRNA primer is thought to be removed by the RT-associated RNase H activity (7-11); thus, the (+) PBS at the 3' end of (+) ssDNA becomes available for hybridization to the (-) PBS synthesized at the 3' end of the (-) strand DNA, and synthesis of both strands resumes with strands copying each other (Fig. 1A).

In this report, we analyzed the following factors that influence the efficiency of HIV-1 (+) strand DNA transfer: the modified nucleosides of primer tRNALys,3, the RNase H activity of RT, and deletions in the (-) PBS of acceptor template. We also examined the fidelity of (+) strand DNA transfer by comparing transfer products obtained with an in vitro system using the following primers: natural tRNALys,3 containing post-transcriptional modifications, synthetic tRNALys,3 lacking modifications, or oligonucleotides (RNA and DNA) complementary to the PBS.

    EXPERIMENTAL PROCEDURES

Oligonucleotides-- All DNA and RNA oligomers are listed in Table I and were purchased from Eurogentec. Radiolabeled oligonucleotides (primers SA-12, R5', and SA-18) were prepared using T4 polynucleotide kinase and [gamma -32P]ATP and purified by 12% PAGE in 7 M urea.

                              
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Table I
DNA and RNA oligonucleotides used in the present study

Proteins and Enzymes-- T7 RNA polymerase, T4 polynucleotide kinase, Taq DNA polymerase, and terminal transferase were purchased from Promega. Sequenase was purchased from Amersham. NCp7 (72 residues) generated by peptide synthesis (12) was provided by D. Ficheux (IBCP, Lyon, France). Recombinant HIV-1 reverse transcriptase (RT p66/p51) and HIV-1 RNase H(-) reverse transcriptase (RT p66/p51; E478Q) (13) were purified from Escherichia coli (14).

tRNALys,3-- Natural tRNALys,3 was purified from beef liver as described (15). Synthetic tRNALys,3 was generated in vitro using T7 RNA polymerase (16).

Cloning-- PCR products were directly cloned into pGEM-T (Promega), which possesses 3'-T overhangs (the pGEMTM-T kit provides an exonuclease-free T4 DNA ligase).

RNA Template-- HIV-1 MAL 5' RNA corresponding to nucleotides 1-311 was generated in vitro as described (17), phenol- and chloroform-extracted, and purified by gel filtration.

tRNALys,3 Reverse Transcription-- Reverse transcription of natural tRNALys,3 or synthetic tRNALys,3 was performed using 5'-32P-labeled primer SA-12. 5'-32P primer SA-12 (3 pmol) was annealed to tRNALys,3 (2 pmol) by incubating with NCp7 (22 pmol; NCp7 to nucleotide ratio was 1:7) for 10 min at 37 °C in 10 µl containing 30 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 30 mM NaCl, 0.2 mM MgCl2, 1 µM ZnCl2, and 2 units of RNasin. Next, RTp66/p51 (5 pmol) was added and the reaction incubated with 0.25 mM each dNTP for 15 or 45 min at 37 °C in 30 mM Tris-HCl (pH 8), 5 mM dithiothreitol, 60 mM NaCl, 3 mM MgCl2 in 25 µl. After phenol and phenol/chloroform extraction, nucleic acid was precipitated with 3 volumes of ethanol, recovered by centrifugation, and resuspended in 6 µl of 87% formamide, Tris borate-EDTA buffer, 0.1% xylene cyanol, and 0.1% bromphenol blue (sample buffer). Half of the mixture was heat-denatured for 2 min at 95 °C and resolved by 7 M urea, 20% PAGE.

tRNALys,3 Gene Sequencing-- Plasmid carrying tRNALys,3 gene (7.5 µg) was alkaline-denatured and ethanol-precipitated in the presence of 5'-32P primer SA-12 (30 pmol). The precipitate was resuspended in H2O (6 µl) and added to a solution containing 10 mM dithiothreitol, 1× Sequenase buffer and 3 units of Sequenase. Sequencing reactions were initiated by addition of this mixture (3.5 µl) to 0.8 µl of dNTP (2.5 mM of each) and 0.5 µl of ddNTP (1 mM). This reaction was performed for each ddNTP. Mixtures were incubated at 37 °C for 15 min and then quenched by addition of 4 µl of sample buffer.

Amplification of Natural tRNALys,3 cDNA-- The cDNA obtained from natural tRNALys,3 was purified following 7 M urea, 16% PAGE. The eluted cDNA was amplified by the RACE (rapid amplification of cDNA ends) method as described (18), 3' tailed with poly(dA) by terminal transferase, and PCR-amplified by using poly(dT) oligonucleotide and the primer SA-12.

Plus Strand DNA Transfer Assay-- The reactions were performed basically as described above for tRNALys,3 reverse transcription. The HIV-1 MAL 5' RNA (311 nt; 1 pmol) was annealed to natural tRNALys,3, synthetic tRNALys,3, RNA PBS primer, or DNA PBS primer (2 pmol) by incubating with NCp7 (44 pmol; NCp7 to nucleotide ratio was 1:7) for 10 min at 37 °C in a total volume of 10 µl. Next, RTp66/p51 (8 pmol), dNTP (0.25 mM of each), 5'-32P primer R5' (3 pmol), and SA-2 MOD or SA-5 MOD (1 pmol) were added and samples incubated 20, 40, or 60 min at 37 °C in a total volume of 25 µl. Products were resolved by 7 M urea, 6% PAGE.

RT RNase H(-) Assay-- ss-cDNA was first synthesized by wild type RT as above, in the absence of 5'-32P primer SA-18 and acceptor template, using a 30-min incubation time. ss-cDNA product was phenol/chloroform-extracted, ethanol-precipitated, and resuspended in 10 µl of a mixture containing 5'-32P primer SA-18 (3 pmol) and NCp7 (44 pmol) and incubated 10 min at 37 °C for annealing. (+) ssDNA synthesis and (+) strand DNA transfer were then initiated by addition of wild type RT or RNase H(-) RT as follows: wild type RT or RNase H(-) RT (8 pmol), dNTP (0.25 mM each), and SA-2 MOD were added, and samples were incubated 30 min at 37 °C in a total volume of 25 µl.

    RESULTS

tRNALys,3 as Template for Reverse Transcription-- Plus strand DNA synthesis, which is primed from a PPT, is believed to proceed as far as the first modified nucleotide of natural tRNA (1-methyladenosine). This nucleoside is localized just downstream of the PBS complementary sequence of tRNALys,3 and may constitute the stop signal for RT preceding (+) strand DNA transfer (Fig. 2B). To investigate this hypothesis, we compared reverse transcription in vitro of the natural tRNALys,3 containing modified nucleosides with that of synthetic tRNALys,3, devoid of modifications. An oligonucleotide (primer SA-12), complementary to the 10 last nucleotides of tRNALys,3, was used to prime reverse transcription of the tRNA by HIV-1 RT in the presence of NCp7. The first three stops observed with both tRNAs (Fig. 2A) are probably due to highly stable tRNA structure (Fig. 2B). Other internal stops are detected only with the natural tRNALys,3; RT is partially stopped by m1A at position 58 and Tm at position 54 and is almost completely blocked by the first hypermodified nucleoside present at position 37 in the anticodon loop: ms2t6A. A high degree of steric hindrance generated by this nucleoside could explain the extent of this block. After 45 min of incubation, a cDNA extended by one nucleotide beyond position 38 can be detected. This may result from the 3' terminal transferase activity of RT, resulting in nontemplated blunt-end 3' addition of nucleotides (19-21). In contrast to natural tRNALys,3, the synthetic tRNALys,3 does not impede RT progress. cDNA synthesized from the synthetic tRNALys,3 is the same length as tRNA and we suppose this product can be self-primed by RNA folding back at the 3' end (22, 23) to yield the additional 125-nucleotide cDNA.

In the absence of NCp7, natural tRNALys,3 reverse transcription is almost completely halted by m1A-58, whereas reverse transcription of synthetic tRNALys,3 remains complete but less efficient (data not shown). These results in the absence of NCp7 are similar to those of Yusupova et al. (24). The data suggest that modified nucleosides stabilizing the natural tRNALys,3 structure (25) impede RT progression.

Sequence Analysis of cDNA from Natural tRNALys,3-- We sequenced the cDNA obtained from reverse transcription of natural tRNALys,3 to determine RT fidelity when reading modified nucleosides. The longest cDNA obtained by reverse transcription of natural tRNALys,3 was purified, amplified, and then cloned for sequencing. Table II reports 36 sequences thus obtained. The most frequently observed error was the reverse transcription of m1A-58 into A (32/36). Surprisingly, Tm-54, which partially arrests RT, analogous to m1A-58 (Fig. 2A), is correctly transcribed into an A. We also found a few errors at m7G-46 (transcribed into T, 4/36) and possible random errors: m5C-50 transcribed into A (1/36) and A-51 transcribed into C (1/36). In addition to these errors, we found three cases of single nucleotide additions at the 3' end of the cDNA (Table II), confirming our hypothesis of nontemplated blunt-end addition of nucleotides by RT. These results indicate that, among modified nucleosides present between positions 38 and 76 of natural tRNALys,3, only m1A-58 is frequently reverse-transcribed incorrectly.

                              
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Table II
Errors upon reverse transcription of the natural tRNALys,3

Analysis of the (+) Strand DNA Transfer Mechanism-- The (+) strand DNA transfer event was studied using an in vitro system schematized in Fig. 1B. The RNA template (HIV-1 5' RNA, 311 nt) for ss-cDNA synthesis contained R, U5, and part of the encapsidation/dimerization signal. ss-cDNA synthesis was primed by natural tRNALys,3, synthetic tRNALys,3, an RNA PBS primer, or a DNA PBS primer. RNA template was preincubated separately with these four primers in the presence of NCp7 and RT and dNTP added to synthesize ss-cDNA. Simultaneous addition of a DNA primer (5'-32P-primer R5') complementary to the 3' end of the ss-cDNA allowed for (+) strand DNA synthesis while the oligodeoxynucleotide ODN 2 (50 nt) was available as the template for strand transfer. Transfer resulted in the synthesis of a 228-nt 5'-32P product. Since all polymerization reactions were performed in a single tube, ODN 2 was 3' blocked by a dideoxy adenosine (ddA) to prevent its use as primer for ss-cDNA synthesis.


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Fig. 1.   A, scheme of retroviral reverse transcription including (+) strand DNA transfer. Steps are explained in more detail in the introduction. 1, strong stop cDNA (ss-cDNA) synthesis is initiated by a specific cellular tRNA at the primer binding site (PBS). 2, (-) strand DNA transfer and (-) strand DNA synthesis. 3, before (+) strand DNA transfer: (+) strong stop DNA ((+) ssDNA) synthesis is initiated by the polypurine tract (PPT) primer. 4, after (+) strand DNA transfer: (+) strand DNA synthesis. B, summary of HIV-1 (+) strand DNA transfer system. 1, the HIV RNA template (R, U5, PBS, and part of encapsidation/dimerization signal) is preincubated in the presence of tRNALys,3 and NCp7. 2, tRNALys,3 is annealed to the PBS of HIV RNA template. 3, ss-cDNA synthesis occurs in the presence of HIV-1 RT and dNTP. 4, the concomitant degradation of the RNA template by the RNase H activity of RT allows annealing of the 5'-32P primer R5' (complementary to the 3' end of ss-cDNA). 5, (+) strand DNA synthesis is initiated by the 5'-32P primer R5'. 6, A second oligodeoxyribonucleotide (ODN 2) acts as the acceptor template for strand transfer by RT. The acceptor template possesses a dideoxyadenosine at its 3' end to prevent its use as primer for ss-cDNA synthesis.

Experiments were first performed without acceptor template ODN 2 to analyze events preceding (+) strand DNA transfer. (+) strand DNA synthesis with natural tRNALys,3 revealed several intermediates (Fig. 3A, lanes 1-3). The 180- and 192-nt products are generated by termination at the 2nd and 14th nt of the PBS (Fig. 3B), respectively, and are probably due to the incomplete unwinding of the tRNA-PBS RNA complex. The three last termination sites (196-, 200-, and 217-nt products) correspond exactly to the previously observed stops of RT during reverse transcription of natural tRNALys,3 (Fig. 2A). Thus, the 196-, 200-, and 217-nt products are due to the presence, in the natural tRNALys,3, of m1A-58, Tm-54, and ms2t6A-37, respectively (Fig. 3B). The same assay performed using the synthetic tRNALys,3 reveals a different pattern (Fig. 3A, lanes 8-10). While 180- and 192-nt products are observed as with natural tRNALys,3, products of 196, 200, and 217 nt are absent, as expected. Instead, a 254-nt product is generated by complete reverse transcription of the synthetic tRNALys,3. Results obtained with the RNA PBS primer (Fig. 3A, lanes 15-17) are similar to those for natural tRNALys,3 except that no product was larger than 196 nt representing complete reverse transcription of the RNA PBS primer. Finally a single product is observed when the DNA PBS primer is used (Fig. 3A, lanes 22-24), reverse transcription terminating at the end of the DNA PBS primer. These data support conclusions made from Fig. 2 concerning the influence of modified nucleosides on reverse transcription of tRNA and show that RT is held up by RNA-RNA duplexes (tRNA-PBS) as opposed to DNA-RNA duplexes (DNA PBS primer-PBS) in agreement with the results of Ben-Artzi et al. (6).


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Fig. 2.   Primer tRNALys,3 as template for reverse transcription by HIV-1 RT. A, natural bovine tRNALys,3 (lanes 1 and 2) or synthetic bovine tRNALys,3 (lanes 3 and 4) were hybridized individually to a 10-mer DNA primer (SA-12, complementary to tRNA positions 67-76) at 37 °C in the presence of NCp7. 5'-32P-labeled primer-tRNA complexes were incubated with HIV-1 RT for 15 and 45 min as described under "Experimental Procedures." Size markers were obtained by tRNALys,3 gene sequencing using 5'-32P-labeled primer SA-12 (see "Experimental Procedures"). B, bovine tRNALys,3 cloverleaf structure.


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Fig. 3.   Analysis of (+) strand DNA transfer by HIV-1 RT. A, (+) strand DNA transfer was performed as described for Fig. 2A. The ss-cDNA primer was natural tRNALys,3 (lanes 1-7), synthetic tRNALys,3 (lanes 8-14), RNA PBS primer (lanes 15-21), or DNA PBS primer complementary to (+) PBS (lanes 22-28). Only the (+) strand DNA primer (primer R5') was 32P-labeled; consequently, only (+) strand DNA synthesis and (+) strand DNA transfer products are labeled. Reactions were performed with (lanes 4-7, 11-14, 18-21, and 25-28) or without acceptor template ODN 2 (lanes 1-3, 8-10, 15-17, and 22-24) and in the presence (lanes 1-6, 8-13, 15-20, and 22-27) or in the absence of NCp7 (lanes 7, 14, 21, and 28). Expected products are (+) strong stop DNA ((+) ssDNA; 196 nucleotides) and the strand transfer product (228 nucleotides). B, scheme of products obtained with natural tRNALys,3 in the absence of acceptor template. The ss-cDNA, including the primary structure of tRNALys,3, is also schematized.

To analyze (+) strand DNA transfer in vitro, the acceptor template ODN 2 was added to the reaction mixtures. (+) strand DNA transfer occurred efficiently only with natural tRNALys,3 (Fig. 3A, lanes 4-6) and RNA PBS primer (Fig. 3A, lanes 19 and 20) reaching 15% and 13% after 60 min of incubation time, respectively. Using synthetic tRNALys,3, (+) strand DNA transfer was hardly detectable (Fig. 3A, lanes 11-13), whereas no transfer product was observed using the DNA PBS primer (Fig. 3A, lanes 25-27). These results clearly indicate a role for modified nucleosides of tRNALys,3 in the (+) strand DNA transfer process. Since (+) strand DNA transfer may be facilitated by the RNase H-catalyzed removal of the tRNA primer (26, 27), we propose that the arrest of RT at m1A-58 of the tRNA is probably required for tRNA cleavage. Even when RT was completely halted at the end of the PBS by use of the DNA PBS primer, (+) strand DNA transfer was not detected while it did occur efficiently with the RNA PBS primer. The above data suggest that removal of the primer for ss-cDNA synthesis by RNase H is an obligatory step for (+) strand DNA transfer and cannot occur if this primer is DNA. Parallel experiments carried out in the absence of NCp7, indicate that NCp7 is an essential component in this in vitro system (Fig. 3A, lanes 7, 14, 21, and 28). All assays shown in Fig. 3A were repeated with an alternative acceptor template (SA-5 MOD, see "Experimental Procedures") composed of the (-) PBS sequence followed by a random sequence, with identical results (data not shown). Thus, the (+) strand DNA transfer mechanism is independent of acceptor template sequence between positions 197 and 228.

RNase H Activity Enhances (+) Strand DNA Transfer in Vitro-- The absence of (+) strand DNA transfer with a DNA PBS primer strongly suggested a role for the RNase H activity of RT in strand transfer. To further investigate the requirement of RNase H for the (+) strand DNA transfer, we used an RNase H-defective RT mutant (mutation E478Q) (13). ss-cDNA was synthesized with natural tRNALys,3 using wild type RT, purified to eliminate the enzyme, and used as template for (+) strand DNA synthesis and (+) strand DNA transfer catalyzed by either RNase H(-) RT or wild type RT (Fig. 4). The amount of (+) strand DNA transfer product was clearly decreased using RNase H(-) RT (Fig. 4, lane 4) compared with that with wild type RNase H(+) RT (Fig. 4, lane 2), thus indicating that RNase H activity of RT is necessary for efficient (+) strand DNA transfer. These results are in agreement with the model proposed above in which tRNALys,3 is released by RNase H activity to facilitate (+) strand DNA transfer. In addition, when RNase H(-) RT was used, the amount of the 200-nt product, generated by RT arrested by Tm at position 54 of primer tRNALys,3 (see Figs. 2 and 3), was remarkably increased. This can be due to an extensive copying of primer tRNALys,3 in the absence of release of tRNALys,3 by RNase H activity of RT.


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Fig. 4.   (+) strand DNA transfer requires RNase H activity. Reactions were carried out with natural tRNALys,3, wild type RT, or RNase H(-) mutant RT. For all assays, ss-cDNA was first synthesized by wild type RT and then purified prior to the (+) ssDNA synthesis and (+) strand DNA transfer.

Sequence Analysis of (+) Strand DNA Transfer Products-- As shown above with natural tRNALys,3, m1A-58 is not an absolute stop site for RT before strand transfer, and in the case of synthetic tRNALys,3 RT transcribes the complete tRNALys,3 (Fig. 3A). We thus sequenced the (+) strand DNA transfer products obtained from natural tRNALys,3, synthetic tRNALys,3, and DNA PBS primer in order to precisely determine the transfer site and eventual nucleotide misincorporations at the transfer site. With acceptor template SA-2 MOD (sequence 179-228) and natural tRNALys,3, there were no errors among 14 clones (Table III). With synthetic tRNALys,3, 2 sequences out of 13 had nucleotide misincorporations located at the transfer site. These errors correspond to the reverse transcription of 3 additional nucleotides of the tRNA (A-58, A-57, and C-56) that replace 3 or 4 nucleotides of the sequence downstream of the PBS. Although no strand transfer product was visible using the DNA PBS primer (Fig. 3), PCR did allow amplification of products. Sequencing of 17 clones revealed 12 wild type sequences and 5 mutated sequences. One of these mutated sequences had an insertion of 9 nucleotides after the PBS, corresponding to the 9 last nucleotides of the PBS. This insertion may have been due to misalignment of annealed PBS sequences. The four other sequences had insertions of one or two complete PBS domains but these products could not have been due to a transfer process. With the other acceptor template SA-5 MOD ((-) PBS followed by a random sequence), sequencing gave generally similar data (Table III). Among 17 clones using natural tRNALys,3 as primer, 2 sequences had one nucleotide misincorporation located at the transfer site, where the A immediately downstream of the PBS had been replaced by G or T. These mutations are probably due to the 3' terminal transferase activity of RT (19-21). With synthetic tRNALys,3, 1 sequence out of 5 was mutated by insertion of 24 nucleotides resulting from the reverse transcription of 11 additional nucleotides of tRNA (A-58 to G-48), followed by partial annealing to the (-) PBS of the acceptor template. Finally, with the DNA PBS primer, 2 sequences out of 6 had one nucleotide deletion at the transfer site and 3 sequences had an insertion of the whole PBS sequence. Taken together, these data confirm that (+) strand DNA transfer is independent of acceptor template sequence, except for the PBS segment.

                              
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Table III
Sequencing data of (+) strand DNA transfer products obtained with three distinct (-) ssDNA synthesis primers and two distinct acceptor templates (ODN 2): underlined sequences are mutated

Effect of Deletions at the 5' End of the Acceptor Template (-) PBS-- Liang et al. (29) showed that HIV-1 containing deletions at the 3' end of the (+) PBS of the viral genomic RNA, exhibited an attenuated replication phenotype. This was shown to be caused by the lower efficiency of tRNALys,3 placement onto viral genomic RNA and the possibility of an effect on (+) strand DNA transfer was also evoked, but not testable in vivo. In order to investigate this possibility in our in vitro system, we used acceptor templates containing 5' (-) PBS deletions (Table I and Fig. 5A). Interestingly, (+) strand DNA transfer was markedly affected by a 2-nt 5' (-) PBS deletion (Fig. 5B, lane 3) or a 4-nt 5' (-) PBS deletion of the acceptor template (Fig. 5B, lane 4) in support of the hypothesis of Liang et al. (29). To determine whether the complete PBS sequence can be restored by the appearance of compensatory deletions downstream of the PBS as observed by Liang et al. (29), we sequenced transfer products. In contrast to the results of Liang et al. (29), we did not find products possessing wild type PBS sequence followed by a deletion (Table IV). Furthermore, we observe a predominance of wild type sequences lacking any deletions. The discrepancy between these in vitro data and the in vivo results of Liang et al. (29) suggests that additional factors may be involved in (+) strand DNA transfer in vivo.


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Fig. 5.   Effect of deletions at the 5' end of the (-) PBS of acceptor template. A, schematic representation of the transfer step of in vitro system and templates with deletion at the 5' end of the PBS. B, (+) strand DNA transfer assays were performed with or without wild type acceptor template (lanes 2 and 1, respectively) or two mutated acceptor templates containing 2- or 4-nt 5' (-) PBS deletions (lanes 3 and 4, respectively) as described in Fig. 1B.

                              
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Table IV
Sequencing data of (+) strand DNA transfer products obtained with two distinct acceptor templates
Bold sequences correspond to the (+) PBS.


    DISCUSSION

Immediatly prior to (+) strand DNA transfer, the 3' end of tRNALys,3, still attached to the 5' end of (-) strand DNA, is reverse-transcribed. To shed light on this specific aspect of (+) strand DNA transfer, we investigated the ability of HIV RT to reverse-transcribe tRNALys,3 alone. The reverse transcription of natural tRNALys,3 shows three RT termination sites due to modified nucleosides m1A-58, Tm-54, and ms2t6A-37, while these stops are not observed with synthetic tRNALys,3. Sequencing of cDNA products revealed that m1A is always transcribed incorrectly into A. In contrast, other modified nucleosides between m1A-58 and ms2t6A-37, comprising Tm, Psi , m5C, D, and m7G are transcribed correctly except for occasional transcription of m7G-46 into T. This indicates that post-transcriptional modifications in natural tRNALys,3 not interfering with Waston-Crick base pairing, as is the case for these five last modified nucleosides, do not affect faithful reverse transcription. For m7G-46, the positive charge due to methylation on its N-7 (30) may occasionally interfere with reverse transcription, giving rise to a T. It should be pointed out that although Tm-54 partially stops RT, it does not cause nucleoside misincorporation. Tm-54 possesses two methylations (one on the base, the other on the ribose) that have no effect on atoms implicated in Waston-Crick base pairing. However stabilization of the C3' endo rather than the usual C2' endo conformer by the 2'-O-methylation on ribose (31) may explain the difficulty of RT to pass through this modified nucleoside. These results are consistent with previous work (32), in which reverse transcription of yeast tRNAPhe by the Klenow fragment of E. coli DNA polymerase I demonstrated that m1A-58 partially prevents elongation and that Y-base (wybutosine), a highly modified nucleoside as ms2t6A, was able to block DNA polymerase. However, the Klenow polymerase nonspecifically transcribed m1A-58 into the four bases while, in our case, HIV RT incorporates only A. Polymerases thus do not read m1A-58 in a universal manner; the positive charge of m1A-58 (30) may interact differently with the active sites of these two enzymes.

(+) strand DNA transfer was reconstituted in an in vitro system using a 3' blocked acceptor template. The advantage of the present system (Fig. 1B) is that the PBS segment of the (+) RNA template remains hybridized to tRNALys,3 as it should be in vivo, while this was not the case in previously used transfer systems (6, 7). Furthermore, this system contains NCp7, known to coat the genomic RNA in virion (33) and to greatly enhance (-) strand DNA transfer (2, 22, 34, 35). ss-cDNA was synthesized by using four distinct primers: natural tRNALys,3, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer. The nature of (+) ssDNA synthesis products was dependent upon the primer used. With natural tRNALys,3, (+) strand DNA synthesis was partially terminated at nucleosides m1A-58 and Tm-54 and was totally halted by ms2t6A-37. As expected, these stops did not occur with synthetic tRNALys,3 where (+) strand DNA synthesis continued as far as the 5' end of the tRNA. With RNA PBS or DNA PBS as primer, full-length (+) ssDNA was obtained. Two common products were synthesized, from all primers except DNA PBS primer, generated by stops occurring at bases 2 and 14 of the PBS. These results indicate that RT stalls, even in the presence of NCp7, at the double-stranded 3' tRNALys,3-PBS RNA complex, but not at the RNA/DNA heteroduplex composed of the DNA PBS primer and viral RNA PBS. The difficulty of RT to catalyze strand displacement synthesis through RNA/RNA duplexes has also been shown by Ben-Artzi et al. (6) in a test performed without NCp7. Thus, while NCp7 is not solely responsible for this phenomenon, its stabilization of intermolecular nucleic acid duplexes (36) might exacerbate it. In contrast, it is clear that NCp7 is essential for synthesis of (+) ssDNA in our in vitro system. This can be explained by the well established ability of NCp7 to promote annealing of the tRNALys,3 to the PBS (reviewed in Ref. 3) as well as to augment efficiency and processivity of RT (23, 34, 37-40).

(+) ssDNA synthesis performed in the presence of acceptor template for strand transfer revealed that stalling of RT at m1A-58 is a prerequisite for efficient (+) strand DNA transfer, since efficiency of transfer was low with synthetic tRNALys,3 relative to natural tRNALys,3. In fact, reverse transcription is not completely arrested by m1A-58, indicating that (+) strand DNA transfer is not sufficiently rapid to prevent continued transcription of the tRNA beyond m1A-58. The use of an RNA PBS primer confirmed this observation since transfer was as efficient as transfer with natural tRNALys,3. In contrast, a DNA PBS primer generated only very low levels of transfer product, suggesting that (+) strand DNA transfer strictly requires RNA as primer for ss-cDNA synthesis, probably to allow removal by RNase H. This hypothesis was confirmed by using an RT mutant deficient of RNase H activity (mutation E478Q) (13). In the absence of RNase H activity, (+) strand DNA transfer occurred but with markedly decreased efficiency. Thus, RNase H activity greatly enhances strand transfer and RT seems to be able to displace the 3' end of tRNALys,3 from the (+) PBS, even without tRNA cleavage. It has been shown that tRNALys,3 is cleaved near DNA/RNA junction, leaving one ribonucleotide A at the 3' end of (-) strand DNA (9) but the mechanism is still unclear. Since polymerase and RNase H active sites are separated by 18 nt, a single RT molecule could simultaneously transcribe the first 18 nt of tRNA and cleave the tRNA. Alternatively, one RT molecule might transcribe tRNA and another might rebind the RNA-DNA duplex to cleave the tRNA, as suggested for PPT release (41). Consistent with data herein for (+) strand DNA transfer, Blain et al. (42) suggested that (-) strand DNA transfer may require not only the RNase H activity of RT to degrade the RNA template but also an unwinding activity of RT to dissociate the small residual RNA fragment remaining annealed to the 3' end of the ss-cDNA that cannot be degraded because of the distance between polymerase and RNase H active sites.

The efficiency of (+) strand DNA transfer is equally dependent on base pairing between the 3' end of (+) ssDNA and acceptor template. Indeed, a two-nucleotide deletion at the 5' end of (-) PBS in the acceptor template that allows mispairing between the last two bases of (+) ssDNA and acceptor template is sufficient to drastically decrease the amount of transfer product. This result is not surprising since a mismatch at the 3' end of a primer affects the kinetics of its extension by RT (23, 43). These observations are consistent with the hypothesis proposed to explain low replication of HIV-1 containing deletions at the 3' end of the PBS (29). However, in contrast to these in vivo results, we did not observe reversion to a wild type PBS sequence in our system in the form of concomitant deletion of nucleotides downstream of the PBS. The efficiency of in vitro (+) strand DNA transfer in our hands (10-15%) is also lower than that observed in infected cells (80-85%) (44). These differences may indicate that other factors influence (+) strand DNA transfer in vivo.

Another aim of this work was to analyze mutagenesis at the (+) strand DNA transfer site. Sequencing of (+) strand DNA transfer products revealed that m1A-58 is also important for the fidelity of transfer. Transfer with natural tRNALys,3 showed only two distinct nucleotide misincorporations immediately downstream of the PBS. These mutations may not result from transfer errors but could instead be due to nontemplated blunt-end nucleotide addition by RT. Such RT terminal transferase activity has been incriminated in nucleotide misincorporation events during (-) strand DNA transfer (19, 20), as well as during internal strand transfers necessary for recombination by a forced copy choice mechanism (21). In contrast, transfer with synthetic tRNALys,3 or a DNA PBS primer revealed more frequent mutations. With synthetic tRNALys,3, we observed insertions of several nucleotides complementary to tRNALys,3 downstream of G-59 that can be reverse-transcribed because of the absence of methylation on A-58. With the DNA PBS primer, we noted single nucleotide deletions downstream of the PBS, possibly due to incomplete displacement of the DNA PBS primer from (+) ssDNA allowing a misalignment between the (+) PBS and acceptor (-) PBS.

Taken together, the data herein suggest that the absence of methylation on A-58 of natural tRNALys,3 would allow mutations contributing effectively to variability of the HIV genome. The interesting observation by Morin et al.2 that tRNALys,3 of chronically HIV infected cells is drastically hypomodified lends support to this hypothesis.

    ACKNOWLEDGEMENTS

We thank Damien Ficheux (IBCP, Lyon, France) for providing nucleocapsid protein NCp7. We thank Michael Rau and Henri Grosjean for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the Agence Nationale de Recherches sur le SIDA, the Association pour la Recherche sur le Cancer, and Sidaction.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.

parallel To whom correspondence should be addressed. Tel.: 33-4-72-72-81-69; Fax: 33-4-72-72-86-86; E-mail: Jean-Luc.Darlix{at}ens-lyon.fr.

The abbreviations used are: RT, reverse transcriptase; PBS, primer binding site; HIV-1, human immunodeficiency virus type 1; PPT, polypurine tract; NCp7, nucleocapsid protein; ODN, oligodeoxyribonucleotide; m1A, 1-methyladenosine; Tm, 5, 2'-O-dimethyluridine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; Psi , pseudouridine; m5C, 5-methylcytidine; D, dihydrouridine; m7G, 7-methylguanosine; Y, wybutosine; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; ss, strong stop; MOD, 3' modified.

2 A. Morin, C. Simon, F. Subra, and H. Grosjean, personal communication.

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Abstract
Introduction
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