Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus

Retroviral virions each contain two identical genomic RNA strands that are stably but noncovalently joined in parallel near their 5' ends. For certain viruses, this dimerization has been shown to depend on a unique RNA stem-loop locus, called the dimer initiation site (DIS), that efficiently homodimerizes through a palindromic base sequence in its loop. Previous studies with Moloney murine leukemia virus (Mo-MuLV) identified two alternative DIS loci that can each independently support RNA dimerization in vitro but whose relative contributions are unknown. We now report that both of these loci contribute to the assembly of the Mo-MuLV dimer. Using targeted deletions, point mutagenesis, and antisense oligonucleotides, we found that each of the two stem-loops forms as predicted and contributes independently to dimerization in vitro through a mechanism involving autocomplementary interactions of its loop. Disruption of either DIS locus individually reduced both the yield and the thermal stability of the in vitro dimers, whereas disruption of both eliminated dimerization altogether. Similarly, the thermal stability of virion-derived dimers was impaired by deletion of both DIS elements, and point mutations in either element produced defects in viral replication that correlated with their effects on in vitro RNA dimerization. These findings support the view that in some retroviruses, dimer initiation and stability involve two or more closely linked DIS loci which together align the nascent dimer strands in parallel and in register.     

Structure and dimerization of HIV-1 kissing loop aptamers

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5' end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers' structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.     

Dimerization of the 3'UTR of bicoid mRNA involves a two-step mechanism.

The proper localization of bicoid (bcd) mRNA requires cis-acting signals within its 3' untranslated region (UTR) and trans-acting factors such as Staufen. Dimerization of bcd mRNA through intermolecular base-pairing between two complementary loops of domain III of the 3'UTR was proposed to be important for particle formation in the embryo. The participation in the dimerization process of each domain building the 3'UTR was evaluated by thermodynamic and kinetic analysis of various mutated and truncated RNAs. Although sequence complementarity between the two loops of domain III is required for initiating mRNA dimerization, the initial reversible loop-loop complex is converted rapidly into an almost irreversible complex. This conversion involves parts of RNA outside of domain III that promote initial recognition, and dimerization can be inhibited by sense or antisense oligonucleotides only before conversion has proceeded. Injection of the different bcd RNA variants into living Drosophila embryos shows that all elements that inhibit RNA dimerization in vitro prevent formation of localized particles containing Staufen. Particle formation appeared to be dependent on both mRNA dimerization and other element(s) in domains IV and V. Domain III of bcd mRNA could be substituted by heterologous dimerization motifs of different geometry. The resulting dimers were converted into stable forms, independently of the dimerization module used. Moreover, these chimeric RNAs were competent in forming localized particles and recruiting Staufen. The finding that the dimerization domain of bcd mRNA is interchangeable suggests that dimerization by itself, and not the precise geometry of the intermolecular interactions, is essential for the localization process. This suggests that the stabilizing interactions that are formed during the second step of the dimerization process might represent crucial elements for Staufen recognition and localization.     

Characteristic of the two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two copies of genomic RNA is a necessary step of retroviral replication. In the case of human immunodeficiency virus type 1 (HIV-1) the process is explored in many details. It is proved that conserved stem-loop structure is an essential element in RNA dimerization. Similar model of two-step dimerization mechanism can be considered for avian sarcoma and leukosis virus group (ASLV) in spite of the absence of homology between dimer initiation site (DIS) of ASLV and that of HIV-1. In this paper, short RNA fragments of two viruses: avian sarcoma virus CT-10 and avian leukosis virus HPRS-103 have been chosen in order to investigate the structural requirements of dimerization process and compare them to that of HIV-1. The rate of spontaneous transition from loose to tight dimer was studied as a function of stem length and temperature. Although both types of dimers were observed for both avian retroviruses chosen, fragments of CT-10 requires much higher RNA concentration to form loose dimer. In spite of identical sequence of the loops (5'-A-CUGCAG-3') avian sarcoma virus CT-10 RNA fragments dimerization was greatly impaired. The differences can be explained by deletion of adenine 271 in avian sarcoma virus CT-10 in the stem and by resulting shortening of the self-complementary loop.     

HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus

Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.     

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.     

The Role of Structural Elements in Dimerization of RNA Fragments in Avian Retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop–loop interaction of two hairpins, or kissing hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

Identification and visualization of the dimerization initiation site of the prototype lentivirus, maedi visna virus: a potential GACG tetraloop displays structural homology with the alpha- and gamma-retroviruses

Dimerization of retroviral genomic RNA is essential for efficient viral replication and is mediated by structural interactions between identical RNA motifs in the viral leader region. We have visualized, by electron microscopy, RNA dimers formed from the leader region of the prototype lentivirus, maedi visna virus. Characterization by in vitro assays of the domains responsible for this interaction has identified a 20 nucleotide sequence that functions as the core dimerization initiation site. This region is predicted to form a GACG tetraloop and therefore differs significantly from the kissing loop palindromes utilized to initiate dimerization in primate lentiviruses. The motif is strongly conserved across the ovine and caprine lentiviruses, implying a critical functional role. Furthermore, the proposed GACG tetraloop exhibits marked structural homology with similar structural motifs present in the leader regions of the alpha- and gamma-retroviruses, and the maedi visna virus dimer linkage region is capable of forming heterodimeric species with the Moloney murine leukemia virus Psi domain. This may be indicative of commonality of origin of the two viruses or convergent evolution.     

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.     

The role of structural elements in dimerization of RNA fragments in avian retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop-loop interaction of two hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

The SL1-SL2 (stem-loop) domain is the primary determinant for stability of the gamma retroviral genomic RNA dimer

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.     

The HIV-1(Lai) RNA dimerization. Thermodynamic parameters associated with the transition from the kissing complex to the extended dimer.

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5' ends. A region upstream from the splice donor comprising an auto-complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV-1(Lai) RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem-loop structure. It has already been shown that this stem-loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop-loop dimer or 'kissing complex' and an extended dimer. We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248-270 of the HIV-1(Lai) SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop-loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to - 29.4 +/- 5 kcal x mol-1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don't indicate a dissociation of the GCGCGC sequence engaged in the loop-loop interaction during the rearrangement of the loop-loop complex into the extended dimer. Our data suggest that the loop-loop interaction is maintained during the temperature dependent conformational change while the intramolecular base-pairing of the stems is disrupted and then reconstituted to form an intermolecular base-pairing leading to an extended dimer.     

Architecture of a gamma retroviral genomic RNA dimer

Retroviral genomes contain two sense-strand RNAs that are noncovalently linked at their 5' ends, forming a dimer. Establishing a structure for this dimer is an obligatory first step toward understanding the fundamental role of the dimeric RNA in retroviral biology. We developed a secondary structure model for the minimal dimerization active sequence (MiDAS) for the Moloney murine sarcoma virus in the final dimer state using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In this model, two self-complementary, or palindromic, sequences (PAL1 and PAL2) form extended intermolecular duplexes of 10 and 16 base pairs, respectively. The monomeric starting state was shown previously to contain a flexible domain in which nucleotides do not form stable interactions with other parts of the RNA. In the final dimer state, portions of this initial flexible domain form stable base pairs, while previously base-paired elements lie in a new flexible domain. Thus, partially overlapping and structurally well-defined flexible domains are prominent features of both monomer and dimer states. We then used hydroxyl radical cleavage experiments to characterize the global architecture of the dimer state. Extensive regions, including portions of both PAL1 and PAL2, are occluded from solvent-based cleavage indicating that the MiDAS domain does not function simply as a collection of autonomous secondary structure elements. Instead, the retroviral dimerization domain adopts a compact architecture characterized by close packing of its constituent helices.     

Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA.

Sequences from the 5' end of type 1 human immunodeficiency virus RNA dimerize spontaneously in vitro in a reaction thought to mimic the initial step of genomic dimerization in vivo. Dimer initiation has been proposed to occur through a "kissing-loop" interaction involving a specific RNA stem-loop element designated SL1: the RNA strands first interact by base pairing through a six-base GC-rich palindrome in the loop of SL1, whose stems then isomerize to form a longer interstrand duplex. We now report a mutational analysis aimed at defining the features of SL1 RNA sequence and secondary structure required for in vitro dimer formation. Our results confirm that mutations which destroy complementarity in the SL1 loop abolish homodimer formation, but that certain complementary loop mutants can heterodimerize. However, complementarity was not sufficient to ensure dimerization, even between GC-rich loops, implying that specific loop sequences may be needed to maintain a conformation that is competent for initial dimer contact; the central GC pair of the loop palindrome appeared critical in this regard, as did two or three A residues which normally flank the palindrome. Neither the four-base bulge normally found in the SL1 stem nor the specific sequence of the stem itself was essential for the interaction; however, the stem structure was required, because interstrand complementarity alone did not support dimer formation. Electron microscopic analysis indicated that the RNA dimers formed in vitro morphologically resembled those isolated previously from retroviral particles. These results fully support the kissing-loop model and may provide a framework for systematically manipulating genomic dimerization in type 1 human immunodeficiency virus virions.     

Solution RNA structures of the HIV-1 dimerization initiation site in the kissing-loop and extended-duplex dimers

Dimer formation of HIV-1 genomic RNA through its dimerization initiation site (DIS) is crucial to maintaining infectivity. Two types of dimers, the initially generated kissing-loop dimer and the subsequent product of the extended-duplex dimer, are formed in the stem-bulge-stem region with a loop including a self-complementary sequence. NMR chemical shift analysis of a 39mer RNA corresponding to DIS, DIS39, in the kissing-loop and extended-duplex dimers revealed that the three dimensional structures of the stem-bulge-stem region are extremely similar between the two types of dimers. Therefore, we designed two shorter RNA molecules, loop25 and bulge34, corresponding to the loop-stem region and the stem-bulge-stem region of DIS39, respectively. Based upon the chemical shift analysis, the conformation of the loop region of loop25 is identical to that of DIS39 for each of the two types of dimers. The conformation of bulge34 was also found to be the same as that of the corresponding region of DIS39. Thus, we determined the solution structures of loop25 in each of the two types of dimers as well as that of bulge34. Finally, the solution structures of DIS39 in the kissing-loop and extended-duplex dimers were determined by combining the parts of the structures. The solution structures of the two types of dimers were similar to each other in general with a difference found only in the A16 residue. The elucidation of the structures of DIS39 is important to understanding the molecular mechanism of the conformational dynamics of viral RNA molecules.     

Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5' noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.