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

This Article Abstract Full Text (PDF) 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 Lanchy, J.-M. Articles by Marquet, R. Search for Related Content PubMed PubMed Citation Articles by Lanchy, J.-M. Articles by Marquet, R. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 16, 12306-12312, April 21, 2000

Dynamics of the HIV-1 Reverse Transcription Complex during Initiation of DNA Synthesis^*

Jean-Marc Lanchy^§, Catherine Isel, Gérard Keith, Stuart F. J. Le Grice^¶, Chantal Ehresmann, Bernard Ehresmann, and Roland Marquet

From the  UPR 9002 du CNRS, IBMC, 67084 Strasbourg cedex, France and ^¶ Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA₃^Lys and reverse transcriptase (RT). The vRNA and the primer tRNA₃^Lys form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA·tRNA₃^Lys complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reverse transcription is a key step in the retroviral replication cycle (1, 2), during which the virus-encoded reverse transcriptase (RT),¹ which possesses RNA- and DNA-dependent DNA polymerase and RNase H activities (3), converts the genomic RNA into double-stranded DNA. In retroviruses, initiation of reverse transcription requires annealing of the 18 3'-terminal nucleotides of a cellular tRNA to the primer binding site (PBS), located near the 5' end of the RNA genome (4-6).

In human immunodeficiency virus type 1(HIV-1), a strong selective pressure maintains tRNA₃^Lys as primer. Mutating the PBS to match the 3' end of other tRNAs dramatically reduces viral replication, and rapid reversion to the wild-type PBS is observed (7-9). A similar situation also prevails in avian leukosis viruses (10). In vitro, HIV-1 RT is able to discriminate against non-self-tRNA primers (7, 11). Interestingly, the specificity of the initiation of HIV-1 reverse transcription correlates with virus-specific interactions between the primer tRNA₃^Lys and the viral RNA (vRNA). Chemical and enzymatic probing revealed intricate intermolecular interactions between the anticodon loop and stem, part of the variable loop of tRNA₃^Lys, and sequences upstream of the PBS (12, 13) (Fig. 1). Extended tRNA-vRNA interactions were also identified in avian retroviruses (14, 15) and in yeast retrotransposon Ty1 (16) and proposed in HIV-2 (17).

In HIV-1, the best-characterized specific intermolecular interaction involves the anticodon loop of tRNA₃^Lys and an A-rich loop conserved in all viral isolates (helix 6C in Fig. 1). In vitro, this loop-loop interaction prevents extension of the HIV-1 vRNA·tRNA₃^Lys complex by heterologous RTs (18, 19), and it is required for efficient transition from the initiation to the elongation of reverse transcription that takes place after the addition of six nucleotides (18, 20). The initiation and elongation phases strongly differ by their polymerization rate and by the dissociation rate of RT from the primer-template complex (20, 21). Interestingly, three-dimensional modeling of the HIV-1 vRNA·tRNA₃^Lys and HIV-1 vRNA·tRNA₃^Lys·RT complexes, based on our probing and footprinting data, indicated that RT does not directly recognize the extended primer-template interactions (22). These interactions favor binding of the homologous RT by preventing steric clashes and ensuring proper orientation of the template domains directly interacting with RT (22).

The extended intermolecular interactions also appeared crucial for the initiation of HIV-1 reverse transcription in cell culture. First, a tRNA₃^Lys with a mutated anticodon was incorporated into the HIV-1 particles but did not prime reverse transcription (23). Second, deletion of the A-rich loop resulted in viruses with diminished levels of infectivity and reduced DNA synthesis, and the A-rich loop was progressively restored upon prolonged cell culture (24). Finally, this interaction is a major determinant of primer usage in HIV-1. Indeed, when the PBS was mutated to match the 3' end of other tRNAs than tRNA₃^Lys, replication of the virus was dramatically reduced, and reversion to the wild-type PBS was rapidly observed (7-9). However, HIV-1 could stably use tRNA^His and tRNA^Met as primers, provided that both the PBS and the A-rich loop were simultaneously mutated to be complementary to these tRNA species (25, 26). When mutated, the other intermolecular interactions (helices 3E and 5D in Fig. 1) were also restored upon prolonged cell culture (27, 28).

In this report, we used enzymatic probing to study the dynamics of the reverse transcription complex and to follow the fate of the interaction between the anticodon and the A-rich loops during addition of the first six nucleotides to the primer tRNA, i.e. during the initiation phase of reverse transcription.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and Enzymes-- RNase T2 and Neurospora crassa endonuclease were from Amersham Pharmacia Biotech. Heparin was from Sigma. Phage T4 polynucleotide kinase was from U. S. Biochemical Corp. [-³²P]ATP (3000 Ci/mmol) and [5'-³²P]cytidine 3',5'-bisphosphate (3000 Ci/mmol) were from NEN Life Science Products. Acrylamide and N,N'-methyl bisacrylamide were from Roth.

RNAs and RTs-- 123-217 vRNA, corresponding to nucleotides 123 to 217 of HIV-1 genomic RNA (Mal isolate), was synthesized and 3' end-labeled as described previously (29). Wild-type and mutant 1-311 vRNA were synthesized as described (18, 30). In the mutant template, 5'-CUAUG-3' was substituted for ¹⁶²GUAAAA¹⁶⁷ (Fig. 1) (18). Natural tRNA₃^Lys was purified from beef liver and, after limited hydrolysis with cobra venom phosphodiesterase, was labeled by reconstituting its 3'-terminal CCA with [-³²P]ATP and unlabeled CTP using tRNA nucleotidyltransferase (31). RNase H() HIV-1 RT bearing the E478Q mutation was purified essentially as described previously (32). This mutant polymerase was used to prevent cleavage of the RNA template by the double-stranded RNase activity associated with HIV-1 RNase H (33). The wild-type and mutant enzymes are equally efficient in initiating reverse transcription (18).

Enzymatic Probing of Viral RNA-- For RNase T2 probing experiments, 3' end-labeled 123-217 vRNA (~50,000 cpm, 4 pmol), either free, in the binary, or in the ternary complex, was incubated for 2 min at 90 °C and cooled on ice. The binary complex was formed by heating vRNA with tRNA₃^Lys (24 pmol) at 70 °C for 20 min in 50 mM Tris-HCl, pH 7.5 (37 °C), 40 mM KCl. After hybridization, samples were incubated in the same buffer supplemented with 5 mM MgCl₂ at 20 °C for 15 min. If performed, extension reactions were initiated by adding 50 pmol of HIV-1 RT and the appropriate dNTP/ddNTP mixtures (dNTPs 100 µM and ddNTP 50 µM) to obtain +1, +2, +3, and +6 extension products. Forty µg of heparin were added to half of the reaction mixtures, which were further incubated for 5 min at 37 °C. RNase T2 probing was with 0.1 units of enzyme for 2.5 and 5 min at 37 °C. After phenol/chloroform extraction and ethanol precipitation, RNA was loaded on a 15% denaturing polyacrylamide gel.

Enzymatic Probing of tRNA₃^Lys-- 3' End-labeled tRNA₃^Lys (~50,000 cpm), either free, in the binary complex formed with 3-fold excess (12 pmol) 1-311 vRNA, or in the ternary complex with 50 pmol of HIV-1 RT, was incubated for 10 min at 37 °C. When appropriate, tRNA₃^Lys was extended by 1, 2, 3, or 6 nucleotides as described above. Half of the reaction mixture was removed, and heparin (40 µg) was added to the remaining solution. Probing of tRNA₃^Lys was with 0.6 units of N. crassa endonuclease for 1 or 2 min. RNA was ethanol-precipitated and analyzed on a 15% denaturing polyacrylamide gel.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental Strategy-- To test the intermolecular interactions between the viral A-rich loop and the anticodon loop of tRNA₃^Lys during initiation of reverse transcription and to monitor translocation of RT along the primer-template complex, we first preformed the vRNA·tRNA₃^Lys ·HIV-1 RT complex. Then we added ddCTP, dCTP + ddTTP, dCTP + dTTP + ddGTP, or dCTP + dTTP + dGTP + ddATP. These nucleotide mixtures allowed extension of tRNA₃^Lys by 1, 2, 3, and 6 nucleotides, respectively (Fig. 1). Half of the reaction mixtures was used to test the vRNA·tRNA₃^Lys ·HIV-1 RT complexes in these registers (0, +1, +2, +3, and +6). Heparin was added to the rest of the reaction mixtures to trap RT, thus allowing probing of the primer-template complexes.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Secondary structure model of the HIV-1 RNA·tRNA3Lys complex, as deduced from enzymatic and chemical probing (12) and site-directed mutagenesis (13). The tRNA sequence is in white on a black background. The region of the vRNA corresponding to nucleotides 123 to 217 of the HIV-1 genome (MAL isolate) is represented. Helices are numbered according to Isel et al. (12). The interaction between the anticodon and the A-rich loops corresponds to helix 6C and that between the 3' end of the tRNA and the PBS forms helix 7F. The other intermolecular interactions correspond to helices 3E and 5D.

Probing the HIV-1 RNA Template in Register Zero-- To probe HIV-1 RNA in the vRNA·tRNA₃^Lys and vRNA·tRNA₃^Lys·RT complexes, we used RNase T2, which cleaves single-stranded regions of RNA, with a slight preference for adenines.

The RNase T2 cleavages observed in 123-217 vRNA either free or bound to tRNA₃^Lys are consistent with the secondary structure models that we previously proposed for the free HIV-1 RNA (34) and for the binary complex (Figs. 1 and 2A) (12, 13). As expected, the single-stranded A-155, A-177, and A-200 were efficiently cleaved in the binary complex (Fig. 2A and Table I). Besides, A-157 and U-161, located in interhelical junctions, were also cleaved, although to a lesser extend. G-159 and A-164 to A-167 were slightly cleaved by RNase T2 (Fig. 2A), due to the dynamic nature of the vRNA-tRNA₃^Lys interactions (12, 13).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Probing of the vRNA in the primer-template and primer-template-RT complexes before and after the addition of 2, 3, or 6 nucleotides. A, 3' end-labeled vRNA, free and in the binary and ternary complexes, was tested with RNase T2 in absence and in the presence of heparin. B, 3' end-labeled vRNA hybridized to tRNA3Lys that was subsequently extended by 2, 3, or 6 nucleotides by HIV-1 RT was probed with RNase T2 in the absence and in the presence of heparin. Lanes 0 are controls incubated in the absence of RNase T2. Lanes 1 and 2 correspond to incubation with 0.1 units of RNase T2 for 2.5 and 5 min, respectively. +Hep corresponds to the addition of heparin before the addition of RNase T2. Lanes L and U2 refer to alkaline ladder and nuclease U2 sequencing, respectively.

We next analyzed cleavage of HIV-1 RNA in the vRNA·tRNA₃^Lys ·RT complex. HIV-1 RT induced strong protection of A-200, A-177, and A-164 to A-167 (Fig. 2A and Table I). Weaker protections were observed at U-161, G-159, and A-157, whereas RNase T2 cleavage at A-155 was strongly enhanced (Fig. 2A and Table I). Protections of A-200 and A-177 were due to direct contacts with or close proximity of the polymerase (22). On the contrary, protections of nucleotides 164-167, G-159, and A-157 were due to stabilization of helices 6C and 5D rather than direct contact with RT (22). The increased reactivity at A-155 most probably reflects a slight reorientation of helix 4 upon RT binding.

Finally, we tested the possibility to trap RT dissociating from the primer-template with heparin, since we wanted to use this technique to probe the vRNA·tRNA₃ complex after extension of the primer (Fig. 2A). After the addition of heparin to the vRNA·tRNA₃^Lys ·HIV-1 RT complex, the RNase T2 profile was essentially identical to that of the vRNA·tRNA₃^Lys complex before the addition of RT (Fig. 2A). This result not only validates the use of heparin to trap RT but also indicates that RT binding did not induce irreversible conformational changes in the vRNA·tRNA₃^Lys complex.

Probing HIV-1 RNA during Initiation of Reverse Transcription-- To analyze the structural changes in the vRNA during initiation of reverse transcription, we used mixtures of dNTPs and ddNTPs, ending extension of tRNA₃^Lys after the addition of 1, 2, 3, and 6 nucleotides. The nucleotide and enzyme concentrations as well as the incubation time of the extension reactions were adjusted to obtain complete extension of the primer (see "Materials and Methods"). The extended tRNA₃^Lys·vRNA complexes were then probed with RNase T2 with or without prior incubation with heparin (Fig. 2B and Table I).

The addition of 1 (data not shown) or 2 nucleotides did not significantly change the reactivity profile of vRNA in the absence of heparin, as compared with the unextended ternary complex (compare Fig. 2, A and B). When tRNA₃^Lys was extended by 2 nucleotides, the A-rich loop and the upstream region were still protected against RNase T2 cleavage in the absence of heparin (Fig. 2B and Table I). The addition of heparin restored cleavage at A-157, G-159, and U-161. Protection of the latter nucleotides by RT was most likely due to stabilization of helices 6C and 5D, as was the case in register zero. The reactivity pattern of stem-loop 8 when tRNA₃^Lys was extended by 1 (data not shown) or 2 nucleotides was unchanged, as compared with the ternary complex in register zero (Fig. 2B and Table I). In the absence of heparin, A-200 was protected from cleavage, but it was strongly cut when RT was trapped by heparin. The only significant change taking place upon the addition of the second nucleotide was the disappearance of RNase T2 cleavage at A-177 in the binary complex (Fig. 2B and Table I). Cleavage at C-175 in the absence of heparin was not reproducibly observed in other experiments.

The cleavage pattern of HIV-1 RNA was significantly modified upon the addition of the third nucleotide (Fig. 2B and Table I). In the absence of heparin, A-164, in the A-rich loop, became moderately accessible to RNase T2, whereas cleavage at A-155 strongly decreased, and U-161, G-159, and A-157 remained fully protected. Simultaneously, loop 8 became significantly cleaved, even in the absence of heparin. Nucleotide A-177, which was now base-paired to the extended tRNA₃^Lys, was totally protected from RNase cleavage. Upon the addition of heparin, cleavage at A-155 was strongly enhanced, as well as cleavages at A-164 and A-165. Comparison of the vRNA·tRNA₃^Lys complexes in registers 2 and 3 is consistent with the unwinding of the distal part relative to the polymerization site of helix 6C upon the addition of the third nucleotide (Figs. 1 and 2B and Table I). In addition, our data indicate that RT interacts with or is close to A-155 in register 3 but not in register 2.

After the addition of the sixth nucleotide and in absence of heparin, all four adenines (A-164 to A-167) that were base-paired with the anticodon loop of tRNA₃^Lys in register 0 became reactive toward RNase T2, as well as A-157, G-159, and U-161 (Fig. 2B and Table I). These cleavages were further increased in the presence of heparin. These data suggest complete unwinding of helix 6C and destabilization of helix 5D at this stage of the initiation of reverse transcription (Fig. 1). Consistent with progression of the polymerase along the template, cleavage of stem-loop 8 in the absence of heparin, which became apparent in register +3, was further enhanced after the addition of the sixth nucleotide (Fig. 2B).

Enzymatic Probing of Primer tRNA₃^Lys in Register Zero-- To monitor the conformation of the anticodon loop of tRNA₃^Lys in binary and ternary complexes, we used the single strand-specific endonuclease from N. crassa (Fig. 3 and Table I). In agreement with our previous studies (12, 31), the only cleavages induced by this nuclease on free tRNA₃^Lys were located in the anticodon loop. Formation of the primer-template complex strongly decreased cleavage of the anticodon loop, whereas G-30 and G-19 and D-20 in the D loop became susceptible to cleavage, in agreement with the secondary structure model of the vRNA·tRNA₃^Lys complex (Fig. 1) (12, 31). The same cleavage pattern was observed when heparin was added to the vRNA·tRNA₃^Lys ·HIV-1 RT complex.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Probing tRNA3Lys in the primer-template and primer-template-RT complexes in registers 0, 1, 2, 3, and 6. 3' End-labeled tRNA3Lys annealed to vRNA was tested with N. crassa endonuclease in the presence of excess RT in the absence and presence of heparin and before and after extension by 1, 2, 3, or 6 nucleotides. Lanes 0 are controls incubated without nuclease. Lanes 1 and 2 correspond to incubation with N. crassa endonuclease for 1 and 2 min, respectively. Lanes denoted T1 and L refer to nuclease T1 sequencing and alkaline ladder, respectively. To facilitate comparison, the three panels of the figure have been aligned in such a way that the unextended and extended uncleaved primers are at the same level. +Hep corresponds to the addition of heparin before the addition of RNase T2.

When the ternary complex was probed in the absence of heparin, cleavages by N. crassa endonuclease were observed at the same positions, but their intensities were reduced, especially for U-35 and G-30 (Fig. 3 and Table I). Protection of the anticodon loop by HIV-1 RT was due to stabilization of helix 6C rather than direct contact with the polymerase (22). Likewise, protection of G-30 was due to steric hindrance rather than direct interaction with RT (22).

Enzymatic Probing of tRNA₃^Lys during Initiation of Reverse Transcription-- Little changes were observed after the addition of the first nucleotide, except that U-35 and G-30 were slightly more protected than in register zero. This difference was observed in the presence as well as in the absence of heparin (Fig. 3 and Table I). Upon the addition of the second nucleotide, cleavages by N. crassa endonuclease in the anticodon loop at G-30 and at G-19 and D-20 all significantly increased, especially in the absence of heparin. Unlike in registers 0 and 1, the anticodon loop of tRNA₃^Lys became more susceptible to cleavage than G-30 in the absence of heparin. Adding this trap has only limited effects, mainly affecting reactivity of G-30.

The addition of the third and sixth nucleotides resulted in the progressive protection of G-30, which became almost unreactive, both in the presence and absence of heparin (Fig. 3 and Table I). At the same time, cleavages in the anticodon loop of tRNA₃^Lys also progressively decreased. Protection of G-30 remained RT-dependent in register 3 but became RT-independent after the addition of the sixth nucleotide, as judged by the absence of heparin-mediated effects in this register. At the opposite, cleavage in the anticodon loop was independent of heparin addition in both registers.

Since, when probing tRNA₃^Lys, the binary complex was formed in the presence of excess vRNA, we performed a control experiment in which the uncomplexed vRNA was removed by chromatography before formation of the ternary complex. Probing data obtained with this material in registers zero and six, with and without heparin, were identical to those obtained with the unpurified vRNA·tRNA₃^Lys complex (data not shown).

Enzymatic Probing of tRNA₃^Lys Annealed to a Mutated vRNA-- To verify that the complex probing pattern of tRNA₃^Lys during initiation of reverse transcription actually reflected alterations of the primer-template interactions, we used a vRNA mutated in the conserved A-rich loop. We previously showed that substituting 5'-CUAUG-3' for ¹⁶²GUAAAA¹⁶⁷ in the vRNA prevents interaction with the anticodon loop of tRNA₃^Lys (13, 18). We also showed that this mutation introduces a defect at the level of the transition between initiation and elongation of reverse transcription (13, 18). Here, we annealed this mutated template to tRNA₃^Lys and tested the primer with the N. crassa endonuclease in registers 0, +3, and +6 in the absence and presence of heparin (Fig. 4).

View larger version (64K): [in this window] [in a new window]   Fig. 4.   Probing tRNA3Lys bound to a mutated vRNA in registers 0, 3, and 6. 3' End-labeled tRNA3Lys annealed to a vRNA mutated in the A-rich loop was tested with N. crassa endonuclease in the presence of excess RT and in the absence and presence of heparin before and after extension by 3 or 6 nucleotides. Lanes 0 are controls incubated in the absence nuclease. Lanes 1 and 2 refer to incubation with N. crassa endonuclease for 1 and 2 min, respectively. Lanes L and T1 correspond to alkaline ladder and nuclease T1 sequencing, respectively. +Hep corresponds to the addition of heparin before the addition of RNase T2.

When the primer was hybridized with the mutated vRNA, cleavage of the anticodon loop in register 0 was very pronounced (Fig. 4), in agreement with the lack of interaction with the A-rich loop (compare with Fig. 3). Indeed, cleavage in the anticodon loop was almost as pronounced as that in the D loop (Fig. 4). Interestingly, the cleavage pattern was not affected by heparin, indicating that HIV-1 RT does not interact with the anticodon loop of tRNA₃^Lys annealed to the mutated vRNA. Furthermore, the relative cleavage intensities at G-30 and in the anticodon and D loops are similar in registers 0, 3, and 6, both without and with heparin. Thus, with the mutant vRNA, the structure of the primer-template did not change during initiation of reverse transcription.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The efficiency and specificity of HIV-1 reverse transcription relies on intricate interactions between the viral RNA and primer tRNA₃^Lys (12, 13, 18, 19, 21, 22, 25-28, 35). Quite surprisingly, in HIV-1 the virus-specific intermolecular interactions (i.e. helices 6C, 5D, and 3E) involve viral sequences located upstream of the PBS (12, 13). Thus, they could potentially hinder reverse transcription and have to unwind as DNA synthesis proceeds (18, 19, 35). The present study shed light into the structural changes occurring within the reverse transcription complex during the initiation of DNA synthesis.

Interestingly, even though the interaction between the anticodon loop of tRNA₃^Lys and the A-rich loop is located 12-17 nucleotides upstream of the PBS, our probing of the vRNA revealed that this interaction progressively unwinds during the addition of the third to the sixth nucleotide to tRNA₃^Lys. Furthermore, detailed analysis indicates that the distal part of helix 6C, relative to the polymerization site, was cleaved before the proximal part; whereas A-164 and A-165 were cleaved in registers 3 and 6, A-166 and A-167 were only cleaved in the latter register. Thus, opening of helix 6C was unlikely due to an unwinding activity associated with RT. Such an activity was proposed on the basis of nuclease footprinting data, indicating that HIV-1 RT may interact with the template up to seven nucleotides upstream from the polymerization site (36). However, detailed kinetics studies suggest that disruption of the template secondary structure during reverse transcription is a passive process (37).

We propose that unwinding of helix 6C at a distance from the polymerization site is a consequence of the tertiary structure of the initiation complex (22). As the first three nucleotides are added to tRNA₃^Lys, the flexible single-stranded junction between helices 2 and 7F is copied by RT and, thus, is transformed into a rigid double-stranded RNA-DNA hybrid (Fig. 1). This flexible region is a crucial hinge that allows proper three-dimensional folding of the vRNA·tRNA₃^Lys complex (22). Its stiffening introduces topological stress in the initiation complex that might induce unfolding of helix 6C.

The progressive unwinding of helix 6C during the addition of the third to the sixth nucleotides might shed a new light on the finding that most annealed tRNA₃^Lys in extracellular viral particles is extended by two nucleotides (11, 38). This observation was difficult to explain on the basis of in vitro kinetics experiments, which showed a strong pausing site after the addition of the third nucleotide (18-20). Our results suggest that the intermolecular interactions might be stabilized in the viral particles and might block reverse transcription when low concentrations of nucleotides are available.

Translocation of RT on the PBS helix during initiation of reverse transcription was visualized by increased accessibility of helix 8 downstream of the PBS as DNA synthesis proceeded. In addition, changes in the RT footprint pattern were observed far upstream of the polymerization site. The most remarkable change was observed at A-155, which became protected by RT in registers 3 and 6. This results suggests that RT can interact with the template strand up to 20 nucleotides upstream of the polymerization site. This observation contrasts with earlier chemical (39) and enzymatic (36, 40) footprinting studies on model primer-template complexes, showing limited interactions between RT and the template upstream of the polymerization site. The difference between our and previous results undoubtedly originates from the complex tertiary structure of the initiation complex, which was absent from the model substrates.

The second partner of the complex, tRNA₃^Lys, also undergoes complex structural rearrangements during initiation of reverse transcription. Whereas cleavage of the anticodon loop gradually increased during the addition of the second and third nucleotide, in keeping with unwinding of helix 6C, its accessibility decreased in register 6. In any case, cleavage of the anticodon loop in register 6 was not affected by heparin, indicating that the protection observed in this register was not due to interaction with RT. The most likely explanation for the progressive protection of the anticodon loop as initiation of reverse transcription proceeded is that this loop became buried inside the vRNA·tRNA₃^Lys tertiary structure. Indeed, the only alternative candidate for a direct interaction with the anticodon loop is the A-rich sequence in loop 8 (Fig. 1). However, our probing data indicate that the accessibility of this stem-loop progressively increased during initiation of reverse transcription, indicating that it did not interact with tRNA₃^Lys.

We previously showed that mutations in the A-rich loop that prevented formation of helix 6C decreased the efficiency of the transition between initiation of reverse transcription (20). However, this transition takes place between addition of the sixth and seventh nucleotides (20, 21), and our probing data indicate that helix 6C does not exist anymore in register 6. Therefore, the effect of these mutations is quite unexpected. Probing of tRNA₃^Lys annealed to the mutated vRNA in registers 0, 1, 2, 3, and 6 brought clues to solve this apparent paradox. In contrast to the situation observed in the presence of wild type vRNA, the anticodon loop of the primer remained accessible at all stages of the initiation of reverse transcription. In register 6, the conformation of tRNA₃^Lys bound to the mutant template significantly differed from that of the primer bound to the wild type HIV-1 RNA. In other words, the conformation of the wild type initiation complex in register 6 depends on intermolecular interactions that were present in register 0 but do not exist in register 6 anymore. This particular conformation might be required for efficient transition from initiation to elongation of reverse transcription. Indeed, detailed studies showed that the specific intermolecular interactions of the initiation complex enhance formation of the vRNA·tRNA₃^Lys ·RT complex rather than the polymerization rate by the ternary complex (20, 21). Since nucleotide addition is distributive during initiation of reverse transcription (20, 21), the structural rearrangements of the wild type complex are probably crucial for efficient rebinding of RT to the partially extended tRNA₃^Lys.

The inability of RT to interact with the anticodon loop of tRNA₃^Lys bound to the mutant vRNA at any stages of the initiation of reverse transcription was rather unexpected. Indeed, HIV-1 RT is able to form a binary complex with its primer by interacting with the anticodon loop of tRNA₃^Lys (35-37). Thus, our present data indicate that the role of the interaction between the anticodon and A-rich loops is not to prevent binding of RT to the anticodon stem of tRNA₃^Lys. As suggested by our recent footprinting and modeling study (23), its function is most likely to ensure proper folding of the primer-template tertiary structure and, hence, to maximize specific interactions with RT while preventing steric clashes.

In summary, our probing data brought several unexpected results. First, they revealed an interplay between the tertiary structure of the vRNA·tRNA₃^Lys complex and RT at a distance from the polymerization site. The structure of the initiation complex allows RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modifies the tertiary structure of the vRNA·tRNA₃^Lys complex at 10-14 nucleotides from the catalytic site. Second, the fate of the anticodon loop and of the A-rich loop as they dissociate is different. Whereas the viral A-rich loop is exposed at the surface of the complex, the anticodon loop of tRNA₃^Lys becomes buried inside it. Finally, our data showed that correct base pairing of the A-rich loop with the anticodon loop in the initial complex is essential for the rearrangement of the complex during initiation of DNA synthesis. This structural rearrangement might be an attractive target for anti-HIV-1 molecules designed against the initiation complex of HIV-1 reverse transcription.

	ACKNOWLEDGEMENTS

Guillaume Bec is acknowledged for help in the tRNA purification, and Matthias Götte is acknowledged for stimulating discussions.

	FOOTNOTES

^* This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS) and by a Biomed II grant from the European Union.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.

^§ Present address: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School,1400 University Avenue, Madison, WI 53706.

To whom correspondence should be addressed: UPR 9002 du CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France. Tel.: 33 3 88 41 70 91; Fax: 33 3 88 60 22 18; E-mail: marquet@ibmc.u- strasbg.fr.

	ABBREVIATIONS

The abbreviations used are: RT, reverse transcriptase; PBS, primer binding site; HIV-1, human immunodeficiency virus; vRNA, viral RNA; d-, deoxy-; dd-, dideoxy-.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Ooms, D. Cupac, T. E. M. Abbink, H. Huthoff, and B. Berkhout The availability of the primer activation signal (PAS) affects the efficiency of HIV-1 reverse transcription initiation Nucleic Acids Res., March 12, 2007; 35(5): 1649 - 1659. [Abstract] [Full Text] [PDF]

				C. Dash, T. S. Fisher, V. R. Prasad, and S. F. J. Le Grice Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference J. Biol. Chem., September 22, 2006; 281(38): 27873 - 27881. [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]

				H. Huthoff, K. Bugala, J. Barciszewski, and B. Berkhout On the importance of the primer activation signal for initiation of tRNAlys3-primed reverse transcription of the HIV-1 RNA genome Nucleic Acids Res., September 1, 2003; 31(17): 5186 - 5194. [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]

				J. G. Julias, M. J. McWilliams, S. G. Sarafianos, E. Arnold, and S. H. Hughes Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo PNAS, July 9, 2002; 99(14): 9515 - 9520. [Abstract] [Full Text] [PDF]

				N. Beerens and B. Berkhout The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription J. Virol., March 1, 2002; 76(5): 2329 - 2339. [Abstract] [Full Text] [PDF]

				J. T. Miller, B. Ehresmann, U. Hubscher, and S. F. J. Le Grice A Novel Interaction of tRNALys,3 with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis J. Biol. Chem., July 13, 2001; 276(29): 27721 - 27730. [Abstract] [Full Text] [PDF]

				M. Lavigne, L. Polomack, and H. Buc Structures of Complexes Formed by HIV-1 Reverse Transcriptase at a Termination Site of DNA Synthesis J. Biol. Chem., August 10, 2001; 276(33): 31439 - 31448. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) 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 Lanchy, J.-M. Articles by Marquet, R. Search for Related Content PubMed PubMed Citation Articles by Lanchy, J.-M. Articles by Marquet, R. Social Bookmarking                      What's this?