Regulation of primate lentiviral RNA dimerization by structural entrapment
© Baig et al; licensee BioMed Central Ltd. 2008
Received: 22 March 2008
Accepted: 17 July 2008
Published: 17 July 2008
Genomic 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.
Although 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.
Despite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1.
The 5' untranslated region of the lentiviral genomic RNA is replete with RNA signals involved in different stages of the replication cycle, such as transcription transactivation, polyadenylation, tRNA primer binding, dimerization, encapsidation, splicing, and translation . The RNA signals mediate viral functions through RNA-protein (genomic RNA encapsidation and reverse transcription) and RNA-RNA interactions (dimerization, tRNA hybridization to PBS). Although most of these signals can be linked to a precise stage of the viral replication cycle, they overlap structurally and functionally ([2–4]). For instance, the stem-loop 1 (SL1) dimerization signal overlaps with the genomic RNA encapsidation signal in HIV-2 ([5–8]). Another interesting characteristic of retroviral leader RNA signals is the fact that their presentation may vary during the different stages of viral replication. For example, the dimerization and encapsidation signals in Moloney murine leukemia virus genomic RNA are proposed to be initially masked and need to be activated by Gag protein-dependent RNA structural rearrangements .
Electron microscopy studies of packaged genomic RNAs revealed that the two RNA molecules are strongly associated with each other through their 5' ends, termed the dimer linkage structure ([10, 11]). In HIV-1, a short sequence located in the 5' untranslated region that promotes dimerization of partial leader transcripts was identified and named the dimerization initiation site (DIS) or stem-loop 1 (SL1) ([12–14]). The SL1 element maintains two RNA molecules in a dimeric state in vitro, either through a kissing-loop interaction, or an extended duplex base pairing arrangement (for review, see [2–4]). Roles of SL1 in the different stages of viral replication have been characterized in cell culture (reviewed in , ). Initially proposed from in vitro results , the genomic RNA dimerization initiation role of SL1 was recently confirmed in HIV-1 . A riboswitch model was proposed in which extensive structural rearrangements of the whole leader region could influence the presentation of SL1 (and other RNA signals) and thus dimerization and packaging efficiencies . In this model the HIV-1 leader RNA can adopt two different conformations: a long-distance interaction (LDI) between the polyA signal and SL1 domains and a distinct branched multiple-hairpin structure (BMH) . Several predicted conformations of the leader region include base pairing between a C-rich sequence in the 5' part of U5 and a G-rich sequence around the gag translation initiation region . Here we call this interaction CGI, for C-box – G-box interaction, to specifically indicate this base pairing rather than an overall conformation of the leader RNA such as LDI or BMH. Formation of CGI favors the BMH conformer in HIV-1 and thus promotes the presentation of SL1 as an efficient dimerization signal ([17, 18]).
To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, we compared the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Overall, it appears that SIV, HIV-1, HIV-2 RNAs have several common features with regard to SL1-mediated dimerization, which points to conserved regulation mechanisms by homologous RNA structures. Truncation analysis revealed that tight dimerization of the three lentiviral leader RNAs is modulated by interactions of nucleotides located between SL1 and the first codons of the gag open reading frame that affect both SL1 presentation and overall leader conformation. Most important, all three lentiviral RNAs tested in this study demonstrated a local regulation of dimerization through the stabilization of SL1 by its lower stem structure called stem B.
Template construction for in vitrotranscription
Cloning oligonucleotides used in this study. The 5' to 3' sequence is indicated from left to right.
TAG GAT CCT AAT ACG ACT CAC TAT AGG TCG CTC TGC GGA GAG
AAG AAT TCA CGC TGC CTT TGG TAC CTC GGC C
AAG AAT TCG CTC CAC ACG CTG CCT TTG
HIV-2 asMUT444Eco a
TTG AAT TCG CTC CTA GAA AGA CGC TGC CTT TGG TAC CTC G
AAG AAT TCA GTT TCT CGC GCC CAT CTC CC
AAG AAT TCA CGC CGT CTG GTA CCG
TTG AAT TCG CTC CTC ACG CCG TCT GG
AAG AAT TCA GTT TCT CAC GCC CAT CTC CC
TAG GAT CCT AAT ACG ACT CAC TAT AGG TCT CTC TGG TTA GAC C
TTG AAT TCT CTT GCC GTG CGC GCT TCA GC
TTG AAT TCT CGC CTC TTG CCG TGC G
TTG AAT TCT CCC CCG CTT AAT ACC GAC
RNA synthesis and purification
The different plasmids were linearized with EcoRI prior to in vitro transcription. RNAs were synthesized by in vitro transcription of the EcoRI-digested plasmids with the AmpliScribe T7 transcription kit (Epicentre). After transcription, the DNA was digested with the supplied RNase-free DNase, and the RNA was purified by ammonium acetate precipitation followed by size exclusion chromatography (Bio-Gel P-4, Bio-Rad).
In vitrodimerization of SIV, HIV-1, and HIV-2 RNAs
The samples were then cooled on ice to stabilize dimers formed during incubation and loaded on a 0.8% agarose gel with 2 μl glycerol loading dye 6× (40% glycerol, Tris-borate 44 mM pH 8.3, 0.25% Bromophenol blue). Electrophoresis was carried out at 3 V/cm for 2 hours at room temperature (26°C) in Tris-borate 44 mM pH 8.3, EDTA 1 mM (TBE). After electrophoresis, the ethidium bromide-stained gel was scanned on a Fluorescent Image Analyzer FLA-3000 (Fujifilm).
Analysis of regulatory RNA signals using antisense oligonucleotides
Antisense oligonucleotides used in this study. The 5' to 3' sequence is indicated from left to right.
CTA GGA GCA CTC CGT CGT GGT TTG
TGG TAC CTC GGC CCG CGC CT
CTA GGA GAG ATG GGA GTA CAC AC
CAT CTC CCA CAA TCT TCT ACC
ATA GGA GCA CTC CGT CGT GGT TGG
TGG TAC CGA CCC GCG CCT
CTA GGA GAG ATG GGA ACA CAC AC
CAT CTC CCA CTC TAT CTT ATT ACC CC
CGA GTC CTG CGT CGA GAG ATC TCC
TGC GCG CTT CAG CAA GCC GAG TCC
AGA CGG GCA CAC ACT ACT TTG AGC A
CAT CTC TCT CCT TCT AGC CTC
Kinetics of tight dimer formation
where Mt is the concentration of monomer at time t, M0 is the initial concentration of dimerization-competent monomer, and kdim is the second order rate constant of dimerization (μM-1min-1).
Effect of the dimerization incubation temperature
Five pmol of RNA were denatured in 8 μl water for 2 minutes at 90°C and cooled on ice for 2 minutes. After addition of 2 μl 5× dimer buffer, dimerization was allowed to proceed for 30 minutes at 24°, 37°, 40°, 45°, 50°, 55°, or 60°C. The samples were then loaded on a 0.8% agarose TBE gel. Electrophoresis was carried out for 2 h at 26°C and 3 V/cm. After electrophoresis, the ethidium bromide-stained gel was scanned on a Fluorescent Image Analyzer FLA-3000 (Fujifilm).
Prediction of secondary structures
Calculated ΔG values (kcal/mol at 37°C) for lentiviral SL1 region folding.
extended stem B
Tight dimerization characteristics of HIV-2, SIV, and HIV-1 leader RNAs
The ability of 1–561 HIV-2, 1–550 SIV, and 1–373 HIV-1 RNAs to form tight dimers when incubated at 55°C in dimerization buffers was compared. All three RNA constructs encompass the 5' untranslated leader region and the first several codons of the gag open reading frame of the genomic RNA (Methods, Figure 1). Tight dimers withstand dissociation during semi-native gel electrophoresis (Tris-borate EDTA (TBE) buffer at 26°C). By contrast, loose dimers are detected only by using native conditions, since they dissociate upon loading in semi-native TBE/26°C gels . Loose dimers are thought to correspond to a loop-loop interaction between two SL1 motifs, whereas tight dimers are thought to represent a more extensive SL1–SL1 interaction due to the intra- to intermolecular conversion of the stems located below the SL1 loop ([2, 4]).
The tight dimerization of HIV-2 1–561 RNA was similar to what has been previously described ([19, 20]). Only a minor fraction of RNA molecules dimerized after 30 or 60 min (Figure 2A, compare lane 1 to lanes 2 and 3). Indeed, the dimerization yield was less than 50% after an eight hour incubation at 55°C (Figure 2A, lane 7). The tight dimerization of 1–550 SIV RNA was similar to HIV-2, with a slightly lower level of tight dimers formed after extended incubation (Figure 2B).
The formation of HIV-1 RNA dimers, measured by the disappearance of the monomer RNA species, was twice as efficient as HIV-2 or SIV over the whole eight hour experiment, although the interpretation of longer time points was made more difficult by the appearance of two other RNA species that appeared after two hours. One band migrated between the monomer and dimer bands and another one was below the monomer band (Figure 2C). Though we cannot rule out that a small fraction of these RNA species represent fast-migrating conformers, electrophoretic analysis of these reactions under denaturing conditions revealed that the majority of these RNA species represent cleaved RNA molecules (data not shown). Significant cleavage was not observed in the HIV-2 or SIV RNAs during extended incubation (Figure 2), which allowed us to rule out an obvious RNase contamination, since most of the buffers, transcription components were shared between all RNAs tested in this study.
The relative inefficiency of HIV-2 and SIV RNA tight dimerization suggested that the presentation of SL1 was not optimal in these RNAs. Though more efficient than HIV-2 and SIV, the yield of HIV-1 tight dimerization during the first hour was low (Figure 2C). Since SL1 presentation is modulated by short- and long-range interactions in HIV-2 ([21, 25–27]) or HIV-1 , we hypothesize that co-incubation of lentiviral leader RNAs with antisense oligonucleotides directed against these modulating interactions may improve SL1 presentation and thus increase tight dimerization.
Analysis of long-range regulatory interactions using antisense oligonucleotides
Similar to SIV, the incubation of 1–373 HIV-1 RNA with the homologous oligonucleotides showed no effect (40% of tight dimers with or without oligonucleotides; Figure 3A, lanes 11 to 15). Experiments using radioactive oligonucleotides or RNase H cleavage assay  confirmed that the oligonucleotides bind the HIV-1 RNA effectively and at the expected locations (data not shown). The lack of effect of asC oligonucleotide binding on HIV-1 dimerization suggests that the formation of the LDI structure was minimal in our experiments. Likewise, the lack of effect of asG oligonucleotide binding suggests that disruption of the BMH structure did not promote either LDI or tight dimer formation. Thus, contrary to HIV-2, disruption of CGI with antisense oligonucleotides did not have an effect on SIV and HIV-1 RNA tight dimerization. This suggests that, besides CGI, stronger local regulatory interactions may impair the use of SL1 as a tight dimer element in SIV and HIV-1.
Analysis of short-range regulatory interactions using antisense oligonucleotides
To test whether local base pairing in the SL1 region affects tight dimerization, we used an antisense oligonucleotide directed against the 5' stem B of SL1 and sequences upstream (as Ψ for HIV-2 and SIV, as246 for HIV-1, Table 2 and Figure 3A). Stem B represents the lower stem of the SL1 structure . The impetus for this experiment came from previous observations of local regulation of SL1-mediated dimerization in HIV-2 RNA . Incubation of HIV-2 1–561 RNA with an oligonucleotide binding to the loop and 5' stem of SL1 induced dimerization mediated by the 10-nt autocomplementary sequence (pal: 5'-392-GGAGUGCUCC-401) located in the HIV-2 encapsidation signal Ψ (Figure 3C, lane 3). Likewise, incubation of SIV and HIV-1 RNAs with the homologous asΨ and as246 oligonucleotides improved tight dimerization (Figure 3C, lanes 7 and 12). Co-incubation with the anti-SL1 oligonucleotide (asSL1, Table 2) confirmed that the increased tight dimerization was SL1-dependent (Figure 3C, lanes 4, 9, 14). Thus, the region upstream of SL1 including stem B has a negative effect on lentiviral tight dimerization. However, the different levels of tight dimers formed in the absence of oligonucleotides, (Figures 2 and 3) indicate that different SL1 sequences may also influence dimerization properties of lentiviral RNAs.
Chimeric RNAs reveal SL1 loop sequence affects tight dimerization
Thus, the difference of only a few nucleotides in the SL1 sequence influences tight dimerization rates in SIV and HIV-2 RNAs. However, the incomplete correlation between the SL1 sequence and the dimerization yield in the chimeric RNAs indicates that other features of the leader RNA, like the stem B element, may influence the formation of tight dimers.
Stem B affects the tight dimerization rate in lentiviral RNAs
Thus, binding of antisense oligonucleotides directed against the 5' side of stem B and nucleotides upstream increased the tight dimerization rate for all three lentiviral RNAs tested in this study. However, the rates without oligonucleotide differ and may be related to individual properties of the different lentiviral SL1s, including the apical loop, stem B, and adjacent sequences (Figure 4).
Stability of stem B modulates the formation temperature of lentiviral RNA tight dimers
Because disruption of stem B was shown to stimulate tight dimerization, we hypothesized that artificially stabilizing stem B would have the opposite effect. We thus substituted nts G437-U438 in 1–444 HIV-2 RNA with the sequence CUUUCUA and called this mutant 1–444 extended SL1 HIV-2 RNA. This mutation extends the stem of the SL1 structure to include stem B through Watson-Crick base pairing (Figure 6C) and consequently doubles the stability of the SL1 region, from -18.2 to -36.4 kcal/mol (ΔGs of wild type and mutated RNAs, respectively; Table 3). The ΔG values correspond to the optimal secondary structure of 396–444 HIV-2 RNAs with or without the mutation using Mfold version 3.2 ([38, 39]). The 1–444 HIV-2 RNA with the extended SL1 was incubated for 30 min at temperatures ranging from 24° to 70°C (Figure 6C). Its tight dimerization yields were very low at almost all temperatures; only the 70°C incubation showed an increase in dimer yield (Figure 6C). Thus, the increase in stability of the SL1 domain in the mutated 1–444 HIV-2 RNA correlated with a decrease in tight dimerization.
The leader RNA of lentiviruses contains signals involved in different stages of the replication cycle and most of these signals are conserved between primate lentiviruses. SL1 is involved in dimerization and encapsidation of the genomic RNA ([2, 4, 15]), and is located between the PBS domain and the major splice donor site in the 5' untranslated leader region of all spliced and unspliced lentiviral RNA species (Figure 1).
Tight dimerization efficiency of primate lentiviral RNAs
We and others previously reported the low efficiency of HIV-2 leader RNA tight dimerization when its 3' end encompasses the gag initiation region ([21, 25]). The impaired dimerization correlated with the formation of the CGI structure (Figure 1) ([21, 25]). We proposed that the formation of HIV-2 CGI favors a structural intramolecular entrapment of SL1 during RNA folding and thus decreases its use as a tight dimerization signal in vitro (Figure 1D) ([25, 26]).
Although extended kinetic analysis revealed an impaired dimerization phenotype of 1–550 SIV RNA, the lack of effect of antisense oligonucleotides directed against CGI underscored an unexpected difference between SIV and HIV-2 leader RNAs, despite their sequence homology. Indeed, CGI-independent intramolecular local folding events may compete with dimerization more effectively in SIV than in HIV-2 RNA. Similar to SIV, 1–373 HIV-1 RNA dimerization was not influenced by the binding of antisense oligonucleotides directed against CGI, although its yield without oligonucleotide was higher than HIV-2 or SIV. Notably, the dimerization rate for each RNA was markedly enhanced by truncation from the full-length leader to shorter versions that end after SL1 (compare Figures 2 and 5) suggesting a conserved modulatory role for downstream leader sequences.
Stem B and lentiviral SL1-mediated dimerization
A unifying principle in the regulation of lentiviral RNA dimerization is the role of the lower stem of SL1, called stem B (Figures 5 and 7). We hypothesized that stem B exerts significant influence on lentiviral RNA dimerization for several reasons. First, stem B is highly conserved in all three lentiviral genomes sequences considered in this study ([22, 41, 42]). Second, recent reports have confirmed that short-distance interactions involving sequences located upstream of SL1 could regulate HIV-2 dimerization in vitro  and in cell culture . Third, stem B was shown to have roles at several stages of HIV-1 replication, including genomic RNA dimerization ([43–45]).
In this study we showed that all three tested lentiviral species RNAs exhibited local regulation of dimerization through the stabilization of SL1 by stem B (Figure 5 and Table 3). We propose that stem B-mediated SL1 entrapment may have twin roles in the regulation of lentiviral leader RNA dimerization (Figure 7). First, it increases the probability of SL1 to fold as a discrete secondary structure, thus favoring SL1 loop/loop (loose) dimerization. It has been shown that HIV-1 stem B promotes SL1-mediated loose dimerization by favoring the correct presentation of the apical autocomplementary sequence ([43, 46]). Second, promoting SL1 presentation by increasing its stability may have a thermodynamic cost for tight dimer formation. The thermodynamic cost might be compensated in vivo by the presence of the loop B structure in SL1. Indeed, several laboratories have shown that loop B (located between stems B and C, Figure 5A) favors temperature- or nucleocapsid-mediated dimerization by helping the transition from loose to tight dimers ([47–49]).
Several studies analyzed the roles of SL1 in genomic RNA dimerization and the nature of SL1–SL1 interaction in vivo but a comprehensive model has not yet been established, due to divergent results from several laboratories. SL1 was shown to promote HIV-1 (reviewed in ) and SIV ([50, 51]) genomic RNA dimerization during viral encapsidation. Accordingly, Laughrea and colleagues proposed a model of HIV-1 genomic RNA dimerization wherein SL1 acts as a genomic RNA dimerization initiation signal that evolves to tight dimers . Alternatively, Hu and colleagues showed that SL1 is involved in HIV-1 genomic RNA selection and recombination through intermolecular base pairing limited to SL1 loop-loop interaction . It is possible that the apparent divergent results of both studies are due to different experimental conditions. Indeed, Laughrea and colleagues showed that SL1 is involved in the early stages of genomic RNA encapsidation, that is, in the first hours of viruses maturation after their release from the cell (five hours or less, ). They suggested that slow-acting dimerization site(s) mask the phenotype of SL1 mutants in "older" viruses (24 hours and older, ). Since Hu and colleagues harvested viruses after 24 hours of culture , the majority of their genomic RNAs may have been matured by slow-acting dimerization site(s), which may explain why most of their SL1 mutations were phenotypically silent. In HIV-2, Lever and colleagues showed that the HIV-2 palindromic sequence pal had more importance for genomic RNA dimerization during viral encapsidation than the palindromic sequence in the loop of SL1 .
Therefore, stem B of SL1 is likely a key regulating element for different conformations of lentiviral leader RNAs, akin to the concept of the riboswitch, proposed in HIV-1 by Berkhout and colleagues . Disrupting stem B is detrimental to many stages of HIV-1 ([43, 45]), HIV-2 ([6, 7]), and SIV ([50, 53]) replication. Remarkably, this extended SL1 structure means that there is a structural overlap between the Ψ encapsidation signal and the core of the SL1 dimerization element in both SIV and HIV-2. Recent reports of stem B effects on dimerization  and encapsidation  support a functional linkage between these two viral functions in HIV-2.
This study demonstrates the ubiquity of structural entrapment as a mechanism of regulation of dimerization among primate lentiviruses and provides a framework for understanding dimerization data in each system through local and long-range RNA arrangements. The 3' leader region exerts an inhibitory effect on lentiviral RNA dimerization by altering the presentation of SL1. Stem B strongly influences SL1 presentation in all three lentiviral RNAs tested in this study. Because a similar set of folding rules can be used to explain the behaviors of three distinct primate lentiviral RNAs in vitro, it is likely that these discrete conformational changes represent a conserved regulatory mechanism in vivo.
In particular, it will be of interest to study the effect of CGI or stem B mutations on packaging and gag translation in mutant viruses in cell culture. Conformational changes like the ones described here may help determine the fate of viral RNA with respect to genomic and/or translational duties. Indeed, such viral riboswitches would be expected to be regulated by RNA chaperones like the viral nucleocapsid or Gag proteins and might be influenced by translational events, like ribosomal scanning.
Human immunodeficiency virus
Simian immunodeficiency virus
C-box – G-box interaction
Branched multiple-hairpin structure.
We acknowledge Quenna N. Szafran for the initial experiments on SIV RNA constructs. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: p239SpSp5' ([28, 29]) and p83-2  from Dr Ronald Desrosiers. The pROD10 plasmids from Drs. J.-M. Bechet and A.M.L. Lever were obtained from the Centralised Facility for AIDS Reagents supported by EU Programme EVA (contract QLK2-CT-1999-00609) and the UK Medical Research Council. This research is supported by the National Institutes of Health grant number AI45388 to J.S.L.
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