Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus

Retroviral virions each contain two identical genomic RNA strandsthat are stably but noncovalently joined in parallel near their5' ends. For certain viruses, this dimerization has been shownto depend on a unique RNA stem-loop locus, called the dimerinitiation site (DIS), that efficiently homodimerizes througha palindromic base sequence in its loop. Previous studies withMoloney murine leukemia virus (Mo-MuLV) identified two alternativeDIS loci that can each independently support RNA dimerizationin vitro but whose relative contributions are unknown. We nowreport that both of these loci contribute to the assembly ofthe Mo-MuLV dimer. Using targeted deletions, point mutagenesis,and antisense oligonucleotides, we found that each of the twostem-loops forms as predicted and contributes independentlyto dimerization in vitro through a mechanism involving autocomplementaryinteractions of its loop. Disruption of either DIS locus individuallyreduced both the yield and the thermal stability of the in vitrodimers, whereas disruption of both eliminated dimerization altogether.Similarly, the thermal stability of virion-derived dimers wasimpaired by deletion of both DIS elements, and point mutationsin either element produced defects in viral replication thatcorrelated with their effects on in vitro RNA dimerization.These findings support the view that in some retroviruses, dimerinitiation and stability involve two or more closely linkedDIS loci which together align the nascent dimer strands in paralleland in register.

INTRODUCTION

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Introduction

Retroviral virions each contain two copies of the single-strandedviral genomic RNA that are stably but noncovalently linked toform a homodimer (17, 42). The dimeric genome is believed topromote retroviral replication and recombination by allowingreverse transcriptase to switch from one viral RNA strand tothe other whenever it encounters a break in its template. Indirectevidence suggests that the dimerized chromosomes are bound togetherby direct RNA-RNA linkages along their entire lengths; thisscenario could explain, for example, how such dimers remainintact even when both RNA strands are extensively nicked andall virion proteins have been removed (39). However, the natureand locations of the imputed dimer contacts are almost entirelyunknown. Indeed, the only site of contact that has been characterizedin detail is a short region, known as the dimer linkage site(DLS), that occurs near the 5' ends of many or all retroviralgenomes (17, 42). The DLS was first defined by electron microscopicexamination of dimers extracted from virions, and its appearanceand location are well conserved among diverse retroviruses (2,22, 28, 37, 47). It is typically the most stable site of linkagebetween strands (often the only such site that can survive denaturationin 75% formamide) and hence may be the first to form duringdimer assembly in vivo. Viruses carrying mutations in the DLShave been found to contain anomalously dimerized or monomericRNA (as judged by nondenaturing gel electrophoresis) and alsoto have impaired replication (3, 12, 24, 30, 31, 35, 40, 41,43), suggesting that accurate DLS-mediated dimerization is criticalfor the retroviral life cycle.

Synthetic RNAs that contain retroviral 5' sequences can dimerizeefficiently in vitro, and this fact has provided a model fordissecting the molecular basis of DLS assembly. Such studieshave revealed that in several retroviruses, DLS dimerizationdepends upon a unique RNA stem-loop, called the dimer initiationsite (DIS), that includes a palindromic (i.e., self-complementary)base sequence in its loop. DIS-mediated dimerization beginswhen the DIS loops of two strands anneal to form a metastable"kissing-loop" complex; in a subsequent isomerization step,the DIS stems in this complex melt and then reanneal to forma single, linear double helix that stably links the two strands.In protein-free artificial systems, isomerization does not occurunless the kissing-loop complex is heated briefly to 50 or 60°C;in the virus, however, it is catalyzed efficiently at room temperatureby the viral nucleocapsid protein (6, 16, 44). Although theirsequences differ widely, analogous DIS stem-loops have beenidentified in a growing number of retroviruses, suggesting thatthey may be a universal feature of these viruses.

Efforts to map the DIS locus of one prototypical retrovirus,Moloney murine leukemia virus (Mo-MuLV), have yielded divergentresults (15, 38, 44). Early attention centered on a potentialstem-loop at residues 283 to 298 that has a palindromic sequenceand was shown to be sufficient to direct RNA dimerization invitro. This candidate DIS, together with two additional stem-loopsimmediately downstream, also form part of the viral packagingsignal (12, 34, 35). Recently, however, other authors identifieda second candidate DIS stem-loop a short distance upstream (atresidues 204 to 228) that reportedly can mediate efficient dimerizationindependently of the known downstream elements (38). While eachof the two proposed DIS elements has the features necessary,in theory, to support kissing-loop interactions, the actualfolded conformations of these elements and the pathways by whichthey may dimerize have not been directly explored. Additionally,as neither locus has been tested for dimerization activity invivo, their biological relevance is as yet unproven, and itis not yet clear to what extent Mo-MuLV RNA dimerization dependson one, the other, or both.

To address these questions, we have carried out studies by usingMo-MuLV RNA sequences that encompass both of the candidate DISloci. Through systematic mutagenesis and antisense interference,we have probed the functional organization of these loci aswell as the contributions that each makes to RNA dimerizationin vitro. In addition, we have also compared the effects ofmutating these elements individually in virions. Our findingsreveal a novel bipartite organization for the signals that initiategenomic dimer assembly in this virus.

MATERIALS AND METHODS

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Materials and Methods

RNA synthesis. DNA templates for in vitro transcription were synthesized byPCR from the vector pINA10 (a kind gift from E. Barklis), whichcomprises a G418 resistance gene flanked by 5' and 3' sequencesfrom Mo-MuLV (35). Each of the forward primers (5'-GAAGATCTTCTAATACGACTCACTATAGGTGGGGGCTCGTCCGGGATC-3'and 5'-GAAGATCTTCTAATACGACTCACTATAGGCCTGCGTCGGTACAGTTAGC-3')incorporated a T7 promoter sequence and a BlgII restrictionsite and, when paired with the reverse primer (5'-CCCAAGCTTGGGCTAATTCTCAGACAAATACAGAAACACAGTCAGAC-3'),amplified residues 146 to 621 and residues 270 to 621, respectively,of the Mo-MuLV genome, where residue 1 is the first transcribedbase. PCR products were purified by using a QIAquick kit (Qiagen)and redissolved in RNase-free water. Transcription reactionmixtures (20 µl) contained 1 µg of DNA template;0.5 mM each unlabeled ATP, GTP, and CTP; 50 µM unlabeledUTP; and 2.5 µM ${alpha}$ -³²P-labeled UTP. Reactions were performedby using a T7 MAXIscript kit (Ambion). Transcripts were purifiedby electrophoresis on 5% polyacrylamide-8 M urea gels in TBEbuffer (90 mM Tris-borate, 2 mM EDTA), ethanol precipitated,and resuspended in RNase-free water.

RNA dimerization assay. For the standard dimerization assay, 8-µl samples containingone or more labeled RNAs (each at approximately 1.5 nM in water)were heat denatured at 95°C for 3 min, immediately snap-cooledon ice, and combined with 2 µl of cold D5X buffer (500mM NaCl, 250 mM Tris [pH 7]). The samples were incubated atvarious temperatures (0 to 70°C) for 1 h, chilled, and analyzedby electrophoresis on nondenaturing 5% polyacrylamide gels inTBE buffer. After being dried, gels were analyzed by using aPhosphorImager (Molecular Dynamics). Antisense inhibition studieswere performed in the same way, except that antisense DNA oligonucleotideswere added (at a final concentration of 60 µM) beforeheat denaturation. For each sample, the fraction of RNA in dimericform was determined from phosphorimager signal intensities asthe ratio of dimeric RNA to the sum of both the monomeric andthe dimeric species.

Cell cultures, transfections, and infections. GP+E86 murine ecotropic packaging cells (ATCC CRL 9642) andNIH 3T3 murine fibroblasts (ATCC CRL 1658) were maintained inDulbecco's modified Eagle's medium with a high glucose concentration(4.5 g/liter) and 10% calf serum (standard medium). Wild-typeor mutant pINA10 DNAs (20 µg) were transfected into 5.0x 10⁵ GP+E86 cells in each 60-mm polystyrene dish by calciumphosphate coprecipitation along with 0.25 µg of the luciferase-encodingvector pGL3 (Promega) as an internal control. Viral titers wereestimated by luciferase assays of the transfected cells as describedpreviously (26, 27), and aliquots of culture supernatants wereused to infect 2.5 x 10⁵ NIH 3T3 cells in the presence of 8µg of Polybrene/ml. Infectious titers were determinedwith these cells by G418-resistant colony formation assays asdescribed previously (26, 27). Producer cell lines were madeby stably transfecting GP+E86 cells with pINA10 vectors containingeither wild-type or mutated DIS sequences and then selectingneomycin-resistant colonies by growth in 500 µg of Geneticin(Gibco-BRL)/ml; several hundred colonies were pooled and usedto produce virus for subsequent experimentation.

Nondenaturing gel electrophoresis. Northern blot analysis of virion RNA on native agarose gelswas performed as described by Fu and colleagues (13, 14). Briefly,5 x 10⁶ cells stably transfected with a neomycin resistancecassette (neo^r) in 40 ml of standard medium containing 500 µgof Geneticin/ml were seeded into T-175 tissue culture flasks(Falcon). Virus supernatants were collected 2 days later andpassed through 0.45-µm-pore-size filters. Virus was pelletedfrom 70 ml of total culture volume for each of the viral constructsby ultracentrifugation in an SW28 rotor (Beckman) at 25,000rpm for 30 min at 4°C. Virion RNAs were then extracted withvirion lysis buffer (0.05 M Tris, 0.01 M EDTA, 0.1 M NaCl, 1%sodium dodecyl sulfate, 0.05 mg of yeast RNA/ml, 0.1 mg of proteinaseK/ml) and resuspended in R buffer (0.01 M Tris, 0.001 M EDTA,1% sodium dodecyl sulfate, 0.05 M NaCl) as described previously(13). A 0.1 volume of resuspended virion RNAs was incubatedat various temperatures for 10 min and placed on ice beforebeing loaded onto 1% agarose gels. RNAs were separated in 1xTris-acetate-EDTA buffer in a cold room (4°C) at 450 V,stained with ethidium bromide (1 µg/ml), and visualized.Nondenaturing Northern blotting was performed essentially aspreviously described (13). A TurboBlotter system was used totransfer RNAs onto Nytran supercharged nylon membranes as describedby the manufacturer (Schleicher & Schuell). RNAs were UVcross-linked by using a Stratalinker (Stratagene) and hybridizedwith 10⁶ cpm of riboprobe containing an antisense sequence ofRNA-4 (see Fig. 2A), which is transcribed from the SP6 promoterof the pSP72-MuLV ${psi}$ vector (unpublished data). Monomeric and dimericRNAs were quantified by phosphorimaging.

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FIG. 2. DIS-1 and DIS-2 each contribute independently to Mo-MuLV RNA dimerization in vitro. (A) Various radiolabeled synthetic RNA fragments containing DIS-1, DIS-2, or both (RNA-1 through RNA-7) were synthesized and tested in vitro. Terminal residues and major features of each are listed. PBS, primer binding site; SD, splice donor. (B) Temperature dependence of in vitro dimer formation. Each RNA was analyzed electrophoretically after preincubation at the temperatures listed above the lanes; RNA-1 and RNA-2 were also tested in combination (lanes 7 and 8). Positions of presumed monomers (M) and dimers (D) are indicated. For lanes 12 to 31, the percentage of dimers in each lane (quantified by phosphorimaging) is shown below the lanes.

RESULTS

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Results

Formation and stability of Mo-MuLV 5' RNA dimers in vitro. DIS elements are defined experimentally by their ability topromote efficient dimerization of a retroviral RNA in its native,highly folded conformation. For in vitro DIS assays, syntheticRNA is first heat denatured and then snap-cooled on ice in orderto simulate native monomeric folding. Studies of human immunodeficiencyvirus type 1 RNA indicate that, if maintained at 40°C orbelow, these monomers form kissing-loop dimers but that suchdimers are too labile to survive electrophoresis on TBE gels.Transient heating to 50 or 60°C, however, provides the energynecessary for isomerization into linear form (see above), yieldingdimers that are stable on TBE gels (4, 10, 23, 36, 46, 48).We applied this same protocol in the current study, synthesizingand refolding portions of the Mo-MuLV 5' sequence, briefly incubatingthese RNAs at various temperatures, and then examining the resultantcomplexes by TBE electrophoresis. As detailed below, our findingsverified the existence of two closely linked DIS elements, oneat residues 204 to 228 and the other at residues 283 to 298,denoted here as DIS-1 and DIS-2, respectively (Fig. 1). Thedimer-sized RNA complexes that we detected electrophoreticallywere proven to be dimers in the course of our work (see Fig.5); for brevity, we refer to these as dimers throughout.

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FIG. 1. Candidate DIS loci in the Mo-MuLV genome. (A) Schematic depiction of the Mo-MuLV 5' leader region, extending from the first transcribed base (residue 1) in the R region through the gag start codon (to residue 621). Locations of the primer binding site (PBS), major 5' splice donor (SD), and palindromic portions of four putative stem-loop structures are shown. Of the latter, DIS-1 and DIS-2 are considered in this study; the two downstream stem-loops appear to serve as the core RNA packaging signal ( ${psi}$ ) (see reference 35). (B) Hypothetical pathways of kissing-loop dimerization for DIS-1 and DIS-2. Each initially homodimerizes through four central palindromic bases in and around its loop, forming a transient kissing-loop complex which then can isomerize into linear form.

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FIG. 5. Heterodimer formation provides direct evidence for a complementary loop-loop interaction at each DIS locus. Mutant RNAs were synthesized in which the four central palindromic nucleotides of DIS-1 or DIS-2 (Fig. 1B) were replaced by the indicated sequences in the context of either RNA-1 (280 bases) or RNA-4 (475 bases). These were then assayed for dimerization alone or in combination (at 1.5 nM each) after preincubation at the temperatures indicated. Above lanes in which two different RNAs were combined, the sequences of their central bases are shown in antiparallel alignment, with dots indicating any expected base pairing. M and D, monomers and homodimers, respectively, of the long (L) and short (S) RNAs; HD, heterodimers. Panels A and B were derived from two separate representative experiments done with different combinations of mutants.

We began by confirming that RNAs containing either DIS-1 orDIS-2 alone could dimerize spontaneously in vitro. We transcribedtwo nonoverlapping portions of the Mo-MuLV genome, which areschematized in Fig. 2A: the first (RNA-1) extended from the5' R region through DIS-1 (i.e., residues 1 to 280), and thesecond (RNA-2) extended from immediately upstream of DIS-2 throughthe start codon of gag (residues 270 to 621). As shown in Fig.2B, each exhibited temperature-dependent homodimer formation.When melted, snap-cooled, and maintained on ice, RNA-1, containingDIS-1, migrated exclusively as a monomer (M₁); however, whenheated to 50 or 60°C, it formed a discrete, dimer-sizedcomplex (D₁) that accounted for more than half of the inputRNA (Fig. 2B, lanes 1 to 3). Precise deletion of the DIS-1 sequencefrom this RNA abolished all apparent dimerization (Fig. 2B,lanes 9 to 11). The somewhat longer RNA-2, which contained DIS-2,also was monomeric at a low temperature (M₂) but formed a dimer(D₂) when heated (Fig. 2B, lanes 4 to 6). When the two RNAswere mixed, snap-cooled, and incubated together, they each efficientlyhomodimerized but failed to produce an intermediate-sized complexthat would suggest heterodimer formation (Fig. 2B, lanes 7 and8). These data were in accord with earlier reports ascribingDIS activity to each of these regions in Mo-MuLV and furthershowed that the two regions do not heterodimerize appreciably.

We next evaluated a longer Mo-MuLV transcript (RNA-4) that extendedfrom the primer binding site through the start codon of gag(i.e., residues 146 to 621) and so encompassed both DIS-1 andDIS-2. We also created three variants of this sequence (RNA-5,RNA-6, and RNA-7) from which DIS-1, DIS-2, or both had beenprecisely deleted (Fig. 2A). As shown in Fig. 2B, the wild-typeRNA-4 remained exclusively monomeric (M) when kept on ice (lane12) but formed a dimer (D) when incubated at 50 to 60°C(lanes 13 to 15). This dimer was most abundant in samples preparedat 60°C, where it accounted for more than half of the totalRNA, but it dissociated back to monomers when heated to 70°C(Fig. 2B, lane 16). The mutants lacking either DIS-1 or DIS-2alone, by comparison, yielded reduced amounts of dimer-sizedcomplexes (accounting for no more than 10 to 30% of the inputRNA) that were approximately equally abundant after preheatingto either 50 or 60°C (Fig. 2B, lanes 17 to 26). When bothDIS-1 and DIS-2 were deleted, no evidence of dimerization wasdetected over the range of temperatures that we tested (Fig.2B, lanes 27 to 31). Taken together, these results indicatethat DIS-1 and DIS-2 each can support dimerization independentlybut that dimers form most efficiently and stably when both lociare present. Deleting either locus alone significantly reducesthe formation and/or thermal stability of the resulting dimers,and deleting both eliminates in vitro dimerization altogether.

Functional mapping of DIS loci by antisense inhibition. As an alternative means to delineate sequences required forMo-MuLV dimer formation, we tested whether antisense DNA oligomersthat targeted various regions of the Mo-MuLV leader sequencecould serve as inhibitors of dimer formation (Fig. 3A). Ourefforts focused mainly on DIS-1, which has been less extensivelycharacterized than DIS-2. We synthesized a series of DNA oligonucleotides,each 18 to 29 bases long (Table 1), which were complementaryto regions of Mo-MuLV RNA in and around the DIS loci (Fig. 3A).Each was then combined, in approximately a 1,000-fold molarexcess, with the dimerization-competent RNA-4 (residues 146to 621). The mixture was heat denatured, snap-cooled, warmedto 60°C, and tested for dimerization. As shown in Fig. 3B,RNA-4 dimerized efficiently in the absence of oligonucleotides(lane 1) and in the presence of DNAs complementary to sequencesimmediately upstream or downstream of DIS-1 (lanes 2 and 7)or to the primer binding site (lane 9). In contrast, oligonucleotidescomplementary to part (Fig. 3B, lanes 3, 5, and 6) or all (lane4) of DIS-1 completely blocked dimer formation, as did the singleoligonucleotide that targeted DIS-2 (lane 8). These resultsfunctionally verify the extent and location of DIS-1 and confirmthat both DIS-1 and DIS-2 are specifically required for theformation and/or stability of Mo-MuLV dimers in vitro.

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FIG. 3. In vitro dimerization of full-length RNA (RNA-4) is inhibited by antisense DNA oligonucleotides complementary to the DIS sequences. (A) Antisense DNA oligomers complementary to various regions of the Mo-MuLV 5' UTR were designed; their properties are detailed in Table 1. PBS, primer binding site. (B) ${alpha}$ -³²P-labeled RNA-4 (1.5 nM) was incubated with 60 µM concentrations of various unlabeled antisense oligomers at 60°C for 1 h and then analyzed on a 5% nondenaturing polyacrylamide gel. Dimerization of RNA-4 was inhibited only by oligomers that overlapped portions of the predicted palindromes in DIS-1 or DIS-2. D, dimeric RNA; M, monomeric RNA. ${alpha}$ DIS-1, ${alpha}$ DIS-2, and ${alpha}$ PBS, antisense oligonucleotides complementary to DIS-1, DIS-2, and PBS sequences, respectively.

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TABLE 1. Antisense oligonucleotides

Mutational evidence of base pairing in the DIS-1 and DIS-2 stems. We used a mutational approach to assess whether the putativestem regions of DIS-1 and DIS-2 fold as predicted. Into thedimerization-competent RNA-4 sequence (residues 146 to 621),we first introduced point mutations designed to disrupt allor most predicted base pairing of the DIS-1 and DIS-2 stems(mutants ${Delta}$ stem1 and ${Delta}$ stem2, respectively) and then created compensatorymutations that could restore base pairing (mutants comp1 andcomp2). As shown in Fig. 4, we found that disrupting eitherstem alone substantially inhibited dimer yield and stabilitybut that compensatory mutations restored dimerization to nearlywild-type levels. These results suggest that the stems of DIS-1and DIS-2 each form and contribute to dimer formation in vitro.

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FIG. 4. The predicted stems of DIS-1 and DIS-2 form and are essential for in vitro dimerization. Wild-type RNA-4 (WT) and four mutant forms of this RNA were tested for dimerization after preincubation at the temperatures indicated. The mutations were designed to either disrupt ( ${Delta}$ stem1 and ${Delta}$ stem2) or maintain (comp1 and comp2) base pairing in the predicted stems of DIS-1 and DIS-2, producing the hypothetical secondary structures shown at the top. D, dimer; M, monomer.

Evidence for homologous loop-loop interactions. To determine conclusively whether the slower-migrating speciesseen on electrophoresis resulted from loop-mediated DIS dimerization,we used a heterodimerization assay described previously (4,27). In this assay, two RNAs of different lengths and each containinga candidate DIS locus are combined and tested for dimerization;the detection of a novel intermediate-sized complex distinctfrom either homodimer implies that the two RNAs have heterodimerized.For the present study, we prepared synthetic Mo-MuLV transcriptsthat were 280 or 475 bases long and that each spanned both DIS-1and DIS-2; we also prepared variants of these transcripts thatcarried specific point mutations in the four central bases ofeach DIS loop palindrome to test their effects on dimer formation.As shown in Fig. 5A, we found that a short (280-base) RNA containingthe wild-type DIS-1 loop palindrome (CCGG) homodimerized efficiently(lane 1), as did a long (475-base) RNA containing the relatedpalindrome GGCC (lane 2). When these two were combined, eachhomodimerized independently, but no heterodimers were observedat either 50 or 60°C (Fig. 5A, lanes 3 and 4). In contrast,when we combined two other RNAs carrying the nonpalindromicDIS-1 loop sequences CCCC and GGGG, each failed to homodimerize,but abundant heterodimers (HD) were observed (Fig. 5A, lanes5 and 6). Similarly, RNAs bearing either the wild-type sequenceAGCU or the variant palindrome UCGA in the DIS-2 loop formedhomodimers but not heterodimers (Fig. 5A, lanes 7 and 8), whereasthe two partially complementary DIS-2 variants UCGA and UGCAwere able to support heterodimer formation (lanes 9 and 10).Identical results were obtained when the loop sequences in thelong and short RNAs were reversed (Fig. 5B). Together, theseresults suggest that dimerization normally depends on homologousbase pairing at the DIS-1 and DIS-2 loops, as would be expectedif each of these loci homodimerized through loop-loop interactions.

Effects of DIS mutations in Mo-MuLV virions. We next explored the effects of mutating the DIS loci on theproperties of Mo-MuLV virions. For this purpose, mutations wereintroduced into the candidate DIS sequences in pINA10, an Mo-MuLV-derivedretroviral vector carrying a neo^r gene. The resulting mutantvectors were transfected into the packaging cell line GP+E86,which is capable of incorporating wild-type pINA10 transcriptsinto Mo-MuLV virion-like particles that are then capable oftransducing the selectable neo^r marker into susceptible targetcells. Enumeration of the resulting neomycin-resistant coloniesprovides a measure of the transduction efficiency of the virus-likeparticles. As summarized in Fig. 6, we found that preciselydeleting either DIS-1 or DIS-2 alone reduced transduction byapproximately 90% compared to the results obtained with wild-typepINA10. Indeed, simply altering two of the four central basesof the DIS-1 loop palindrome to create the nonpalindromic sequenceCCCC or GGGG inhibited transduction to nearly the same degreeas deleting DIS-1 entirely. In contrast, four other mutationsthat preserved the palindromic natures of the DIS-1 or DIS-2loops caused somewhat lesser deficits, in most cases inhibitingtransduction by only 30 to 40%. Thus, in general, the relativeimpact of each mutation on transduction efficiency in vivo correlatedwith its effect on RNA dimerization in vitro.

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FIG. 6. Relative titers of Mo-MuLV virus-like particles carrying DIS mutations. Particles were harvested from the supernatants of cells transfected with derivatives of the vector pINA10 that contained either wild-type (WT) or mutant forms of the Mo-MuLV leader region. The samples were assayed for transducing titers by using a colony-forming assay (see Materials and Methods). The mutations tested included precise deletions of DIS-1 or DIS-2 ( ${Delta}$ DIS-1 or ${Delta}$ DIS-2, respectively) or substitutions of the indicated four-base sequences for the four central bases of the palindrome in DIS-1 or DIS-2. Titers were normalized to that of the WT vector, which averaged 5.7 x 10⁵ CFU/ml in the experiment shown here. Data were from three independent assays. Error bars show standard deviations.

To determine whether deleting the DIS loci affects RNA dimerizationin these virion-like particles, we extracted RNA from particlescarrying both DIS-1 and DIS-2 mutations, incubated it at varioustemperatures, and then analyzed it by nondenaturing gel electrophoresisand Northern blotting. As shown in Fig. 7, wild-type RNA containingboth DIS loci migrated primarily as dimer-sized complexes (D)that dissociated into apparent monomers (M) at a melting temperatureof 60°C (lanes 1 to 3). The RNA lacking both DIS loci, incontrast, yielded dimers with a melting temperature of lessthan 55°C. These results closely parallel those that weobtained for the in vitro dimerization of the same mutant constructs(Fig. 2) and confirm the contributions of these DIS loci toMo-MuLV RNA dimerization in vivo.

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FIG. 7. Nondenaturing Northern blots of virion-derived RNAs. Deproteinized RNAs extracted from Mo-MuLV virion-like particles containing either wild-type (WT) pINA10 or a mutant that lacked both DIS-1 and DIS-2 ( ${Delta}$ DIS-1 ${Delta}$ DIS-2) were preheated to the indicated temperatures for 10 min, electrophoresed on a 1% agarose gel at 4°C, blotted onto a nylon membrane, and probed for pINA10 sequences. All lanes shown are from a single blot, but exposure times for the two constructs varied to compensate for differences in image intensity; these differences may reflect disparities in RNA packaging, virion release, or other, undetermined factors. Results shown are typical of three independent experiments. D, dimer; M, monomer.

DISCUSSION

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Discussion

The importance of Mo-MuLV as both a model retrovirus and a versatilegene transfer vector has prompted numerous studies of the cis-actingdimerization signals in its genome. Early electron microscopicimages of dimers from Mo-MuLV virions identified the DLS asthe most stable point of contact between RNA strands, suggestedthat it linked the strands in parallel (i.e., 5' to 5'), andindicated its location as approximately 400 bases from the 5'end (2, 22, 28, 37, 47). Later, artificial DLS dimers formedfrom synthetic Mo-MuLV sequences showed a similar morphology,a finding that helped confirm the validity of the in vitro dimerizationassay (44). The in vitro DLS could also be mapped more preciselyto residues 215 to 420, a region previously shown to encompasscritical packaging signals (29, 45), and this finding raisedthe possibility that RNA dimerization and packaging are mechanisticallylinked (44). Subsequent research on both processes has tendedto focus primarily on this region of the genome.

Chemical probing, computerized structure prediction, and mutationalanalyses all indicate that Mo-MuLV residues 215 to 420 can foldinto at least three separate stem-loops (1, 7, 15, 51). Themost 3' of these, which we term DIS-2 (other authors refer toit as stem-loop B or H1), is centered around residues 283 to298 and has been shown to be essential for dimerization in vitro(7, 15, 16) as well as to enhance the efficiency of genomicpackaging into Mo-MuLV virions (12, 34, 35). The palindromicsequence of its loop, together with thermodynamic, kinetic,and chemical evidence that it undergoes a conformational changeduring dimerization (51), suggests that DIS-2 dimerizes througha kissing-loop pathway similar to that described for other retroviruses(4, 8, 9, 11, 19, 20, 25, 26, 33, 39). Two other presumed stem-loopsdownstream of DIS-2 (at residues 310 to 352 and residues 355to 374) appear to function primarily in packaging (12, 34, 35)but also reportedly enhance the kinetics of DIS-2-dependentdimer formation (7), perhaps by homodimerizing weakly throughtheir loops (21). Similarly, the inclusion of additional sequencesfarther downstream (residues 364 to 565) was also found to stabilizeDIS-2-dependent dimers (16), though the exact sequences involvedhave not been further characterized.

Nearly all of the foregoing results were derived from studiesof RNAs that lacked most sequences upstream of DIS-2. Oroudjevand colleagues (38), in contrast, recently reported that thoseupstream sequences alone could dimerize efficiently in vitroand that this scenario depended on a hypothetical stem-loopat positions 204 to 228. This upstream element, which we termDIS-1, is well conserved among murine C-type retroviruses, includingthose that lack any apparent ortholog of DIS-2. Data from Mikkelsenand coworkers (32) further suggested that DIS-1 might serveas a hot spot for template switching by reverse transcriptase,an activity similar to that reported for DIS-2 of Mo-MuLV andfor DIS elements in certain other retroviruses. In light ofthose findings, it became unclear whether DIS-1 or DIS-2 wasthe primary initiator of Mo-MuLV dimerization.

Our study reconciles and extends the earlier reports by showingthat both proposed DIS loci contribute to dimer formation. Byanalyzing longer RNAs that span the entire 5' untranslated regionand so encompass both DIS-1 and DIS-2, we found that preciselydeleting either locus alone partially inhibits in vitro dimerformation but that deleting both eliminates dimerization altogether(Fig. 2B). We observed that each locus contributes independentlynot only to the yield but also to the thermal stability of invitro dimers: whereas the wild-type dimer was stable at 60°C,dimers lacking either DIS-1 or DIS-2 or the loop sequences ofthese two structures were generally more abundant at 50°Cthan at 60°C (Fig. 5 and data not shown). These findingsare in accord with published melting data for shorter RNAs thatcontained one or the other DIS (38, 44). Our data can also accountfor the earlier observation that deleting DIS-2 along with twodownstream stem-loops reduced in vitro dimer yield by only about40%, provided that upstream sequences remained intact (7).

Although the detailed structures of DIS-1 and DIS-2 remain unknown,the results of our site-directed mutagenesis studies help confirmthat each folds as predicted and that their folded structuresare important for efficient dimer formation. Mutations thatdisrupt the proposed stem structures markedly reduced dimerizationin our in vitro assay, whereas compensatory mutations designedto restore those stems yielded nearly wild-type levels of dimerization(Fig. 4). Similarly, a mutational analysis of the predictedloop regions indicated that the in vitro DIS activity of eitherlocus depends upon Watson-Crick complementarity between theloops (Fig. 5 and data not shown). To our knowledge, these arethe first reported results of point mutagenesis for either locus,and they strongly support the assumption that each dimerizesindependently through a kissing-loop pathway.

We also tested the effects of DIS mutations on RNA dimerizationwithin Mo-MuLV virion-like particles in vivo by using a truncatedrecombinant Mo-MuLV genome in a previously validated cell culturemodel (33). As shown in Fig. 7, we found that deproteinatedRNA extracted from these particles was present primarily inthe form of a dimer that melted at 55 to 60°C; these temperaturesare comparable to the melting temperatures previously reportedfor genomic dimers extracted from authentic Mo-MuLV (13, 28).In agreement with our in vitro data, we found that deletingboth DIS-1 and DIS-2 reduced dimer stability, so that meltingwas essentially complete at 55°C. It is not surprising thatthis double deletion did not abolish in vivo dimer formationcompletely, since dimer contacts are believed to occur alongthe entire length of a genomic strand once it has been encapsidated(39). Similar instability has been reported for some DIS mutantsof human immunodeficiency virus type 1 (4, 5, 18, 24). In ourseries, mutations that inhibited dimer formation in vitro alsotended to reduce viral titer in vivo (Fig. 6), but is not clearto what extent these infectivity defects can be attributed toaberrant dimerization, since the mutated regions also influencepackaging, reverse transcription, and possibly other aspectsof Mo-MuLV replication (12, 34, 35).

Our findings indicate that two separate stem-loop elements,situated 55 bases apart in the Mo-MuLV leader region, each contributeto genomic dimer initiation through independent, homologouskissing-loop interactions. Although this may be the first documentedexample of dimer initiation through dual kissing loops, thenotion that two or more adjacent loci can act in concert toachieve dimer linkage is not unprecedented and may prove tobe a common theme among retroviruses (11, 26, 49, 50). In murinesarcoma virus, for example, the genomic strands are thoughtto associate through specific G-rich elements, which then facilitatedimerization through an adjacent kissing loop (25, 26). Evenin Mo-MuLV itself, additional elements downstream of the bipartiteDIS can enhance the kinetics and stability of dimer formation(7, 16). Although not yet proven, it seems likely that the twoconsecutive stretches of interstrand base pairing created bythe bipartite DLS of Mo-MuLV (Fig. 8) may appear as a singleDLS structure when viewed at the resolution of the electronmicroscope. This topology illustrates how two or more locallyantiparallel linkages can give rise to a globally parallel genomicdimer.

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FIG. 8. Proposed model of the globally parallel genomic dimer formed by two locally antiparallel DIS loci in Mo-MuLV.

ACKNOWLEDGMENTS

We thank E. Barklis for the generous gift of pINA10 vectors,J. L. Clever for technical advice and many stimulating discussions,and D. Miranda and Y. Liang for help with artwork.

This research was supported by NIH grants AI37036 and AI40317to T.G.P. H.L. was supported in part by UCSF AIDS postdoctoraltraining grant NIH AI07395.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Box 0511, University of California, San Francisco, CA 94143-0511. Phone: (415) 476-1015. Fax: (415) 514-3165. E-mail: parslow{at}cgl.ucsf.edu.

REFERENCES

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