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Nucleic Acids Research, 2003, Vol. 31, No. 19 5764-5775
© 2003 Oxford University Press

Structure–function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNAHis as primer

Mickaël Rigourd, Valérie Goldschmidt, Fabienne Brulé, Casey D. Morrow1, Bernard Ehresmann, Chantal Ehresmann and Roland Marquet*

Unité Propre de Recherche 9002 du CNRS conventionnée à l‘Université Louis Pasteur, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France and 1 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

*To whom correspondence should be addressed. Tel: +33 3 88 41 70 91; Fax: +33 3 88 60 22 18; Email: r.marquet{at}ibmc.u-strasbg.fr
Present addresses:
Mickaël Rigourd, Department of Genetics and Microbiology, Faculty of Medicine, University of Geneva, Switzerland
Fabienne Brulé, Institut de Transgénose, Laboratoire de Génétique Moléculaire et Expérimentale, CNRS FRE 2358, Orléans, France

Received April 16, 2003; Revised July 11, 2003; Accepted August 5, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Discussion
 REFERENCES
 
Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNAHis as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNAHis, only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNAHis as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNAHis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 Discussion
 REFERENCES
 
Reverse transcription of the single-stranded genomic RNA into a double-stranded DNA with duplicated long terminal repeats is a key event in the retroviral replication cycle (1,2). This process is performed by reverse transcriptase (RT), an RNA- and DNA-dependent DNA polymerase that also harbors an RNase H domain. RT utilizes a cellular tRNA that is selectively encapsidated into the viral particles to prime DNA synthesis (3–5). The natural primer used by most immunodeficiency viruses, including the type 1 human immunodeficiency virus (HIV-1), is tRNA3Lys (3–5).

In all retroviruses, the 18 3' terminal nucleotides of the primer tRNA become annealed to the complementary primer binding site (PBS), located in the 5' region of the genomic RNA. Additional ‘virus-specific’ interactions have been described in several retroviruses and LTR-containing retrotransposons including avian retroviruses (6–8), HIV-1 (9–20), HIV-2 (21,22), FIV (23), Ty1 (24,25) and Ty3 (26).

In HIV-1, chemical and enzymatic probing of the viral RNA (vRNA) and of the primer tRNA3Lys (9,10,15), combined with site-directed mutagenesis of the former (14,19,27) provided strong evidence for the existence of additional interactions between tRNA3Lys and the genomic RNA in vitro. These experimental data were used to construct secondary (10) and tertiary (15) structure models of the initiation complex of the HIV-1 MAL isolate. In these models, parts of the anticodon arm and of the variable loop of tRNA3Lys interact with viral sequences upstream of the PBS (Fig. 1A). Of particular importance is the interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop. The same interactions were observed when the primer–template complex was annealed by heat or by the nucleocapsid protein (28).



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  		Figure 1. The initiation complex of reverse transcription. (A) Secondary structure model of the complex formed between the HIV-1 MAL RNA and tRNA3Lys, as deduced from structural probing (10). The PBS is highlighted in yellow and the sequences of the viral A-rich loop and of the anticodon loop of tRNA3Lys that form an intermolecular interaction are in green. (B) Sequence alignment of the HIV-1 MAL RNA and of the WT and mutant RNAs derived from HIV-1 HXB2 used in this study. The PBS complementary to tRNA3Lys and tRNAHis are highlighted in yellow and in light pink, respectively. The A-rich loops complementary to tRNA3Lys and tRNAHis are in green and violet, respectively, and the mutations selected during cell culture are in blue. Numbering according to the MAL and HXB2 sequences is shown above and below the sequence alignment, respectively. (C) Secondary structure of tRNAHis. Nucleotide numbering follows international conventions, so that the anticodon corresponds to nucleotides 34–36.

 
Evidence suggesting that these interactions also take place in cell culture was obtained from studies in which the identity of the primer tRNA was switched. Several groups showed that mutant viruses whose PBS had been changed to be complementary to a variety of non-cognate primer tRNAs are unstable and rapidly revert to the wild type (WT) PBS sequence (29–31). However, HIV-1 can replicate by stably using either tRNAHis (32–35), tRNAMet (36,37) or tRNA1,2Lys (38) as primers, provided that the viral PBS and A-rich loop are simultaneously mutated to be complementary to these tRNA species.

However, the implications of these results are not as straightforward as it might seem. As already pointed out (18,39), their interpretation is complicated by the presence of overlapping sequence motifs involved in the integration of the viral DNA (40–42). Even though the mutant viruses stably use the non-cognate primer tRNAs, they replicate more slowly than the WT virus (32,33,35,36,38). In addition, proviruses with deletions in the U5 region are replication competent (42). Therefore, it was proposed that mutations of the A-rich loop allowed long term usage of non-cognate tRNAs by disfavoring tRNA3Lys, rather than favoring the alternate primers (43). Finally, even though mutant viruses in which the PBS and the A-rich loop were made complementary to tRNAHis, tRNAMet or tRNA1,2Lys maintained these tRNA species as primer, they rapidly accumulated additional mutations that improved their replication capabilities (32,33,36,38,44). These observations suggest that the interaction between the anticodon loop of the primer tRNA and the viral A-rich loop may not be sufficient for optimal initiation of reverse transcription.

Here, we studied the in vitro reverse transcription of HIV-1 mutants in which either the PBS alone or both the PBS and the A-rich loop were complementary to tRNAHis. In the latter case, we compared the initial mutant with mutants having accumulated adaptative mutations during prolonged cell culture. In order to better understand the structure–function relationships of the reverse transcription initiation complex, we also investigated its structure by chemical and enzymatic probing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 Discussion
 REFERENCES
 
Primers, templates and RT
Natural tRNA3Lys was purified from beef liver as described (45). Its sequence and post-transcriptional modifications are identical to those of human tRNA3Lys. The gene coding for human tRNAHis was constructed under the control of the T7 RNA polymerase and inserted into the EcoRI and XmaI sites of plasmid pUC18 using oligonucleotides 5'-AAT TCT AAT ACG ACT CAC TAT AGG CCG TGA TGA TCG TAT AGT-3', 5'-GGT TAG TAC TCT GCG TTG TGG CCG CAG CAA-3', 5'-CCT CGG TTC GAA TCC GAG TCA CGG CAC CAT GCA TC-3', 5'-TAC TAA CCA CTA TAC GAT CAC GGC CTA TAG TGA GTC GTA TTA G-3', 5'-AAC CGA GGT TGC TGC GGC CAC AAC GCA GAG-3' and 5'-CCG GGA TGC ATG GTG CCG TGA CTC GGA TTC G-3'. The resulting pFBHIS plasmid was cut with NsiI and transcribed in vitro with the T7 RNA polymerase as described (46). Alternatively, tRNAHis was purified from beef liver to >60% homogeneity using established methods (47). When required, tRNAs were dephosphorylated with calf intestine phosphatase and labeled at the 5'-end with [{gamma}-32P]ATP and phage T4 polynucleotide kinase according to published procedures (15).

In order to synthesize the WT and mutant RNA templates used in this study, nucleotides 1–732 of plasmids pHXB2, pHXB2(His), pHXB2(His-AC), pHXB2(His-AC-GAC) and pHXB2(His-AC-AT-GAC) (35), were amplified using oligonucleotides 5'-ACG TGA ATT CTA ATA CGA CTC ACT ATA GGT CTC TCT GGT TAG ACC A-3'and 5'-TGC ACC CGG GTA ATT TTG GCT GAC CTG-3'. The PCR products were cleaved with EcoRI and XmaI, and inserted into the corresponding site of pUC18. The resulting plasmids pHXB2(1–732)WT, pHXB2(1–732)His, pHXB2(1–732)His-AC, pHXB2(1–732)His-AC-GAC and pHXB2(1–732)His-AC-AT-GAC were linearized with RsaI and transcribed using previously described conditions (46), yielding vRNAs encompassing nucleotides 1–295 of HXB2.

The plasmid used for production of the heterodimeric WT HIV-1 RT was kindly provided by Dr Torsten Unge (Uppsala, Sweden), together with the protocols for protein overexpression and purification.

Annealing of tRNA3Lys and tRNAHis to the vRNAs
Viral RNA and primer tRNA3Lys or tRNAHis were first denatured in water for 2 min at 90°C and chilled on ice. Annealing was performed at 70°C for 20 min in sodium cacodylate (pH 7.5) 50 mM, KCl 300 mM. An aliquot of each annealing reaction was analyzed on a native 8% polyacrylamide gel to determine the annealing efficiency. The reaction products were discarded if <95% of the primer were annealed to the vRNA. A functional and structural comparison of the WT MAL initiation complexes formed using this protocol or nucleocapsid protein mediated annealing at 37°C revealed no differences between the two complexes (28). In addition, we previously showed that this protocol allows quantitative annealing of both native and synthetic tRNAs (9).

Minus strand strong stop DNA synthesis
In a standard experiment, vRNA (100 nM final concentration) was annealed with 32P-labeled tRNA3Lys or tRNAHis (2 nM) as described above, and pre-incubated for 4 min at 37°C with 25 nM RT in 50 mM Tris–HCl pH 8.0, 50 mM KCl, 6 mM MgCl2, 1 mM DTE. Reverse transcription was initiated by adding a mixture of the four deoxynucleoside triphosphates (50 µM each) pre-incubated at 37°C in the same buffer. Formamide containing 50 mM EDTA was added to aliquots of the reaction mixture at times ranging from 15 s to 30 min, and the reaction products were analyzed on 8% denaturing polyacrylamide gels, and quantified with a BioImager BAS 2000 (Fuji) using the MacBas software.

In order to compare the native and synthetic tRNAHis, a 5-fold molar excess of unlabeled tRNAHis was annealed to 10 nM of His-AC-GAC RNA as described above. DNA synthesis was performed as described above except for the following modifications. The RT concentration was 27 nM, the dATP, dGTP and dTTP concentrations were 150 µM, and the dCTP concentration was 15 µM (10 µCi in a final volume of 100 µl).

Chemical probing of vRNA
After hybridization, the vRNA/tRNA complexes were incubated at 20°C for 15 min in the annealing buffer supplemented with 5 mM MgCl2 before probing with dimethyl sulfate (DMS). Hybridization and probing buffers were the same for 1-cyclohexyl 3-(2 morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT), except that they contained 50 mM sodium borate (pH 8.0) instead of sodium cacodylate (pH 7.5).

After addition of 1 µg of yeast total tRNA, RNA was modified by addition of 1 µl of 10-fold diluted DMS in ethanol for 5 or 10 min, or 2 µl of CMCT (40 mg/ml in water) for 10 or 20 min. RNA modification was stopped with 200 µl ethanol and 50 µl sodium acetate 0.3 M (pH 5.3) containing 1 µg glycogen. Modified bases between positions 120 and 220 were detected by primer extension with RT as previously described (48).

Enzymatic and chemical probing of tRNAHis
Viral RNA (12 pmol) and 50 000 c.p.m. of 5'-end-labeled tRNAHis (0.4 pmol) were hybridized as described above. The tRNAHis, in its free form or involved in the binary complexes, was cleaved with 0.06 and 0.1 U of RNase V1 and RNase T1, respectively, for 7.5 or 15 min at room temperature. Free and hybridized tRNAHis was cleaved with 200 U of nuclease S1 from Aspergillus orizae for 7.5 or 15 min at room temperature. After phenol/chloroform and ethanol precipitation, tRNA3Lys fragments were analyzed on 15% denaturing polyacrylamide gels.

For chemical probing, 12 pmol of vRNA and 0.4 pmol of tRNAHis were hybridized as described above, except for the pH of the sodium cacodylate buffer, which was decreased to 7.0. After addition of 1 µg of yeast total tRNA, tRNAHis in its free form or involved in the binary complexes was modified with 3 µl of kethoxal (20 mg/ml) diluted in ethanol 20% for 7 and 15 min. Reactions were stopped with 200 µl ethanol and 50 µl sodium acetate 0.3 M (pH 5.3) containing 1 µg glycogen. Modified bases were detected by primer extension with RT as previously described (48).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
Discussion
 REFERENCES
 
The vRNA mutants studied
Among all mutant that replicate using alternate tRNAs as primers, those using tRNAHis are the best characterized in cell culture (32–35). Unlike HXB2(His), which possessed a mutated PBS, HXB2(His-AC), in which both the PBS and A-rich loop were mutated, stably used tRNAHis as primer for reverse transcription (32) (Table 1, Fig. 1B and C). However, analysis of the U5-PBS region of this virus revealed multiple nucleotide changes in U5 and around the PBS (32,33). Three nucleotide substitutions predominated in the adapted HXB2(His-AC-GAC) virus: U174G, G181A and U200C (Table 1). Note that nucleotides 174 and 181 are located between the PBS and the A-rich loop, and nucleotide 200 is the first nucleotide 3' to the PBS (Fig. 1B). Importantly, substitution of a 200-nucleotide fragment encompassing the U5-PBS region of HXB2(His-AC) and HXB2(His-AC-GAC) into the WT proviral clone (pHXB2), conferred the capacity of the resulting viruses, named pHXB2(His-AC) and pHXB2(His-AC-GAC), respectively, to utilize tRNAHis to prime reverse transcription (33). These experiments provided evidence that the U5-R region of the genomic RNA plays a key role in the primer utilization. Following extensive cell culture of pHXB2(His-AC-GAC), two main additional nucleotide substitutions were selected: C152A and C160U (33) (Table 1 and Fig. 1B). Subcloning of the U5-PBS region of this virus into pHXB2 yielded pHXB2(His-AC-AT-GAC) (35).


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  		Table 1. Characteristics of the viral RNAs used in this study
 
We have cloned the R, U5, PBS and leader region, as well as part of the gag coding region of pHXB2, pHXB2(His), pHXB2(His-AC), pHXB2(His-AC-GAC) and pHXB2(His-AC-AT-GAC) immediately downstream of a T7 RNA polymerase promoter. In vitro transcription allowed us to synthesize RNAs corresponding to the first 295 nucleotides of the vRNAs and containing the mutations mentioned above (Table 1 and Fig. 1B).

Efficiency of (–) strand strong stop DNA synthesis
The (–) strand strong stop DNA results from the extension of a primer bound to the PBS up to the 5'-end of the genomic RNA. Its synthesis can be divided into two steps: initiation, which corresponds to the slow and distributive addition of the first six nucleotides (in the case of the WT template) to the primer tRNA, followed by a fast and processive elongation (27,49,50).

Kinetics of (–) strand strong stop DNA synthesis from tRNA3Lys annealed to WT RNA, and from tRNAHis annealed to His, His-AC, His-AC-GAC and His-AC-AT-GAC RNAs are shown in Figure 2. When the WT RNA was reverse transcribed using tRNA3Lys as primer, (–) strand strong stop DNA appeared after 10 min and gradually increased to reach 21% of the initial amount of primer after 30 min. The amount of (–) strand strong stop DNA synthesized from tRNAHis on His RNA was much lower (Fig. 2) and only amounted to 5% after 30 min. (Table 2). More unexpectedly, the amount of (–) strand strong stop DNA was hardly increased on the His-AC RNA, but was dramatically raised by mutations U174G, G181A and U200C present in His-AC-GAC RNA (Fig. 2 and Table 2). Surprisingly, the amount of (–) strand strong stop DNA synthesized after 30 min was equal to 31%, corresponding to a 50% increased efficiency, as compared to the WT RNA (Table 2). This efficiency was not significantly affected by the additional mutations C152A and C160U that are present in His-AC-AT-GAC RNA (Fig. 2 and Table 2).



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  		Figure 2. Kinetics of (–) strand strong stop DNA synthesis. Labeled native tRNA3Lys annealed to WT RNA (left panel in the upper row), and labeled synthetic tRNAHis annealed to the mutant templates indicated above the gels were extended for 0, 15, 30, 45 s, 1, 2.5, 5, 10, 15, 20, 25 and 30 min (lanes 1–12, respectively) as described in Materials and Methods. The extension products were analyzed on an 8% denaturing polyacrylamide gel. The labels on the right of the gels indicate the position of the primer and some of the extension products. The +181 product corresponds to the (–) strand strong stop DNA.

 

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  		Table 2. Efficiency of the (–) strand strong stop DNA synthesis and of the initiation of reverse transcription of WT and mutant RNA templates
 
Kinetics of primer tRNA extension and pausing during reverse transcription
In order to directly access the efficiency of the initiation of reverse transcription, we determined the fraction of unextended primer remaining over time (Fig. 3). As previously observed, the kinetics of the initial tRNA extension are best described as a bi-exponential process (19,49). Indeed, previous pre-steady and steady-state kinetics conducted on the WT HIV-1 MAL RNA/tRNA3Lys complex indicated that the fast reaction corresponds to extension of the preformed primer–template:RT complex, whereas the slow step requires RT recycling and a conformational change of the primer–template complex (49,50).



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  		Figure 3. Kinetics of the initiation of reverse transcription. The amount of unextented and extended primer in the experiments shown in Figure 2 were quantified, and the fraction of unextended primer was plotted as a function of time. The experimental data obtained with WT (open circles), His (closed circles), His-AC (open squares), His-AC-GAC (closed squares) and His-AC-AT-GAC (open triangles) RNAs were fit with the Igor Pro Carbon software for Mac OS X using the equation where funextended is the fraction of unextended primer, Afast and Aslow are the amplitudes of the fast and slow phases, respectively, and kfast and kslow correspond to the rate constants of these phases. The values of Afast and kfast obtained from these fits are listed in Table 2.

 
When using the WT RNA, 71% of the primer tRNA3Lys was extended with a rate constant kfast of 0.025 s–1 (Fig. 3 and Table 2). The remaining 29% of the primer were extended slowly, and 10% were not extended after 30 min (Fig. 3). The kfast of tRNAHis extension on the His RNA was decreased 5-fold, and the amplitude of the fast phase (Afast) was reduced to 20% (Fig. 3 and Table 2). Even though similar amounts of (–) strand strong stop DNA were generated on His and His-AC templates, the initiation of reverse transcription appeared more efficient with the latter RNA (Fig. 3). This was not due to an increase in kfast, but to a 2-fold increase of Afast (Table 2).

The efficiency of the initiation of reverse transcription was dramatically improved with His-AC-GAC (Fig. 3). Indeed, the kfast value measured on this template was ~4.5-fold higher than that for the WT RNA (Table 2). Surprisingly, kfast was decreased to 1.25-fold the WT value with His-AC-AT-GAC, indicating that mutations C152A and C160U decreased the efficiency of the initiation of reverse transcription (Fig. 3 and Table 2). Worth noticing, primer extension was complete on His-AC-GAT and His-AC-AT-GAC templates. At the opposite, ~10, 25 and 50% of the primer was not extended after a 30 min reaction on the WT, His-AC and His templates, respectively (Fig. 3). The complete primer extension on His-AC-GAT and His-AC-AT-GAC RNAs suggests that the initiation complexes formed by annealing tRNAHis to these templates were structurally homogeneous, while a fraction of the WT complex adopted a non-functional structure. This feature likely contributes to the increased amount of (–) strand strong stop DNA observed with these mutant RNAs.

During (–) strand strong stop DNA synthesis, a distinct pattern of RT pausing sites was observed for each template (Fig. 2). As previously observed, the WT HIV-1 HXB2 RNA gave a pausing pattern similar to the WT HIV-1 MAL RNA (19,27,51), the main pausing sites being at position +3 and +5 upstream of the PBS and in the A-rich loop. The pausing patterns observed with the His and His-AC RNAs were unique, whereas those observed with His-AC-GAC and His-AC-AT-GAC were very similar. The only distinct feature was weak pausing at position +1 at long incubation times during reverse transcription of His-AC-AT-GAC RNA that was not detected with His-AC-GAC (Fig. 2). This observation suggested that the structures of the initiation complexes involving these templates were similar. On the contrary, the pausing patterns observed with the His-AC and His-AC-GAC RNAs were very different (Fig. 2). Since most of the differences were located at a distance from the three mutations that distinguish these RNAs, they indicated that the corresponding initiation complexes most likely adopted different secondary or/and tertiary structures (see below).

Effect of the post-transcriptional modifications of tRNAHis
Using the wild type HIV-1 MAL RNA, we previously showed that the efficiency of the initiation of reverse transcription was strongly dependent on the presence of the post-transcriptional modifications of tRNA3Lys. In their absence, the interaction between the viral A-rich loop and the tRNA anticodon loop was unstable (9,10,52), and initiation of DNA synthesis was inefficient (27,49).

Therefore, we compared the (–) strand strong stop DNA synthesis using His-AC-GAC RNA as template and tRNAHis that was either synthesized in vitro or purified from beef liver, as primer (Fig. 4). Since purification of the natural tRNAHis was not complete, we used unlabeled tRNA primers and included a radioactive dNTP in the DNA synthesis reaction. Under these conditions, only the (–) strand strong stop DNA and the longest pausing products were detected (Fig. 4). Interestingly, we noticed little difference between the two primers, both in the kinetics of synthesis and in the final amount of (–) strand strong stop DNA synthesized. By comparison, a 5- to 10-fold difference in the efficiency of (–) strand strong stop DNA synthesis could be observed between natural and in vitro synthesized tRNA3Lys when using the WT HIV-1 MAL RNA as template (27).



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  		Figure 4. Comparison of synthetic and natural tRNAHis. Minus strand strong stop DNA was synthesized using His-AC-GAC as template and either synthetic or natural unlabeled tRNAHis. A radioactive nucleotide was included in the reaction, which was stopped after 5, 10, 15, 20, 25 and 30 min (lanes 1–6, respectively), and the products were analyzed by polyacrylamide gel electrophoresis.

 
Probing of the His-AC and His-AC-GAC RNA templates in their initiation complexes
The kinetics of reverse transcription described in the previous sections indicated that the efficiency of the initiation step of reverse transcription, as well as that of the (–) strand strong stop synthesis, dramatically increased with the accumulation of mutations U174G, G181A and U200C in the RNA template. We therefore examined the structure of the initiation complexes formed with His-AC and His-AC-GAC RNAs, using chemical and enzymatic probes.

In order to compare the structure of His-AC and His-AC-GAC RNAs, either in their free form, or in complex with tRNAHis, we used DMS and CMCT (Fig. 5). DMS methylates positions N1 of adenines and N3 of cytosines, and CMCT modifies positions N1 of guanines and N3 of uridines. Watson–Crick base pairs and non-canonical interactions involving these positions protect them from chemical modification.



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  		Figure 5. Chemical probing of His-AC and His-AC-GAC RNAs. His-AC and His-AC-GAC RNAs, either free or in complex with tRNAHis, were submitted to modification by DMS (A) or CMCT (B) for 0 (control lane), 5, 10 or 15 min, as indicated above the lanes. Lanes marked U, G, C and A correspond to dideoxy sequencing reactions. The position of the PBS and of the sequence complementary to the anticodon loop (AC) of tRNAHis is indicated on the right of the gels.

 
Comparison of the reactivity profiles of His-AC and His-AC-GAC RNAs in their free form indicated that the U174G, G181A and U200C mutations affected their structure. Differences in the modification patterns could be observed in the vicinity of the mutated residues, but also at remote positions. For instance, U199 and U201 were modified by CMCT in His-AC RNA, but not in His-AC-GAC-RNA (Fig. 5B). Similarly, U174 was modified, but the corresponding G174 was not reactive in His-AC-GAC (Fig. 5B), and C179 was modified only in His-AC RNA (Fig. 5A).

As RNAs His-AC and His-AC-GAC both possess a sequence complementary to the anticodon loop of tRNAHis, we were particularly interested in potential modifications in the reactivity profile that could take place at and around nucleotides 167–172 upon annealing of tRNAHis. In the free form of these RNAs, several nucleotides were modified by CMCT and DMS. Upon formation of the tRNAHis-vRNA complexes, we observed several modifications in the reactivity patterns in this region. Regarding modifications of the U and G residues by CMCT, we observed no striking differences between His-AC and His-AC-GAC RNAs. In both templates, primer annealing increased the reactivity of U159 and U165 towards CMCT, while the reactivity of U163 decreased (Fig. 5B). However, marked differences appeared in the DMS reactivity patterns of His-AC and His-AC-GAC RNAs upon tRNAHis annealing. In His-AC RNA, the strong modifications of A171, A172 and A177, and the weak modifications of C173 and C175 were not affected by the primer binding. On the other hand, tRNAHis induced strong or complete protection of A169, C170, A171, A172 and C173, which were strongly modified in the free form of His-AC-GAC RNA (Fig. 5A).

Thus, our probing data indicated that the interaction between the anticodon loop of tRNAHis and the mutated A-rich loop of His-AC RNA did not take place, or that it was very unstable, even though the mutations in the viral RNA were designed to allow it. In contrast, our data suggested that, in the case of His-AC-GAC RNA, this interaction was stable and conferred a strong protection against chemical modification to nucleotides 169–173.

In addition to the experiments described above, which were performed in the annealing buffer (see Materials and Methods), we also performed DMS probing of His-AC and His-AC-GAC RNAs, either free or in complex with tRNAHis, in the absence and in the presence of a 3-fold molar excess of RT, in the reverse transcription buffer (data not shown). Identical DMS modification patterns were obtained under all experimental conditions. The absence of an RT footprint on the initiation complex when using DMS is not new. We previously observed a footprint of RT on the WT MAL initiation complex when using enzymatic probes, but not chemical ones (15).

Probing of the primer tRNAHis in the initiation complexes formed by His-AC and His-AC-GAC RNA templates
In order to corroborate these conclusions, we analyzed the accessibility of tRNAHis in its free form and annealed to either His-AC or His-AC-GAC RNA, using nuclease S1, RNase T1 and RNase V1 (Fig. 6A and data not shown). Nuclease S1 and RNase T1 cleave single-stranded regions of the RNA molecules, the latter being specific for G residues, while RNase V1 specifically cuts into base-paired and stacked RNA regions (53).



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  		Figure 6. Enzymatic and chemical probing of tRNAHis. (A) 3'-end labeled tRNAHis, free or annealed to His-AC, His-AC-GAC or His RNA was submitted to hydrolysis with nuclease S1. Reactions were for 0 (control lane), 7.5 or 15 min, as indicated above the lanes. Lanes marked T1 and U2 correspond to sequencing reactions with T1 and U2 RNases, respectively. (B) Unlabeled tRNAHis, free or annealed to His, His-AC or His-AC-GAC RNA was modified with kethoxal for 0, 7 or 15 min. The modified guanines were identified by primer extension. The position of the anticodon loop (AC) and of the sequence complementary to the PBS (anti-PBS) are indicated.

 
As expected, the cleavage pattern of tRNAHis was dramatically affected upon annealing to the viral templates. However, the cleavage patterns of tRNAHis hybridized either to His-AC RNA or to His-AC-GAC RNA looked very similar (Fig. 6A and data not shown). Nevertheless, minor differences in the cleavage pattern of the D arm of tRNAHis (nucleotides 10–25) by nuclease S1 (Fig. 6), RNase T1 and RNase V1 (data not shown) suggested that the initiation complexes formed by these viral RNAs adopted slightly different structures. Surprisingly, the anticodon loop of tRNAHis, which was cut preferentially 3' of nucleotides 34 and 33, and weakly at positions 31, 32 and 36 by nuclease S1 in the free form of the primer, was strongly protected by hybridization to either His-AC or His-AC-GAC RNA (Fig. 6). A similar observation was made with RNase T1 (not shown).

These results seem to contradict the DMS probing results indicating that only the mutated A-rich loop of His-AC-GAC RNA became protected upon tRNAHis annealing. Further more, when we annealed tRNAHis to His RNA and submitted the complex to nuclease S1 probing, we observed a strong protection of the anticodon loop of tRNAHis (Fig. 6A), even though the A-rich loop of His RNA cannot interact with this region of the primer (Fig. 1B and Table 1). Two alternative explanations can be put forward. Due to the size of the enzymatic probes, protection of the anticodon loop of tRNAHis might result from steric hindrance and might not be a reliable indicator of its interaction with the viral A-rich loop. Alternatively, the anticodon loop of tRNAHis might establish tertiary interactions with all the templates (His, His-AC and His-AC-GAC), but these interactions would differ from one vRNA to the other. Only with the His-AC-GAC RNA would the tRNAHis anticodon loop interact with the A-rich loop.

In order to test this possibility, we probed tRNAHis with kethoxal, which modifies positions N1 and N2 of unpaired guanines. Unlike nuclease S1 mapping, probing with kethoxal is not sensitive to steric hindrance. Interestingly, nucleotide G34 was strongly modified in the isolated tRNAHis, but protected in all tRNAHis/vRNA complexes (Fig. 6B). Thus, the tRNAHis anticodon loop did establish tertiary interactions in all complexes. A computer analysis using Mfold (54) indicated that nucleotides 0–57 of tRNAHis could fold into a stable intramolecular structure upon annealing its 3' end to the PBS. However, this structure is not consistent with the kethoxal probing data, since G34 is predicted to be unpaired (data not shown). This analysis strongly suggests that the tRNAHis anticodon loop interacted with the template RNAs. Indeed, several sequences partially complementary to the tRNAHis anticodon loop were identified in the vRNA sequences, outside the region probed in this study.

A complete analysis of the kethoxal modification patterns reveals that tRNAHis adopted different structures in the complexes with His RNA and His-AC-GAC RNA. For instance, tRNAHis G4 and G6 were more strongly modified in the complex with His-AC-GAC RNA than in the complex with His RNA, while the reverse situation was observed for G17, G18, G22 and G43 (Fig. 6B). An intermediate pattern was observed when tRNAHis was annealed to His-AC RNA (Fig. 6B), suggesting that this complex existed as a mixed population or equilibrium between two structures.


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Discussion
REFERENCES
 
The mutant HIV-1 viruses that use alternate tRNAs as primer provide a unique opportunity to establish direct relationships between viral replication, efficiency of the initiation of reverse transcription and the structure of the initiation complex in vitro. This is particularly true for the mutants using tRNAHis because their evolution during prolonged cell culture is well documented (32–35).

The pHXB2(His) virus, in which a PBS complementary to tRNAHis has been substituted for the PBS complementary to tRNA3Lys is unstable in cell culture and rapidly reverts to the WT virus (31,32). Here, we showed that the initiation of reverse transcription of the corresponding RNA (His RNA) is very inefficient. The kfast value was decreased 5-fold as compared to the WT RNA, resulting in a 4-fold decrease of (–) strand strong stop DNA. These data explain why the pHXB2(His) virus is unstable in cell culture. Indeed, even though the PBS in the WT and mutant PBS differ by seven nucleotides, reversion is a one step process (29–31). If a tRNA3Lys molecule packaged into the virion is hybridized, although imperfectly, to the mutant PBS, its 18 3' terminal nucleotides are copied during synthesis of the (+) strand strong stop DNA, regenerating the WT PBS. Given the differences in the (–) strand strong stop DNA synthesis, the WT virus rapidly outgrows the pHXB2(His) virus.

The pHXB2(His-AC) virus does not revert to the WT virus, but rapidly selects three additional mutations (T174G, G181A, T200C) (32,33). The resulting virus, pHXB2(His-AC-GAC) replicates more rapidly than pHXB2(His-AC). Our data showed that even though the initiation of reverse transcription of pHXB2(His-AC) is slightly more efficient than that of pHXB2(His) (Fig. 3), equal amounts of (–) strand strong stop DNA are synthesized. Thus, the reason why pHXB2(His) reverts to the WT virus, while pHXB2(His-AC) maintains tRNAHis as primer cannot be found in their relative efficiency in initiating reverse transcription. Rather, it stems from the fact that incorporation of only three point mutations in pHXB2(His-AC) allows this virus to initiate reverse transcription and synthesize (–) strand strong stop DNA more efficiently than the WT virus. At the opposite, reversion of pHXB2(His-AC) to the WT virus would not only require the reversion of the PBS, but also of the four point mutations in the A-rich loop.

Even though we cannot exclude that other viral functions are affected, our data showed that the mutations introduced in pHXB2(His) and pHXB2(His-AC) dramatically affect the initiation of reverse transcription and the (–) strand strong stop DNA synthesis. More important, our work revealed that the first mutations selected by pHXB2(His-AC) restore these reverse transcription steps. Thus, despite the possible overlap with integration signals (40–42), viral evolution was dictated by the initiation of reverse transcription. Our data show that, contrary to previous proposals (43), the mutations in the A-rich loop allow maintaining tRNAHis as primer by increasing the efficiency of this primer, rather than solely disfavoring tRNA3Lys. The rapid selection of the T174G, G181A and T200C mutations reveals a strong selection pressure to restore the initiation of reverse transcription, and thus indicates that this process constitutes a valuable target for anti-HIV agents.

Upon prolonged cell culture, pHXB2(His-AC-GAC) slowly selects two additional mutations (33,44). However, our data revealed that these mutations did not increase reverse transcription. On the contrary, initiation of reverse transcription of His-AC-AT-GAC RNA was reduced >3-fold relative to His-AC-GAC RNA, but this resulted in no significant reduction of (–) strand strong stop DNA. Thus, our data indicate that the virus has no pressure to optimize initiation of reverse transcription above a given threshold, because this does not translate into a significant increase of the longer reverse transcription products. The data we obtained with His-AC-AT-GAC RNA also confirm that restoring the initiation of reverse transcription is the first problem pHXB2(His-AC) has to deal with, while optimizing other replication steps occurs at later stages. It is presently unclear whether mutations C152A and C160T affect other steps of the reverse transcription process, integration, or another replication step.

Even though both pHXB2(His-AC) and pHXB2(His-AC-GAC) viruses can potentially interact with the anticodon loop of tRNAHis, the latter replicate more efficiently (33) and initiate reverse transcription much faster. This observation raised the possibility that the interaction between the anticodon loop of tRNAHis and the viral RNA was not sufficient to allow efficient initiation of reverse transcription. However, enzymatic and chemical probing of the primer/template complexes revealed structural differences between the complexes containing His-AC and His-AC-GAC complexes. Most importantly, DMS probing of the vRNA revealed that tRNAHis interacts with the mutated A-rich loop of His-AC-GAC RNA, but not that of His-AC-RNA. Mutation T174G likely plays an essential role in this interaction by extending the complementarity between the mutated A-rich loop and the anticodon stem of tRNAHis.

Thus, in the mutant HXB2 viruses utilizing tRNAHis as primer, efficient replication and initiation of reverse transcription correlate with a stable interaction between the primer anticodon loop and the viral RNA. This finding extends the conclusions of our previous studies conducted on the HIV-1 MAL isolate which showed that efficient in vitro initiation of HIV-1 MAL RNA reverse transcription requires the interaction between the viral A-rich loop and the anticodon of tRNA3Lys (19,27,49,50). Collectively, our present and past results suggest that an intermolecular interaction between the viral RNA and the primer anticodon loop is a widespread feature in natural HIV-1 isolates and mutant HIV-1 adapted to utilize alternate primers. However, this does not appear to be an absolute requirement, as mutant viruses with two PBS that lack any complementarity with the anticodon loop of the two potential primer tRNAs have been observed (55).

Berkhout and co-workers recently proposed an interaction between a viral sequence named Primer Activation Signal (PAS) and the complementary anti-PAS sequence located in the T{Psi}C arm of tRNA3Lys (17,18,56). However, analysis of the in vivo evolution of HIV-1 mutants using either tRNAHis or tRNAMet as primer revealed no adaptation of the PAS to the anti-PAS sequence of these primers (51).

Stabilization of the interaction between the A-rich loop of HIV-1 MAL RNA and the U-rich anticodon of tRNA3Lys (9,52,57), and hence efficient initiation of reverse transcription (27,49), was shown to require the post-transcriptional modifications of the tRNA, especially the mcm5s2U nucleotide at position 34. This was expected as the thio moiety of U34 was known to stabilize the otherwise unstable A-U base pairs (58). By comparison, His-AC-GAC RNA forms a G-C rich interaction with the anticodon stem of tRNAHis (Fig. 1B and C). Therefore, our finding that the post-transcriptional modifications of tRNAHis are not required to promote the efficient initiation of reverse transcription of His-AC-GAC RNA is not very surprising. However, this result is of great importance for future structural studies (X-ray crystallography or/and NMR) of the initiation complex, which will be greatly facilitated by the use of an in vitro transcribed tRNA, instead of natural tRNA purified from mammal cells.


   Acknowledgements
 
We are grateful to G. Bec and G. Keith for the tRNA purification, to P. Walter for the gift of HIV-1 RT and to Dephine Richer for technical assistance. We thank Catherine Isel and Jean-Christophe Paillart for critical reading of the manuscript. This work was supported by the ‘Agence Nationale de Recherches sur le SIDA’ (ANRS). F.B. was supported by fellowships from Sidaction and ANRS.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 Discussion
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