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.     

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.     

Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.     

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.     

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg²⁺, but not by K⁺ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg²⁺ ions.     

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.     

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.     

Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging

The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5' region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.     

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.     

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.     

HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses

A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.     

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.     

Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site

We describe the crystal structures of the RNA dimerization initiation sites (DIS) of HIV-1 subtypes A and B. Both molecules adopt a hairpin conformation, with loop sequences consisting of 272-AGGUGCACA-280 and 272-AAGCGCGCA-280, respectively. This includes a six-base self-complementary stretch (underlined) that allows homodimerization through the formation of a loop-loop, or 'kissing-loop', complex. The DISs for the two sequences have identical conformations, and the two interacting hairpins show a perfect coaxial alignment. The conserved purines, A272 and R273, are stacked in a bulged-out conformation and symmetrically join the upward and downward strands of each hairpin by crossing the helix major groove. There is good agreement between these structures and previous results from chemical probing in solution, as well as with differences in magnesium dependence for dimerization. The overall shape of the kissing-loop complex is very similar to that of the previously determined subtype A DIS duplex form. Unexpectedly, the purine R273 is the only base seen at a different position and is responsible for the difference in topology between the two forms. We propose that the transition from kissing-loop duplex could occur by a recombination mechanism based on symmetrical chain cleavage at R273 of each hairpin and subsequent cross-religation, rather than by base-pair melting and subsequent reannealing.     

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.     

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.     

A human immunodeficiency virus type 1-infected individual with low viral load harbors a virus variant that exhibits an in vitro RNA dimerization defect

We investigated the in vitro RNA dimerization properties of the untranslated leader RNA derived from human immunodeficiency virus type 1 variants circulating in an individual with a low viral load and slow disease progression. The leader sequences of these viruses contain highly unusual polymorphisms within the dimerization initiation site (DIS): an insert that abolishes dimerization and a compensatory substitution. The dimerization of leader RNA from late stages of infection is further improved by additional mutations outside the DIS motif that facilitate a secondary structure switch from a dimerization-incompetent to a dimerization-competent RNA conformation.     

Dominant role of the 5' TAR bulge in dimerization of HIV-1 genomic RNA, but no evidence of TAR-TAR kissing during in vivo virus assembly

The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The ΔUCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than ΔUCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.