©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dimerization of HIV-1 RNA at Low Ionic Strength
AN AUTOCOMPLEMENTARY SEQUENCE IN THE 5` LEADER REGION IS EVIDENCED BY AN ANTISENSE OLIGONUCLEOTIDE (*)

(Received for publication, December 7, 1994; and in revised form, January 25, 1995)

Delphine Muriaux (§) Pierre-Marie Girard Bénédicte Bonnet-Mathonière Jacques Paoletti (¶)

From the Unité de Biochimie-Enzymologie, URA 147 CNRS, Laboratoire de Pharmacologie et Physicochimie des Macromolécules, Rue Camille Desmoulins, Institut Gustave Roussy, 94805 Villejuif, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genomic human immunodeficiency virus type 1 (HIV-1) RNA consists of two identical RNA molecules joined noncovalently near their 5` ends in a region called the dimer linkage structure (DLS). Previous work has shown that the putative DLS is localized in a 113-nucleotide domain encompassing the 5` end of the gag gene. This region contains conserved purine tracks that are thought to mediate dimerization through purine quartets. However, recently, an HIV-1 RNA dimerization model was proposed as the HIV-1 RNA dimerization initiation site, involving another region upstream from the splice donor site and possibly confined within a stem-loop. In the present study, we have investigated the dimerization of HIV-1 RNA, using in vitro dimerization assays under conditions of low ionic strength, predictive RNA secondary structures determined by computer folding, and antisense DNA oligonucleotides in order to discriminate between these two models. Our results suggest that purine quartets are not involved in the dimer structure of HIV-1 RNA and have led to the identification of a region upstream from the splice donor site. This region, comprising an autocomplementary sequence in a possible stem-loop structure, is responsible for the formation of dimeric HIV-1 RNA.


INTRODUCTION

All retroviruses have a common feature, namely a genome consisting of two identical unspliced RNA molecules noncovalently linked(1) . It has been shown by electron microscopy that this junction is located close to their 5` ends, in a structure termed the dimer linkage structure(2, 3, 4) .

Dimerization of retroviral RNA is thought to play a crucial role in several steps of the retroviral life cycle. RNA dimerization seems to be closely related to the encapsidation of retroviral RNA, since cis-elements such as the dimer linkage structure (DLS) (^1)and the encapsidation site (E) are localized within the same region in the genome of MoMuLV(5) , RSV(6) , REV(7) , BLV(8) , and HIV-1(9) . It has also been suggested that dimerization of retroviral RNA is associated with reverse transcription through interstrand switching(10) , genomic recombination(11, 12, 13) , and down-regulated translation(6) .

Several attempts have been made to investigate factors responsible for the dimerization event. Reports describe a crucial role for a trans-acting factor, the HIV-1 nucleocapsid protein (NCp7) in the formation of RNA dimers(9, 14) . It has also been demonstrated that in the absence of any cellular or viral protein, HIV-1 RNA is able to spontaneously dimerize in vitro under conditions of high ionic strength(15, 16, 17, 18) . HIV-1 sequences able to support this spontaneous dimerization have been localized between nucleotides 311 and 415 encompassing the 5` end of the gag gene(9) . Published results indicate that (i) the HIV-1 RNA dimer is very stable, (ii) antisense RNA cannot form a dimer, and (iii) a consensus sequence, PuGGAPuA, is found in the putative dimerization-encapsidation region of HIV and other retroviral genomes(15) . Marquet et al.(15) and others (16, 17, 18) have suggested that the structural motif mediating the association of two identical viral RNA molecules should involve purine quartets. This model is based on the dimerization of telomeric DNA which occurs via the formation of unusual intermolecular quadruple helical structures that are stabilized by guanine base tetrads(19) .

However, dimerization of viral RNA by purine quartets has recently been discussed(20, 21, 22, 23) . First, Berkout et al.(21) reported that the HIV-2 mutant RNA, bereft of all PuGGAPuA sites, could dimerize in vitro. Second, Skripkin et al.(22) recently postulated that the linking of the two HIV-1 RNA molecules is initiated at the level of a short RNA region, located upstream from the splice donor site and consisting of a palindromic sequence in a stem-loop structure. However, this process requires high ionic strength and the presence of MgCl(2) in the electrophoresis gel in order to be observed(22, 23) . Similarly, based on an RNA-RNA recognition model, Girard et al.(^2)identified a possible stem-loop structure containing a palindromic sequence, which is probably responsible for the formation of dimeric MoMuLV RNA.

In this report, we analyze the spontaneous in vitro dimerization process of HIV-1 RNA, under conditions of low ionic strength, in an attempt to enhance our understanding of the nature of the interactions leading to dimerization and to distinguish between the above described dimerization models. We have characterized the dimer linkage structure by using HIV-1 RNA fragments of different lengths, complementary DNA oligonucleotides, and heterodimer formation.


EXPERIMENTAL PROCEDURES

Materials

BssHII, HaeIII, RsaI, SacI, and SmaI restriction enzymes were obtained from New England Biolabs and EcoRI, HindIII, and PstI restriction enzymes and T4 Polynucleotide Kinase from Boehringer Mannheim. Standard procedures and sequencing were used for restriction enzyme digestion and plasmid construction(25) . The Escherichia coli strain DH5alpha was used for plasmid manipulation and preparation. All the constructions were sequenced (United State Biochemical Corp. Kit 70770, Sequenase Version 2.0 DNA Sequencing Kit) in order to verify the exact sequence of the synthesized RNA fragment(26) . The constructed plasmids were transcribed, after appropriate linearization, with T7 RNA polymerase under conditions provided by the RiboMAX Large Scale RNA production System (Promega).

Plasmid Construction and Digestion

Derivatives of the plasmid pGEM-3zf (Promega) were constructed to generate viral RNA by in vitro transcription.

Plasmid pBRU2 (a gift of Dr. F. Subra) contains the HIV-1 cDNA in the pKP59 vector with XbaI-AatII as the cloning site. This plasmid has a deletion of 2701 base pairs from +5367 to +8061 to prevent it from being infectious.

Plasmid pDM2 and HIV-1 RNA Fragments from Nucleotides 224 to 402 and 224 to 296

The pBRU2 plasmid was digested with SacI (position +224) and PstI (position +960) to produce the viral fragment 224-960 which was inserted into pGEM-3zf (sites SacI/PstI) and resulted in pDM2. The pDM2 plasmid was digested either by HaeIII or RsaI and transcribed by T7 RNA polymerase giving rise to transcripts starting from position 224 of the genomic HIV-1 RNA sequence and ending at positions 402 and 296, respectively. These fragments will be referred to as fragments 224-402 and 224-296.

Plasmid pDM3 and HIV-1 RNA Fragments from Nucleotides 77 to 402 and 77 to 256

The pBRU2 plasmid was cleaved with HindIII, and the viral fragment from positions +77 to +631 was inserted into pGEM-3zf previously digested by HindIII and resulted in pDM3. The plasmid pDM3 was digested either by HaeIII or BssHII and transcribed by T7 RNA polymerase giving rise to transcripts starting from position 77 of the genomic HIV-1 RNA sequence and ending at positions 402 and 257, respectively. These fragments will be referred to as fragments 77-402 and 77-257.

Plasmid pDM6 and HIV-1 RNA Fragment from Nucleotides 296 to 402

The pDM3 plasmid was cleaved with RsaI and HindIII: the viral fragment from position 296 to 631 was isolated and inserted into pGEM-3zf previously digested with SmaI and HindIII, and resulted in pDM6. The pDM6 plasmid was digested by HaeIII and transcribed by T7 RNA polymerase giving rise to transcripts starting from position 296 of the genomic HIV-1 RNA sequence and ending at position 402. This fragment will be referred to as fragment 296-402.

Plasmid pDM7 and HIV-1 RNA Fragment 224-402 Lacking Nucleotides 257-266

Polymerase chain reactions (27) were performed on pDM2 with two sets of oligonucleotides. The first set contained the T7 universal primer and oligonucleotide D1, corresponding to the complementary positions +256(5`) to +236(3`) of the HIV-1 genome. The second set contained oligonucleotide D2 corresponding to positions +263(5`) to +280(3`) of the HIV-1 genome and the complementary sequence of the SP6 universal primer. The first polymerase chain reaction product was digested by the EcoRI enzyme and the second by PstI. Both DNA fragments obtained were purified by agarose gel electrophoresis. These two fragments were ligated at their blunt ends and inserted between EcoRI and PstI sites of pGEM-3zf(-). One clone of interest was recovered, pDM7, which contained a viral fragment from position 224 to 960 lacking nt 257-266 of the HIV-1 genome. The pDM7 plasmid, first digested by HaeIII, was transcribed with T7 RNA polymerase and gave rise to a RNA fragment 224-402 bereft of nt 257-266.

In Vitro RNA Synthesis and Purification

Under the conditions stipulated by the RiboMAX Large Scale RNA Production System, 5 µg of the linearized plasmids were transcribed with T7 RNA polymerase. The sample was subjected to three phenol chloroform extractions after DNase treatment, and the RNA transcripts were ethanol precipitated in the presence of 0.3 M sodium acetate, pH 5.2. The RNA precipitate was dissolved in double-distilled sterile water (20 µl) and microdialyzed (Millipore filters type V6, 0.025 µm) for 2 h against sterile double-distilled water. The purity and integrity of the RNA samples were checked by denaturing polyacrylamide gel electrophoresis. If the purity of the RNA was insufficient, the sample in water was loaded on 1.5% agarose gel and electrophoresed as described above. The corresponding RNA fragment was excised from the agarose gel and purified using a spin-X column (Costar). Ethidium bromide was extracted from the sample with one volume of butanol-1, and RNA was ethanol-precipitated. The concentration was determined by UV spectroscopic measurement at 260 nm.

RNA in Vitro Dimerization

RNA dimerization assays were performed on 0.5 µg of RNA generated in vitro, in a buffer containing 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, to a final volume of 10 µl, for 45-60 min at 37 °C. RNA in water was heated for 2 min at 90 °C, chilled on ice for 2 min before adding the buffer. Monomeric and dimeric RNA fragments were analyzed by 1.5% agarose gel electrophoresis at 5V/cm in a buffer containing 50 mM Tris-borate, pH 8.3, and 1 mM EDTA, at 4 °C. Ethidium bromide (0.2 µg/ml) was added to the buffer.

Tm Determination of Dimer Dissociation

After denaturation at 95 °C for 2 min in nuclease-free water, the RNA was incubated in a buffer containing 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, at a concentration of 1 µM (molecules), at 55 °C for 50 min for optimal dimerization. The resulting dimer was then microdialyzed (Millipore filters type V6, 0.025 µm) for 2 h at 4 °C against a buffer composed of 40 mM Tris-HCl, pH 7.5, 10 mM NaCl, and 1 mM EDTA. 10-µl aliquots of each sample were then incubated for 5 min at varying temperatures ranging from 20 to 70 °C and electrophoresed as described above. After fluorescent scanning of the gels, the percentage of dimer and monomer was estimated. The percentage of the dimer was defined as the area of the dimer peak divided by the sum of the areas of monomer and dimer peaks. The melting temperature (Tm) was estimated from the plot of the amount of the dimer as a function of temperature.

Antisense and Control DNA Oligonucleotides

Synthetic DNA oligonucleotides complementary to positions 251-267 (oligonucleotide 257B), 312-326 (oligonucleotide 320B), 322-336 (oligonucleotide 327B), 336-350 (oligonucleotide T), 360-374 (oligonucleotide 365B), and 377-391 (oligonucleotide 382B) of the HIV-1 sequence were made using an Applied Biosystem Synthesizer. Another oligonucleotide, 257 M, complementary to positions 268-284 of the HIV-1 sequence(29) , was used as a control oligonucleotide. It differs from 257B by four nucleotides.

All these oligonucleotides were loaded on an 18% polyacrylamide gel in a buffer containing 4 M urea, 50 mM Tris-borate, pH 8.3, and 1 mM EDTA, electrophoresed, and purified by excision from the gel by UV shadowing. DNA oligonucleotides were 5`-P-labeled with [P]ATP (Amersham, United Kingdom) and T4 polynucleotide kinase. The specific radioactivity was about 10^7 cpm/µg DNA oligonucleotide. RNA 224-402 was heat denatured in the presence of the [P]DNA oligonucleotide before starting in vitro dimerization process. [P]DNA oligonucleotidebulletRNA complexes were analyzed by agarose gel electrophoresis. The level of hybridization of the [P]DNA oligonucleotide to monomer and dimer RNA 224-402 was detected by autoradiography of the corresponding gel.

Determination of RNA Secondary Structure by Computer Folding

Free energy minimization predictions were made using PCFOLD software (version 4.0) written by M. Zucker in accordance with parameters for the prediction of RNA structures described by Turner et al.(28) . The 5` sequence of HIV-1 has been reviewed (29) and, in an attempt to determine the secondary structure of our RNA fragments, sequences corresponding to these fragments were folded.


RESULTS

Spontaneous Dimerization of HIV-1 RNA in Vitro

To obtain a more detailed characterization of the DLS and the RNA dimerization process of HIV-1, a RNA fragment spanning nucleotides 77-402, which contains the leader region (without the TAR domain) and the 5` gag sequence (Fig. 1a), was prepared by in vitro transcription and analyzed by agarose gel electrophoresis. After heat denaturation, the RNA is in a monomeric form (Fig. 1c, lane 1), while upon incubation at 37 °C in a buffer composed of 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, dimeric RNA appears (Fig. 1c, lane 2). As reported(30) , this spontaneous dimerization is specific to retroviral RNA. Similarly, we produced two more RNA fragments: RNA 77-257 and RNA 224-402 (Fig. 1b, RNAs 2 and 3). RNA 77-257, shortened at the 3` end, lost its ability to dimerize (Fig. 1c, lanes 3 and 4), while RNA 224-402, shortened at the 5` end, dimerized efficiently (lanes 5 and 6). We obtained up to 80% of the dimer under conditions of low ionic strength. We therefore used the latter RNA as the reference fragment for the remaining experiments.


Figure 1: Mapping of the sequences required for HIV-1 RNA dimerization by deletion mutagenesis. a, representation of the 5` end of HIV-1 DNA. Numbering is relative to the genomic RNA cap site (+1).The restriction sites of interest are indicated: HindIII (+77), SacI (+224), and RsaI (+296). PBS and ATG indicate, respectively, the primer tRNA-binding site and initiation codon for Pr55gag synthesis. b, HIV-1 RNAs generated in vitro and used in this study. RNAs were generated in vitro by transcription of pDM2, pDM3, pDM6, and pDM7 linearized with the appropriate enzyme. RNA 1 and 2 begin at position +77, RNAs 3-5 at position +224, and RNA 6 at position +296. The deletion in RNA 5, between nt 257-266 is represented by the broken line. The column of symbols, headed RNA Dimer, indicates the level of dimeric RNA. -, 0-5%; ±, 10-15%; +++, 70-90% (means value from at least three experiments). c, agarose gel electrophoresis of HIV-1 RNAs. For each sample, the RNAs are numbered as in b. Heat-denatured RNAs (M) are shown in lanes 1, 3, 5, 7, 9, and 11, and RNAs (D) in dimerization conditions are shown in lanes 2, 4, 6, 8, 10, and 12. Lane MK, 0.16-1.77-kb RNA ladder (Life Technologies, Inc.).



Thermal Stability of the HIV-1 RNA Dimer

Thermal stability of the HIV-1 70 S genomic RNA dimer has been previously determined: the melting temperature of HIV-1 genomic RNA purified from HIV-1 virions was around 50 °C(9, 31) . For comparison, we studied the thermal denaturation of the RNA 224-402 under the same experimental conditions. Once formed at 55 °C in a buffer containing 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, the dimer was subjected to thermal denaturation in a buffer containing 40 mM Tris-HCl, pH 7.5, 10 mM NaCl, and 1 mM EDTA(9) . The results obtained after thermal denaturation of the RNA 224-402 dimer are shown on agarose gel (Fig. 2) together with the denaturation curve representing the relative percentage of the dimer as a function of temperature. Thermal transition from the dimer to the monomer occurred at 53 °C. The same Tm value was obtained for dimer RNA 77-402 (data not shown). This Tm value is close to that of entire HIV-1 viral RNA(9, 31) . The correspondence between these two Tm values suggests that the nature of the interactions between the in vitro generated HIV-1 RNA fragments is comparable to that of the 70 S natural dimer.


Figure 2: Thermal stability of the dimer RNA 224-402 as a function of temperature. Samples were analyzed by 1.5% agarose gel electrophoresis and the percent of each species determined from the gel. The temperatures on the plot correspond to the temperatures shown above the gel.



The same behavior observed for RNA 77-402 and 224-402 justified our choice of RNA 224-402 as the reference fragment for the following studies.

Computer-assisted Analysis of HIV-1 RNA 224-402 Folding

To further define the precise sequence involved in HIV-1 dimerization, we applied free energy minimization computer analysis to examine the RNA 224-402 secondary structure. As shown in Fig. 3, the predicted structure appears to be in the form of three stem-loops branched together. Stem-loops II and III, consisting of nt 296-402, correspond to the putative DLS 311-415 of HIV-1 previously described by Darlix et al.(9) , and contain three purine tracks. The remaining region, spanning nucleotides 224-296, exhibits an autocomplementary stem-loop (loop I in Fig. 3). Based on the HIV-1 dimerization initiation site recently described by Skripkin et al.(22) , we speculate that the presence of this autocomplementary GCGCGC sequence in loop I could be responsible for the recognition of two identical RNA molecules through a loop-loop interaction. Only one autocomplementary loop was found in the HIV-1 RNA region investigated.


Figure 3: RNA secondary structure predicted for HIV-1 RNA 224-402 by computer-assisted energy minimization analysis. The full lines represent the antisense DNA oligonucleotides, used in this work, which are complementary to the RNA sequences covered.



It is noteworthy that our computed model is in accordance with the one proposed by G. P. Harrison for HIV-1(32) which comprises this stem-loop with the same primary sequence.

Mapping of the Sequence Involved in the Formation of the HIV-1 RNA Dimer using Complementary DNA Oligonucleotides

To test the validity of this assumption, suggested by computer-assisted analysis of RNA 224-402 folding, we hybridized a set of synthetic DNA oligonucleotides to RNA 224-402. The DNA oligonucleotides chosen were complementary to small sequences of RNA 224-402 (Fig. 3). The selected oligonucleotides (see ``Experimental Procedures'') were 5`-Plabeled and heat denatured with RNA 224-402 before initiating the dimerization process.

The first question to be addressed was: is the autocomplementary stem-loop I, which encompasses nt 251-266, involved in the dimerization of HIV-1 RNA?

We studied the dimerization process of RNA 224-402 when incubated with increasing concentrations of oligonucleotide 257B. As shown in Fig. 4a, antisense oligonucleotide 257B, which targets the autocomplementary sequence 257-262, completely blocks dimerization. Total inhibition of dimerization is observed for RNA 224-402 when the oligonucleotide 257B ratio was equal to 1:1. The affinity constant can be estimated to be 0.1 to 1 µM of the antisense DNA oligonucleotide 257B for the RNA 224-402 target (for a strand concentration of 1 µM). Autoradiogram (Fig. 4b) shows that oligonucleotide 257B only hybridizes to RNA 224-402 in its monomeric form.


Figure 4: Inhibition of HIV-1 RNA 224-402 dimer formation in the presence of increasing oligonucleotide 257B concentrations. Fixed concentrations of RNA 224-402 (0.5 µg of heat denatured RNA/assay) were incubated in a buffer with 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl, for 45-60 min at 37 °C, in the presence of (lanes 2-11) 0, 0.05, 0.075, 0.1, 0.5, 0.75, 1, 2, 5, and 7 molar equivalent(s) of the [P]DNA oligomer 257B, complementary to nucleotides 256-336. Lane 1 shows the monomeric form of HIV-1 RNA 224-402 (heat denatured for 5 min at 95 °C), lane MK, as in Fig. 1. m and d indicate monomeric and dimeric RNAs, respectively. a, 1.5% agarose gel electrophoresis. The samples are visualized by ethidium bromide staining. b, autoradiogram of a.



Two antisense DNA oligonucleotides were used as controls. Oligonucleotide 257 M, differing from oligonucleotide 257B by four nucleotides in its sequence, is unable to prevent the dimer formation of RNA 224-402 (Fig. 5A). It converts 50% of the dimer into the monomer when its concentration is 5-fold higher. The other control oligonucleotide T (see ``Experimental Procedures'') does not inhibit the RNA 224-402 dimerization process, even at high concentrations (Fig. 5B). It should be noted that oligonucleotide 257M is not bound to the RNA fragment since it migrates as a free oligonucleotide in the gel. Consequently, it did not anneal RNA 224-402 because of the presence of its four mutated nucleotides. Such was not the case with oligonucleotide T which hybridizes to the RNA target 224-402 (in Fig. 5B a difference can be observed in dimer shift mobility in the gel when oligonucleotide T concentrations increase).


Figure 5: Analysis of HIV-1 RNA 224-402 dimer formation in the presence of oligonucleotides 257 M (A) and T (B) as controls. As described in Fig. 4, fixed concentrations of RNA 224-402 were incubated in the presence of (lanes 2-8) 0, 0.05, 0.1, 0.5, 1, 2, and 5 molar equivalent(s) of the DNA oligonucleotide 257M or T. Lane 1, monomeric form of HIV-1 RNA 224-402. Lane MK, as in Fig. 1.



The second question was: Were antisense DNA oligonucleotides 320B, 327B, 365B, and 382B, which are complementary to polypurine tracks, able to interfere with dimer formation?

Oligonucleotides 327B, 365B, and 382B are complementary to PuGGAPuA sequences, and oligonucleotide 320B targets a GGAGG sequence which was proposed by Sundquist and Heaphy (16) as an attractive candidate for a purine-rich region involved in dimerization. As shown in Fig. 6A, neither completely inhibited RNA 224-402 dimer formation, and oligonucleotide 327B never reduced the amount of dimer by more than 50%. In every case, each [P]DNA oligonucleotide annealed to both monomer and dimer RNA 224-402 (see autoradiographies, Fig. 6A).


Figure 6: Analysis of HIV-1 RNA 224-402 dimer formation in the presence of each oligonucleotide 320B, 327B, 365B, and 382B (A) or the four together (B). As described in Fig. 4, fixed concentrations of RNA 224-402 were incubated in the presence of (lanes 1-6) 0, 0.1, 0.5, 1, 2, and 5 molar equivalent of each [P]DNA oligonucleotide (A) or in the presence of the four together, each of them at a molar equivalent of RNA (B). Oligonucleotides 320B, 327B, 365B, and 382B which correspond, respectively, to nucleotides 312-326, 322-336, 360-374, and 377-391 of the HIV-1 sequence, are indicated in A. Lane M shows monomeric form of HIV-1 RNA 224-402. Monomer (m), dimer (d) of RNA 224-402, and free DNA oligonucleotides are indicated. The results are visualized on 1.5% agarose gel electrophoresis by ethidium bromide staining (gels above) and autoradiograms (views below).



Each RNA 224-402 molecule contains four polypurine sequences. We postulate that any of these sequences are able to interact indifferently with any of those in a second RNA 224-402 molecule. We therefore incubated the four oligonucleotides 320B, 327B, 365B, and 382B together with the RNA 224-402 (Fig. 6B) and found that dimerization still occurred. [P]DNA oligonucleotides marked monomeric and dimeric forms of the RNA 224-402 equally (see autoradiogram, Fig. 6B).

A New Cis-acting Element Required for HIV-1 RNA Dimerization in Vitro

Based on the results of DNA oligonucleotide mapping, we synthetized three new shorter HIV-1 RNA fragments, derived from RNA 224-402: RNA 224-296, which only contains the autocomplementary stem-loop I, RNA 296-402, which contains the four polypurine tracks mentioned above, and RNA 224-402DEL bereft of the GCGCGCACGG sequence (Fig. 1b, RNAs 4-6). We analyzed their ability to dimerize spontaneously in vitro (Fig. 1c). We found that RNA 224-296 dimerized very efficiently (Fig. 1c, lanes 7 and 8), while RNA 296-402 was unable to dimerize (lanes 11 and 12) under the salt conditions used to study the dimerization process(30) .^2

To confirm the role of the autocomplementary GCGCGC sequence in HIV-1 dimerization, nucleotides 257-266 were deleted from RNA 224-402. This deleted RNA was analyzed by agarose gel electrophoresis to estimate the degree of dimerization (Fig. 1b, RNA 5). RNA 224-402DEL lost the capacity to form dimeric RNA (Fig. 1c, lanes 9 and 10) while RNA 224-402 formed up to 80% of the dimer under the same experimental conditions (Fig. 1c, lane 6). The 10-nucleotide deletion did not alter the overall predicted secondary structure of the molecule in spite of a modification in the primary sequence of the stem-loop which was no longer autocomplementary (Fig. 8c). Furthermore, it is noteworthy that RNA 77-257 corresponds to an RNA molecule spliced in loop I: it had lost the capacity to dimerize efficiently (Fig. 1, b and c, lanes 3 and 4).


Figure 8: Model of HIV-1 dimerization process. a, predicted structure of stem-loop I, encompassing nucleotides 257-262, implicated in the recognition of the two HIV-1 RNA molecules. This structure was determined on RNAs 77-402, 224-402, and 224-296 using PCFOLD software(28) . b, formation of a double-stranded helix by the opening of the predicted stem-loop structure. Nucleotides spanning 240 through 280 are indicated. c, predicted structure of the stem-loop lacking nucleotides 257-266 which contain the GCGCGC sequence.



We therefore conclude that the 257-266 sequence is involved in the formation of dimeric HIV-1 RNA.

Analysis of Heterodimer Formation of HIV-1 RNAs

Finally, to determine whether dimerization of HIV-1 RNA 77-402 and RNAs 224-402 and 224-296 occurred via the same mechanism, we tried to form heterodimers containing one HIV-1 RNA 77-402 molecule and one molecule of the shorter HIV-1 RNAs. When an in vitro dimerization reaction was performed with RNA 77-402 and RNA 224-402, or RNA 224-296, we were able to detect heterodimers. The new band between the monomeric and dimeric forms of RNA 77-402 was clearly visible (Fig. 7, a and b, lanes HD). By contrast, no intermediate band could be detected with RNA 296-402 (Fig. 7c, lane HD) or RNA 224-402DEL (Fig. 7d, lane HD): indeed no heterodimer was formed.


Figure 7: Analysis on agarose gel electrophoresis of HIV-1 RNA heterodimers between RNA 77-402 and RNAs 224-402 (a), 224-296 (b), 224-402DEL lacking nt 257-266 (c) and 296-402 (d). Lanes M show heat-denatured RNA 77-402 and lanes D show RNA 77-402 in dimerization conditions. Coincubations of the same amount of each of the different HIV-1 RNAs and RNA 77-402 in dimerization conditions are shown in lanes HD. Monomer (m) and dimer (d) of the different RNA fragments are indicated. Numbering corresponds to that given for RNA fragments in Fig. 1b.



Thus, the mechanism of HIV-1 RNA dimerization is most probably common to RNAs of different sizes, only if they contain the 257-266 sequence. The heterodimers have a dimerization region similar to that of the homodimers.


DISCUSSION

We present here data on the in vitro dimerization of HIV-1 RNA transcripts (strain Lai) under conditions of low ionic strength. As shown in Fig. 1, HIV-1 RNA 77-402, a 5` leader RNA fragment, can efficiently dimerize in vitro. This RNA does not contain the TAR domain but, according to Berkout et al.(21) , who has implicated TAR inverted sequences in the dimerization process, this domain should not play any role. Likewise, shorter RNAs 224-402 and 224-296 can dimerize up to 80-90% in low ionic strength buffer whereas RNAs 77-257 and 296-402 cannot. RNA 224-402 was chosen as the reference fragment for the study. Computer folding analysis of RNA 224-402 showed that an autocomplementary sequence could be a part of a stem-loop structure (loop I in Fig. 3). When this sequence is deleted from this region, the RNA (224-402DEL) is unable to dimerize (Fig. 1) although the predicted stem-loop structure may be conserved (Fig. 8c). Furthermore, and consistent with this finding, a DNA oligonucleotide, complementary to this region, totally inhibits the HIV-1 dimerization process (Fig. 4), whereas control oligonucleotides 257M and T do not (Fig. 5).

A hypothetical mechanism for RNA dimerization, based on an extensive comparison of sequences in the leader regions of 30 retroviral genomes, has been proposed(15) . This comparative analysis suggested that the consensus sequence PuGGAPuA participates in the dimerization process through the formation of purine quartets involving both adenine(s) and guanine(s). As HIV-1 RNA 224-402 contains three such sites as well as the GGAGG sequence described by Sundquist and Heaphy (16) , we tested the role of these elements in the dimerization process. Surprisingly, RNA 224-296, derived from RNA 224-402 but totally bereft of purine consensus sites, was able to dimerize spontaneously in vitro with the same efficiency (Fig. 1). Furthermore, this RNA 224-296 was able to form a heterodimer with the leader RNA 77-402. RNA 224-296 therefore acts as an antisense RNA since we observed more heterodimer than the homodimer 77-402 (Fig. 7b). The remaining RNA region 296-402, which corresponds to the DLS 311-415 of HIV-1, previously shown to support the necessary sequence for HIV-1 dimerization(9) , was neither able to dimerize in vitro under conditions of low ionic strength (Fig. 1), nor to form a heterodimer with RNA 77-402 (Fig. 7d). We therefore failed to inhibit the formation of RNA dimer 224-402 in the presence of the antisense DNA oligonucleotides 320B, 327B, 365B, and 382B (Fig. 6) which target the purine tracks. We observed that the oligonucleotide 327B reduced the amount of the dimer by only 50% (Fig. 6A). The partial inhibition of dimerization by the oligonucleotide 327B probably implicates another region that may participate, to a certain extent, in the stabilization of the final structure of the dimer. However, the total inhibition observed with oligonucleotide 257B indicates that this stabilization, in itself, is not sufficient enough to lead to the formation of the dimer.

Our results suggest that a recognition mechanism between the two identical RNA molecules is operating via a loop-loop interaction through nucleotides GCGCGC in loop I (Fig. 3). We postulate that a transient complex is formed between complementary nucleotides in loops. The opening of both stem-loops during the interaction could lead to a double-stranded region via Watson-Crick base pairing, as shown in Fig. 8, a and b. This mechanism is proposed in the light of two similar mechanisms described for (i) the formation of a duplex between RNA I and RNA II from plasmid ColE1 (33) and (ii) the antisense RNA CopA and its target RNA CopT in the replication of plasmid R1(34) .

Such a mechanism for the HIV-1 DLS signal is in good agreement with recent analytical studies on HIV-1 RNA dimerization(22) , except for a difference observed by us in dimer stability. This difference could be attributed to a sequence modification when it passes from HIV-1 to HIV-1 RNA (5` . . . GAAGCGCGCACGG . . . 3` to 5` . . . GAGGUGCACACAG . . . 3`). The HIV-1 RNA 224-402 dimer is strongly stabilized through the GC residues present in the dimerization sequence (Fig. 8). It dissociates into the monomer at 53 °C (Fig. 2). This Tm value is in good agreement with that found by Darlix et al.(9) with the full-length HIV-1 viral RNA isolated from wild-type virus particles.

The nucleotides GCGCGC are remarkably conserved in 18 of the 21 HIV-1 strains observed(29) . Interestingly, mutations observed in the stem-loop of various HIV-1 strains were offset so that the structure and the autocomplementary sequence were maintained (except for HIVHXB2 strain).

A potential link between dimerization and encapsidation of HIV-1 genomic RNA has been proposed(7, 9, 31) . Kim et al.(35) have recently constructed several recombinant HIV-1 proviral DNA clones. One of them, lacking 13 bases upstream from the splice donor site, produced a virus with less efficient packaging of its genomic RNA than a virus bearing mutations between the 5` splice donor site and the gag gene. This deletion encompasses nucleotides 241-253 which are a part of stem-loop I shown in Fig. 3and Fig. 8a. Understanding the structures present in dimeric retroviral RNAs is therefore an essential prerequisite for antiviral strategies. We therefore propose stem-loop I as a potential target in attempts to interfere with HIV-1 replication. Since one of the current antisense strategies is based on targeting oligonucleotides to complementary sequences of an RNA molecule, oligonucleotide 257B is proposed for this function. HIV-1 RNA is totally inhibited in vitro, under conditions akin to physiological ones, by this antisense DNA oligonucleotide complementary to the putative stem-loop I which contains the autocomplementary GCGCGC sequence.


FOOTNOTES

*
This work is dedicated to the memory of Claude Paoletti.

* This work was supported by the Agence Nationale de la Recherche sur le SIDA (ANRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Student of the Institut de Formation Supérieure Biomédicale (IFSBM) and supported by a Synthelabo fellowship.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: DLS, dimer linkage structure; HIV-1 or or , human immunodeficiency virus type 1 (strains Lai or Mal or NL43); MoMuLV, Moloney murine leukemia virus; RSV, Rous sarcoma virus; BLV, bovine leukemia virus; REV, reticuloendotheliosis virus; TAR, trans-activation response element; nt, nucleotide.

(^2)
P.-M. Girard, B. Bonnet-Mathonière, D. Muriaux, and J. Paoletti, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Frédéric Subra (IGR, Villejuif) for the gift of plasmid pBRU2, Philippe Fossé (IGR, Villejuif) for helpful discussions, and Filippo Rusconi di Lugano (CNRS, Gif-sur-Yvette) and Lorna St-Ange (IGR, Villejuif) for critical reading of the manuscript.

Addendum-While this manuscript was reviewed, Laughrea et al.(24) have speculated that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA. Our results and theirs are complementary and mutually supportive: we reached the same postulated dimerization model of HIV-1 RNA.


REFERENCES

  1. Coffin, J. M. (1984) in RNA Tumor Viruses (Weiss, R., Teich, N., Varmus, H., and Coffin, G., eds) Vol. 1, pp. 261-368, Cold Spring Harbor, Cold Spring Harbor, NY
  2. Kung, H. J., Hu, S., Bender, W., Bailey, J. M., Davidson, N., Nicolson, M. O., and Mc Allister, R. M. (1976) Cell 7, 609-620 [Medline] [Order article via Infotrieve]
  3. Bender, W., Chien, Y. H., Chattopadkyay, S., Vogt, P. K., Gardner, M. R., and Davidson, N. (1978) J. Virol. 25, 888-896 [Medline] [Order article via Infotrieve]
  4. Murti, K. G, Bondurant, M., and Tereba, A. (1981) J. Virol. 37, 411-419 [Medline] [Order article via Infotrieve]
  5. Prats, A. C., Roy, C., Wang, P., Erard, M., Housset, V., Gabus, C., Paoletti, C., and Darlix, J. L. (1990) J. Virol. 64, 774-783 [Medline] [Order article via Infotrieve]
  6. Bieth, E., Garbus, C., and Darlix, J. L. (1990) Nucleic Acids Res. 18, 119-127 [Abstract]
  7. Darlix, J.-L., Gabus, C., and Allain, B. (1992) J. Virol. 66, 7245-7252 [Abstract]
  8. Katoh, I., Yasunaga, T., and Yshinaka, Y. (1993) J. Virol. 67, 1830-1839 [Abstract]
  9. Darlix, J. L., Gabus, C., Nugeyre, M. T., Clavel, F., and Barré-Sinoussi, F. (1990) J. Mol. Biol. 216, 689-699 [Medline] [Order article via Infotrieve]
  10. Panganibam, A., and Fiore, D. (1988) Science 241, 1064-1069 [Medline] [Order article via Infotrieve]
  11. Hu, W. S., and Temin, H. M. (1990) Proc. Natl. Acad. Sci. U. S. A 87, 1556-1560 [Abstract]
  12. Temin, H. M. (1991) Trends Genet. 7, 71-74 [Medline] [Order article via Infotrieve]
  13. Stuhlmann, H., and Berg, P. (1992) J. Virol. 66, 2378-2388 [Abstract]
  14. Weiss, S., Köning, B., Morikawa, Y., and Jones, I. (1992) Gene (Amst.) 121, 203-212 [Medline] [Order article via Infotrieve]
  15. Marquet, R., Baudin, F., Gabus, C., Darlix, J. L., Mougel, M., Ehresmann, C., and Ehresmann, B. (1991) Nucleic Acids Res. 18, 2349-2357
  16. Sundquist, W., and Heaphy, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3393-3397 [Abstract]
  17. Awang, G., and Sen, D. (1993) Biochemistry 32, 11453-11457 [Medline] [Order article via Infotrieve]
  18. Weiss, S, Häusl, G, Famulok, M., and König, B. (1993) Nucleic Acids Res. 21, 4879-4885 [Abstract]
  19. Williamson, J. R., Raghuraman, M. K., and Cech, T. R. (1989) Cell 59, 871-880 [Medline] [Order article via Infotrieve]
  20. Sakagushi, K., Zambrano, N., Baldwin, E. T., Shapiro, B. A., Erickson, J. E., Omichinski, J. G., Clore, G. M., Gronenborn, A. M., and Appela, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5219-5223 [Abstract]
  21. Berkout, B., Oude Essink, B. B., and Schoneveld I. (1993) FASEB J. 7, 181-187 [Abstract/Free Full Text]
  22. Skripkin, E., Paillart, J. C., Marquet, R., Ehresmann, B., and Ehresmann, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4945-4949 [Abstract]
  23. Marquet, R., Paillart, J. C., Skripkin, E., Ehresmann, C., and Ehresmann, B. (1994) Nucleic Acids Res. 22, 145-151 [Abstract]
  24. Laughrea, M., and Jetté, L. (1994) Biochemistry 33, 13464-13474 [Medline] [Order article via Infotrieve]
  25. Maniatis, T., Fritch, E. F., and Sambrock, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor University Press, Cold Spring Harbor, NY
  26. Haseltine, W. A., Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 989-993 [Abstract]
  27. Milligan, J. F., Groebe, D., Whiterell, G., and Uhlenbeck, O. (1987) Nucleic Acids Res. 15, 8783-8799 [Abstract]
  28. Turner, D. H., Sugimoto, N., Jaeger, J. A., Longfellow, C. E., Freier, S. M., and Kierzek, R. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 123-133 [Medline] [Order article via Infotrieve]
  29. Myers, G., Korber, B., Berzofsky, J. A., Smith, R. F., and Pavlakis, G. N. (eds) (1992) Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences , Los Alamos National Laboratory, Los Alamos, NM
  30. Roy, C., Tounekti, N., Mougel, M., Darlix, J. L., Paoletti, C., Ehresmann, C., Ehresmann, B., and Paoletti, J. (1990) Nucleic Acids Res. 18, 7287-7292 [Abstract]
  31. Fu, W., Gorelick, R. J., and Rein, A. (1994) J. Virol. 68, 5013-5018 [Abstract]
  32. Harrison, G. P., and Lever, A. M. L. (1992) J. Virol. 66, 4144-4153 [Abstract]
  33. Eguchi, Y., Itoh, T., and Tomizawa, J. I. (1991) Annu. Rev. Biochem. 60, 631-652 [CrossRef][Medline] [Order article via Infotrieve]
  34. Persson, C., Gerhart, E., Wagner, H., and Nordström (1990) EMBO J. 9, 3767-3775 [Abstract]
  35. Kim, H.-J., Lee, K., and O'Rear, J. J. (1994) Virology 198, 336-340 [CrossRef][Medline] [Order article via Infotrieve]

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