RNA Dimerization Research Articles

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7–p1–p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7–p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1–p6) and tertiary (p7–p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7–p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.

Noncovalent probes for the investigation of structure and dynamics of protein‐nucleic acid assemblies: The case of NC‐mediated dimerization of genomic RNA in HIV‐1

The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as noncovalent probes. Small-molecule ligands make contact with their target substrates through complex combinations of H-bonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies. In the case of SL1, dimerization and isomerization create unique structural features capable of sustaining stable interactions with classic nucleic acid ligands. The binding modes exhibited by intercalators and minor groove binders were adversely affected by the significant distortion of the duplex formed by palindrome annealing in the kissing-loop (KL) dimer, whereas the modes observed for the corresponding extended duplex (ED) confirmed a more regular helical structure. Consistent with the ability to establish electrostatic interactions with highly negative pockets typical of helix anomalies, polycationic aminoglycosides bound to the stem-bulge motif conserved in all SL1 conformers, to the unpaired nucleotides located at the hinge between kissing hairpins in KL, and to the exposed bases flanking the palindrome duplex in ED. The patterns afforded by intercalators and minor groove binders did not display detectable variations when the corresponding NC-SL1 complexes were submitted to probing. In contrast, aminoglycosides displayed the ability to compete with the protein for overlapping sites, producing opposite effects on the isomerization process. Indeed, displacing NC from the stem-bulges of the KL dimer induced inhibition of stem melting and decreased the efficiency of isomerization. Competition for the hinge region, instead, eliminated the NC stabilization of a grip motif formed by nucleobases of opposite strands, thus facilitating the strand-exchange required for isomerization. These noncovalent probes provided further evidence that the structural context of the actual binding sites has significant influence on the chaperone activities of NC, which should be taken in account when developing potential drug candidates aimed at disrupting genome dimerization and isomerization in HIV-1.

Synthesis of RNA Dimer and Trimer Blocks and Their Uses

Since RNA interference was first observed in plants, worms, and then mammalian cells, siRNA becomes a potent therapeutic approach to cure diseases. However, the synthesis of RNA oligonucleotide usually gives lower purities and yields than DNA oligonucleotides due to 2′- hydroxyl group. To be an affordable medicine, many approaches for higher purities and yields have been attempted in RNA oligoncucleotide synthesis. New RNA phosphoramidites and other types of monomer, solution phase synthesis, hybrid of solid-solution phase synthesis, and novel supports are among them. We have developed an efficient method to synthesize siRNA using RNA dinucleotide blocks in solid phase synthesis. Various RNA dimer phosphoramidites were synthesized and applied for siRNA synthesis giving the highly pure RNA oligonucleotides comparable to DNA oligonucleotides. The RNA trimer blocks were also synthesized and its application will be discussed as well.

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag–Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90), most of the Gag and Gag–Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.

Regulation of primate lentiviral RNA dimerization by structural entrapment

BackgroundGenomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1) element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1. To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, here we compare the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Prior studies from several groups have employed a variety of constructs and experimental conditions.ResultsAlthough some idiosyncratic differences in the dimerization details were observed, we find unifying principles in the regulation strategies of the three viral RNAs through long- and short-range base pairing interactions. Presentation and efficacy of dimerization through SL1 depends strongly upon the formation or dissolution of the lower stem of SL1 called stem B. SL1 usage may also be down-regulated by long-range interactions involving sequences between SL1 and the first codons of the gag gene.ConclusionDespite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1.

2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.

Binding of Aminoglycoside Antibiotics to the Duplex Form of the HIV‐1 Genomic RNA Dimerization Initiation Site

Targeting HIV RNA: High-resolution X-ray structures of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex bound to several aminoglycoside antibiotics have been obtained (see ribostamycin complex as an example). They show the feasibility of targeting viral RNA with small drugs, and open up the possibility of structure-based rational drug design aimed against viral replication.

Binding of Aminoglycoside Antibiotics to the Duplex Form of the HIV‐1 Genomic RNA Dimerization Initiation Site

Binding of Aminoglycoside Antibiotics to the Duplex Form of the HIV‐1 Genomic RNA Dimerization Initiation Site

Conformational transitions of flanking purines in HIV‐1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

Dimerization of HIV-1 genomic RNA is initiated by kissing loop interactions at the Dimerization Initiation Site (DIS). Dynamics of purines that flank the 5' ends of the loop-loop helix in HIV-1 DIS kissing complex were explored using explicit solvent molecular dynamics (MD) simulations with the CHARMM force field. Multiple MD simulations (200 ns in total) of X-ray structures for HIV-1 DIS Subtypes A, B, and F revealed conformational variability of flanking purines. In particular, the flanking purines, which in the starting X-ray structures are bulged-out and stack in pairs, formed a consecutive stack of four bulged-out adenines at the beginning of several simulations. This conformation is seen in the crystal structure of DIS Subtype F with no interference from crystal packing, and was frequently reported in our preceding MD studies performed with the AMBER force field. However, as CHARMM simulations progressed, the four continuously stacked adenines showed conformational transitions from the bulged-out into the bulged-in geometries. Although such an arrangement has not been seen in any X-ray structure, it has been suggested by a recent NMR investigation. In CHARMM simulations, in the longer time scale, the flanking purines display the tendency to move to bulged-in conformations. This is in contrast with the AMBER simulations, which indicate a modest prevalence for bulged-out flanking base positions in line with the X-ray data. The simulations also suggest that the intermolecular stacking between purines from the opposite hairpins can additionally stabilize the kissing complex.

Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging

Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity, and the nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) facilitates HIV-1 gRNA dimerization. To identify the relevant and dispensable positions of NC, 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants prepared by site-directed mutagenesis. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The N-terminal and linker segments appeared to play predominantly electrostatic and steric roles, respectively. Mutating the hydrophobic patch of either zinc finger, or substituting alanines for their glycine doublet, was as disabling as deleting the corresponding finger. Replacing the CysX2CysX4HisX4Cys motif of either finger by CysX2CysX4CysX4Cys or CysX2CysX4HisX4His, interchanging the zinc fingers or, replacing one zinc finger by a copy of the other one, had generally intermediate effects; among these mutations, the His23→Cys substitution in the N-terminal zinc finger had the mildest effect. The charge of NC could be increased or decreased by up to 18%, that of the linker could be reduced by 75% or increased by 50%, and one or two electric charges could be added or subtracted from either zinc finger, without affecting gRNA dimerization. Shortening, lengthening, or making hydrophobic the linker was as disabling as deleting the N-terminal or the C-terminal zinc finger, but a neutral and polar linker was innocuous. The present work multiplies by 4 and by 33 the number of retroviral and lentiviral NC mutations known to inhibit gRNA dimerization, respectively. It shows the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein. Most, but not all, mutations inhibited gRNA packaging; some had a strong effect on virus assembly or stability.

Role of the 5′ TAR Stem−Loop and the U5-AUG Duplex in Dimerization of HIV-1 Genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).

Long-range Recombination Gradient between HIV-1 Subtypes B and C Variants Caused by Sequence Differences in the Dimerization Initiation Signal Region

HIV-1 intersubtype recombinants have an increasingly important role in shaping the AIDS pandemic. We sought to understand the molecular mechanisms that generate intersubtype HIV-1 recombinants. We analyzed recombinants of HIV-1 subtypes B and C, and identified their crossover junctions in the viral genome from the 5′ long terminal repeat (LTR) to the end of pol. We identified 56 recombination events in 56 proviruses; the distribution of these events indicated an apparent recombination gradient: there were significantly more crossover junctions in the 3′ half than in the 5′ half of the region analyzed. HIV-1 subtypes B and C have different dimerization initiation signal (DIS). We hypothesized that the inability of subtype B and C RNAs to form perfect base-pairing of the DIS affects the dimeric RNA structure and causes a decrease in recombination events at the 5′ end of the viral genome. To test this hypothesis, we examined recombinants generated from a subtype C virus and a modified subtype B virus containing a subtype C DIS. In the 56 proviruses analyzed, we identified 96 recombination events, which are significantly more frequent than in the B/C recombinants. Furthermore, these crossover junctions were distributed evenly throughout the region analyzed, indicating that the recombination gradient was corrected by matching the DIS. Therefore, base-pairing at the DIS has an important function during HIV-1 reverse transcription, most likely in maintaining nucleic-acid structure in the complex. These findings reveal elements important to retroviral recombination and provide insights into the generation of HIV-1 intersubtype recombinants that are important to the AIDS epidemic.

Protein–RNA tethering: The role of poly(C) binding protein 2 in poliovirus RNA replication

The exploitation of cellular functions and host proteins is an essential part of viral replication. The study of this interplay has provided significant insight into host cell processes in addition to advancing the understanding of the viral life-cycle. Poliovirus utilizes a multifunctional cellular protein, poly(C) binding protein 2 (PCBP2), for RNA stability, translation and RNA replication. In its cellular capacity, PCBP2 is involved in many functions, including transcriptional activation, mRNA stability and translational silencing. Using a novel protein–RNA tethering system, we establish PCBP2 as an essential co-factor in the initiation of poliovirus negative-strand synthesis. Furthermore, we identified the conserved KH domains in PCBP2 that are required for the initiation of poliovirus negative-strand synthesis, and showed that this required neither direct RNA binding or dimerization of PCBP2. This study demonstrates the novel application of a protein–RNA tethering system for the molecular characterization of cellular protein involvement in viral RNA replication.

Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase

An artificial phosphodiesterase () bearing two types of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration. The rate constants for the hydrolysis reaction of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and RNA dimer (ApA) with and without an effector metal ion were evaluated; the k(obs) value of HPNP hydrolysis for .(Zn(2+))(3) (2.0 x 10(-4) s(-1)) is 3.3 times larger than that for .(Zn(2+))(2). In the case of and Cu(2+), a 19.4 times larger k(obs) value was obtained for .(Cu(2+))(3) (1.2 x 10(-3) s(-1)) against .(Cu(2+))(2). The increase in the catalytic activity is ascribed to the allosteric conformational transition of induced by the coordination of effector metal ion to the Bpy moiety. A detailed investigation revealed that a conformational change of induced by the third M(2+) complexation enhances the rate of hydrolysis rather than a change in the substrate affinity.

Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity

BackgroundRetroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication.ResultsUsing a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores.ConclusionIn addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (Kd ∼ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (Kd ∼ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Characterization of a natural heterodimer between MLV genomic RNA and the SD′ retroelement generated by alternative splicing

Murine leukemia virus (MLV) specifically packages both genomic RNA (FL RNA) and a subgenomic RNA, which we call SD'. SD' RNA results from alternative splicing of FL RNA. It is reverse-transcribed, and its DNA copy, integrated into the host genome, constitutes a splice donor-associated retroelement. FL and SD' RNAs share a common 5'-UTR that includes the packaging/dimerization signal (Psi). To investigate whether the mechanism of copackaging of these two RNAs involves RNA heterodimerization, we examined the spontaneous dimerization capacity of the two RNAs as large synthetic RNAs transcribed in vitro. We showed that SD' RNA not only formed homodimers with similar efficiency as the FL RNA, but that FL and SD' RNAs also formed FL/SD' heterodimers via Psi sequences. Comparison of the thermostabilities determined for these different dimeric species and competition experiments with Psi RNA fragments indicate the recruitment of similar dimer-linkage interactions within the Psi region. To validate these results, the dimeric state of the SD' RNA was analyzed in MLV particles. RNA capture assays performed with the FL RNA as bait revealed that SD', and not the host packageable U6 or 7SL RNAs, was associated with the FL RNA in virions. Heterodimerization of SD' RNA with FL RNA may argue for the recent concept of a nuclear dimerization at or near the site of transcription and raises the new hypothesis of RNA dimerization during splicing. Furthermore, FL/SD' heterodimerization may have leukemogenic consequences by influencing the pool of genomic dimers that will undergo recombinogenic template switching by reverse transcriptase.

In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs

The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 5' untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop-loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.

Guanine tetrad and palindromic sequence play critical roles in the RNA dimerization of bovine foamy virus

Retroviruses are unique in having a diploid genome. However, the RNA sequences and structures that link the two RNA molecules are different. To identify the dimer linkage site of bovine foamy virus (BFV), complementary DNAs were used to interfere with RNA dimerization of BFV. We found that two sites, designated SI and SII, within a 53-base RNA fragment, were essential for BFV dimerization in vitro. SI consists of a potential guanine tetrad (GGGGC), which overlaps the primer binding site, while SII contains 15 nucleotides including a palindromic sequence, UCCCUAGGGA. Masking either of the sites completely abolished RNA dimer formation. Furthermore, a deletion of SII was introduced into a BFV infectious DNA clone; we found that deletion of SII significantly increased expression of BFV transactivator Borf-1. Interestingly, we also found that this deletion abolished viral infectivity. These results suggest that dimerization might play a unique role in the BFV life cycle.

Structural Requirements for Nucleocapsid Protein-mediated Dimerization of Avian Leukosis Virus RNA

The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5′-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem–loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem–loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the μΨ RNA packaging signal. Results show that conservation of a long stem–loop structure and a loop–loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem–loop is sufficient to form an extended duplex and the whole stem–loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.