An additional dimer linkage structure in moloney murine leukemia virus RNA1

Article No. jmbi.1999.2984 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 291, 603±613

An Additional Dimer Linkage Structure in Moloney Murine Leukemia Virus RNA Emin M. Oroudjev1, Peter C. E. Kang1 and Lori A. Kohlstaedt1,2* 1

Department of Chemistry and

2

Program in Biochemistry and Molecular Biology, University of California, Santa Barbara CA 93106, USA

We have identi®ed an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 50 ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identi®ed at nucleotides 278-303. Here, we describe RNA dimers formed from sections of the MoMLV 50 untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199-243. This sequence contains a stem-loop structure (nucleotides 204-227) much like the 278-303 region. We describe the chemical and thermal stability of dimers containing the 204-227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 50 untranslated region may contain separately folding and dimerizing domains. # 1999 Academic Press

*Corresponding author

Keywords: retrovirus; RNA structure; RNA dimerization; recombination; reverse transcription

Introduction The virions of retroviruses contain two copies of the viral genome. These genomic RNAs are linked near their 50 ends in an apparently parallel orientation. When viewed by electron microscopy under partially denaturing conditions, the link appears as a chromatin-like particle (Bender & Davidson, 1976; Bender et al., 1978). Dimers remain linked even in completely deproteinized samples. In fact, RNA fragments containing critical sequences can dimerize spontaneously in vitro without the assistance of any protein (Beith et al., 1990; Prats et al., 1990; Roy et al., 1990). In vivo or under physiological conditions, nucleocapsid protein or the gag precursor is necessary to complete dimerization (Beith et al., 1990; Prats et al., 1990; Roy et al., 1990; De Rocquigny et al, 1992; Lapadat-Tapolsky et al, 1995). Abbreviations used: DEPC, diethylpyrocarbonate; PEG, polyethylene glycol; MoMLV, Moloney murine leukemia virus; HIV-1, human immunode®ciency virus type 1; DLS, dimer linkage structure; PBS, primer binding site; gRNA, full-length genomic RNA. E-mail address of the corresponding author: [email protected] 0022-2836/99/330603±11 $30.00/0

The RNA sequences responsible for the dimerization of Moloney murine leukemia virus (MoMLV) genomic RNA (gRNA) have been investigated. Early experimentation localized dimerization to a region called PSI, nucleotides 215-565, which was shown to contain sequences necessary for encapsidation (Mann & Baltimore, 1985; Mougel & Barklis, 1997). Later, deletion analysis and study of RNAs synthesized in vitro allowed identi®cation of 278-303 as a sequence that can nucleate dimerization (Prats et al., 1990; Tounekti et al., 1992; Girard et al., 1995). This latter region is called the DLS and is also part of the encapsidation signal. The DLS can form a hairpin, and dimerization is nucleated through base-pairing of a palindrome sequence in the hairpin loop (Girard et al., 1995, 1996). More recently, it has been realized that dimerization contacts are quite extensive, and that other regions in the 50 untranslated region participate in dimerization. Two other stem-loops adjacent to DLS278-303, at 310-352 and 355-374, participate in dimerization, enhancing the activity of DLS278-303 (De Tapia et al., 1998). A region farther 30 , between nucleotides 364 and 565, can dimerize independently of DLS278-303 (Girard et al., 1996). De Tapia et al. (1998) showed that regions both 50 and 30 of 215-565 can dimerize, although the 50 end of the # 1999 Academic Press

604 genome dimerized only weakly and neither region formed heterodimers with longer RNAs. Indeed, since the encapsidation signal PSI is thought to extend between nucleotides 205 and 1035 (Bender et al, 1978), other functions may extend throughout the region. Parallel ®ndings with other retroviruses show that it is common for multiple regions to participate in dimerization (Feng et al., 1995; Torrent et al., 1994; Katoh et al., 1993). RNA dimers formed by the MoMLV 50 untranslated region have a number of distinctive characteristics. The DLS is embedded in an area rich in secondary structure (Tounekti et al., 1992; Mougel et al., 1993), which is one of at least three RNA folding domains present in the 50 untranslated region (Tounekti et al., 1992; Mougel et al., 1993; De Tapia et al., 1998). Dimerization results in changes to structure that re¯ect both secondary and tertiary structure (Tounekti et al., 1992); contacts in the mature dimer are thought to be mediated by Watson-Crick base-pairing and by other sorts of interactions. In particular, dimerization is enhanced in the presence of Mg2‡ (Roy et al., 1990), suggesting that some structural contacts are mediated through metal ion coordination. The dimerization interaction is quite stable. The melting temperature of the genomic dimer is estimated to be about 60  C (Bender et al., 1978), and some dimer contacts persist even in 50 % (v/v) formamide and 2.5 M urea (Murti et al., 1981). A fragment of the 50 untranslated region containing the DLS retains most of this stability, but other dimerizable fragments that have been tested do not make such stable contacts (De Tapia et al., 1998). In the work described here, we show that another sequence can nucleate dimerization with an ef®ciency and stability comparable to that of the whole 50 region or the previously identi®ed DLS278303. This region contains a sequence that can form a hairpin loop containing a palindrome and most likely nucleates RNA dimerization by the same mechanism as DLS278-303. This newly identi®ed sequence brings the number of probable sites for the nucleation of dimerization to at least three. These three sequences may represent separate folding domains in the mature genome dimer. Moreover, this newly identi®ed dimer linkage structure lies near the primer binding site, raising the possibility that the template for initiation of reverse transcription is locally a dimer.

Results A dimerization signal exists in the region 50 to the previously identified DLS The ®rst RNA we studied was a fragment from the 50 untranslated region of MoMLV gRNA corresponding to nucleotides 28-258, which we call TR(28-258). This transcript included the U5 region along with the primer binding site (PBS) and part of the leader sequence (Figure 1). We noted an anomalous mobility for this RNA on agarose gel

Moloney Murine Leukemia Virus RNA Dimerization

Figure 1. A diagram of in vitro transcribed RNAs containing various portions of the MoMLV 50 untranslated region and summary of the ability of each of these to dimerize. The positions of 50 and 30 ends for each transcript relative to full-length MoMLV gRNA are indicated by the numbers in parentheses. The right-hand column indicates the percentage of each RNA converted to the dimeric form after incubation at standard dimerization conditions for one hour. The top line shows the positions of the various functional regions of the 50 untranslated region relative to these transcripts. R ‡ U5, the R and U5 regions; PBS, the tRNA primer binding site; 50 -DLS, the predicted dimer linkage structure at nucleotides 204-228 (DLS204-228); 30 -DLS, the well-studied dimer linkage structure at nucleotides 278-303 (DLS278-303).

electrophoresis. Under non-denaturing conditions, electrophoresis revealed that freshly synthesized TR(28-258) RNA contained two major bands with mobilities of 270 nucleotides and 450 nucleotides relative to DNA markers (Figure 2, lane 1). The true size of the transcript is 255 nucleotides (see Materials and Methods); RNAs have lower mobilities relative to DNAs of the same length. When the RNA from the low-mobility band was extracted from the gel, heated to 95  C and quickly cooled on ice, all the RNA was converted to a single fastmobility band with the size expected for the RNA transcript (Figure 2, lane 2). Suspecting that the low-mobility band might represent a dimer, we treated the previously heated TR(28-258) RNA with conditions reported to cause dimerization of MoMLV gRNA (Roy et al., 1990). Under these ``standard'' dimerization conditions (100 mM NaCl, 10 mM MgCl2, pH 7.6, 16 hours, 37  C) TR(28-258) was able spontaneously to reform the low-mobility band (Figure 2, lane 3), suggesting that the RNA does indeed dimerize. Formation of heterodimers with transcripts of different lengths con®rms this interpretation (see below). A minor fraction (20 %) of the monomeric form of TR(28-258) RNA was detected even after prolonged (16 hours) incubation under dimerization conditions (Figure 2, lane 3). To con®rm that all

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Moloney Murine Leukemia Virus RNA Dimerization

Figure 2. An RNA consisting of nucleotides 28-258 from the 50 untranslated region of MoMLV RNA can dimerize. TR(28-258) RNA samples were separated on 2.5 % agarose gel and stained as described in Materials and Methods. Lane 1, total unpuri®ed TR(28-258) RNA directly after in vitro synthesis. Lane 2, the same RNA after gel puri®cation and heat denaturation (95  C for ®ve minutes). Lane 3, puri®ed RNA as in lane 2 but after incubation for 16 hours at standard dimerization conditions. The positions of DNA molecular mass markers are shown.

RNA in our sample was chemically although not conformationally equivalent, we puri®ed the residual monomer from a sample of RNA that was mostly converted to dimer. This residual monomeric sample was refolded by heating to 95  C for ®ve minutes followed by slow cooling to room temperature. When reincubated under the dimer formation conditions given above, this fraction converted, in large part, to dimer, exhibiting the same monomer-dimer equilibrium seen in the original sample (data not shown). A number of other groups have observed a fraction of remaining monomer in dimerization experiments with gRNA or RNA fragments containing the DLS at nucleotides 303-378 (Girard et al., 1995; De Tapia et al., 1998). They attributed a slow-dimerizing fraction to the misfolding of a fraction of the RNA into conformations where sequences that nucleate dimerization are inaccessible.

from the 50 end and 64 nucleotides from the 30 end relative to TR(28-258), has a dramatically decreased ability to form dimers. Two RNAs, TR(28-196) and TR(79-258), both of which have one end in common with TR(28-258), have very disparate abilities to dimerize. TR(79-258), with its 30 end in common with the longer RNA, still dimerizes well; TR(28196) with the 50 end in common dimerizes only weakly. An RNA containing only PBS and a portion of the leader up to nucleotide 258, TR(140258), also dimerizes well, showing that the sequence responsible for dimerization resides in the 30 third of the original TR(28-258). Further truncation analysis from either the 50 -end (TR(163-258), TR(182-258), TR(199-258)), the 30 -end (TR(79-243), TR(79-230), TR(79-213)) or from both ends (TR(199243)) mapped this novel site where dimerization can occur to between nucleotides 199 and 243. Secondary structure analysis of this section of MoMLV gRNA shows that the RNA sequence between 204 and 228 can adopt a stem-loop structure containing a ten nucleotide long palindrome (Figure 3). The potential to form such a structure suggests that dimerization occurs at 204-228 through the same mechanism as that of the previously recognized DLS at 278-303 (Girard et al., 1995, 1996; De Tapia et al., 1998). To distinguish between these sequences that can independently nucleate dimerization, we will designate these two DLS sequences as DLS204-228 and DLS278-303. Formation of heterodimers We were able to form heterodimers between RNAs of different lengths that contain DLS204-228 (Figure 4). A portion (0.4 mg) of TR(199-258) in monomeric form was mixed with 0.2 mg of monomeric TR(28-258) RNA. These RNAs were able to form heterodimers when incubated under standard dimerization conditions for 60 minutes. Two other RNAs (TR(79-258) and TR(79-230) ) were tested and were able to form heterodimers with TR(28258) (data not shown). In addition to con®rming the dimeric nature of the low-mobility bands in our RNA preparations, these experiments show

Mapping a novel region involved in dimerization To map the region in TR(28-258) responsible for dimerization, we generated a number of shorter RNAs (Figure 1). We assessed the ability of these truncated RNAs to form dimers and compared the extent of dimer formation to full-length gRNA. In each case, the general protocol described above was followed, and dimer formation was assessed by measuring the amount of RNA in the dimer and monomer bands after three hours of incubation under standard dimerization conditions. TR(79-196), an RNA shortened by 51 nucleotides

Figure 3. Predicted secondary structure for nucleotides 200-230 of MoMLV gRNA. Bold type indicates the palindrome sequence inside the stem-loop structure.

606

Figure 4. Formation of heterodimers between TR(28258) and TR(199-258) RNA transcripts. RNAs were incubated at standard dimerization conditions for 16 hours and then analyzed by agarose gel electrophoresis. Positions of DNA molecular mass markers are indicated. Lane 1, TR(199-258) RNA alone. Lane 2, TR(199-258) RNA mixed with TR(28-258) RNA. Lane 3, TR(28-258) RNA alone.

that the ability to form dimers is not signi®cantly affected by changing the overall length of the RNA containing DLS204-228, provided that the 204-228 hairpin is intact.

Moloney Murine Leukemia Virus RNA Dimerization

at each temperature, samples were quickly quenched by chilling to preserve their dimerization state. The results of this melting experiment in the absence of Mg2‡at several RNA concentrations are shown in Figure 5. The apparent tm for the dimer was highly dependent upon the concentration of RNA. This marked concentration dependence can be attributed to a rapidly established dimer/monomer equilibrium at the higher concentrations. Consequently, the true tm in the absence of magnesium is probably best estimated from the lowest concentration and is approximately 52  C. In the presence of 10 mM MgCl2, the TR(28-258) dimer is resistant to thermal denaturation at all tested concentrations (data not shown). The only loss of dimer that occurred in the presence of 10 mM MgCl2 was due to spontaneous RNA degradation at temperatures above 80  C. The in¯uence of Mg2‡ on dimer stability was further investigated by subjecting preformed dimer to formamide denaturation. The presence of magnesium in millimolar concentrations greatly increased the stability of dimers toward formamide denaturation. Preformed dimer (0.05 mg/ml) was incubated for 60 minutes at 37  C either in magnesium-free dimerization buffer (100 mM NaCl, 30 mM bistrispropane buffer, pH 7.6) or in the same buffer with 10 mM MgCl2. The concentration of formamide was varied (Figure 6(a)). In the absence of MgCl2, 30 % formamide was suf®cient to transform almost all the dimer back into monomeric form. Addition of MgCl2 to 10 mM, however, made this dimer almost completely resistant to high concentrations of formamide.

Optimum temperature for dimer formation We compared dimers formed from the region in MoMLV containing DLS204-228 to dimers of the whole 50 untranslated region and to dimers containing the previously studied DLS278-303. To do so, we studied a number of physical parameters of dimer formation. These experiments were performed with TR(28-258). RNA was puri®ed, denatured, and then allowed to dimerize at a concentration of 0.02 mg/ml in standard dimerization buffer for one hour. We tested the temperature-dependence of dimer formation. Although TR(28-258) RNA was able to dimerize to a great extent at 37  C, we found that the optimum temperature was near 55  C (data not shown). The same temperature optimum was found for dimerization of the entire 50 untranslated region (Roy et al., 1990). Dimer stability We analyzed the heat-stability of dimers using a thermal cycler to apply a step temperature gradient. Varying concentrations of preformed TR(28-258) dimer were subjected to incrementally increased temperature. After allowing equilibration

Figure 5. Temperature-induced dissociation of preformed TR(28-258) dimers at varying RNA concentrations. Preformed dimers of TR(28-258) at concentrations of 100 nM (!), 400 nM (^), 2 mM (*) and 4 mM ( & ) were subjected to incremental temperature increase (see Materials and Methods), and the percentage dimer remaining at each temperature was determined.

607

Moloney Murine Leukemia Virus RNA Dimerization

Kinetics of dimerization RNA sequences containing DLS204-228 form dimers at a similar rate compared to the whole 50 untranslated region or RNAs containing DLS278-303. Dimerization of TR(28-258) RNA, which contains DLS204-228, is a relatively slow, two-phase process (Figure 7(a)). To study the time-dependence of dimer formation, we ®rst denatured TR(28-258) and placed the sample on ice. RNA was then diluted to 0.02 mg/ml in standard dimerization buffer and 10 ml aliquots were placed at 37  C or 55  C to allow dimerization. Aliquots were transferred back to ice for each time-point; a control remained on ice at all times. During the ®rst 10-15 minutes, dimer formation at 37  C or 55  C is relatively rapid; this initial phase is followed by a ``slow'' phase, where dimer continues to accumulate. These two kinetic phases can be explained if there are multiple conformations present in the monomer, not all of which are capable of dimerization, and interconversion of monomer conformations is slow relative to dimer formation. The extent of dimerization is greater at 55  C, possibly because the interconversion of monomer conformations is faster at the elevated temperature. Equilibrium character of dimers containing DLS204-228

Figure 6. Stability of TR(28-258) RNA dimers toward formamide denaturation. The percentage of RNA in the dimer form was determined as described in the text. (a) Effect of formamide concentration on dimer stability in the presence (*) or absence ( & ) of 10 mM MgCl2. For each sample, 0.05 mg/ml preformed dimer was incubated for one hour at 37  C under the given conditions. (b) In¯uence of varying MgCl2 concentration on denaturation by 50 % formamide. For each sample, 0.05 mg/ ml preformed dimer was incubated for one hour at 37  C in 50 % formamide with the indicated concentration of MgCl2.

The dimer formed from sequences containing DLS204-228 is in dynamic equilibrium. TR(28-258) RNA starts to dissociate back to monomer when diluted to a concentration of 40 nM or less (data not shown). Therefore, we decided to estimate the dissociation constant (Kd) for TR(28-258) RNA dimers. A constant amount of 32P-labeled monomeric TR(28-258) RNA (0.4 nM) was mixed with 1210 nM of unlabeled monomeric RNA in 10 ml reactions. After incubation at standard dimerization conditions for two hours, the samples were separated by electrophoresis and the amount of monomeric and dimeric RNA was determined (Figure 7(b)). The Kd for TR(28-258) was estimated as 10-20 nM by the method of Paillart et al. (1996). This Kd is very similar to that for dimers of short RNAs carrying the previously identi®ed MoMLV DLS278-303 sequence (8-40 nM) as well as for dimers of the entire MoMLV 50 untranslated region (40 nM) (De Tapia et al., 1998). Effect of Mg2‡ on dimerization

The effect that magnesium exerts on the stability of the RNA dimer is dependent on the Mg2‡ concentration. We incubated 0.6 mM preformed dimer for one hour at 37  C in the presence of 50 % formamide and different concentrations of MgCl2 (Figure 6(b)). A dramatic increase in stability occurs, starting at about 10 mM MgCl2, which does not reach saturation as the MgCl2 concentration is raised to 100 mM.

Dimerization for the entire 50 untranslated region and for the PSI domain alone is sensitive to the concentration of salts of various monovalent and divalent metals (Roy et al., 1990; De Tapia et al., 1998). Magnesium seems to play a special role in dimerization of both the PSI region and the whole 50 untranslated region of MoMLV. In the absence of Mg2‡, high concentrations of KCl (2501000 mM) were able to increase signi®cantly the ability of TR(28-258) RNA to dimerize (Figure 7(c)).

608

Moloney Murine Leukemia Virus RNA Dimerization

Figure 7. Details of the formation of TR(28-258) dimers. In all cases, the percentage of RNA in the dimeric form was determined from the relative intensity of the dimer and monomer gel bands as described in the text. (a) Kinetics of TR(28-258) RNA dimerization. Denatured RNA (0.02 mg/ml) was incubated at standard dimerization conditions at 37  C (^) or 55  C (*) for the indicated times. (b) Formation of dimer as a function of RNA concentration. Monomeric RNA, supplemented with a constant amount of 32P-labeled RNA, was incubated at varied concentrations at standard dimerization conditions for two hours. The amount of dimer was determined by measuring the presence of the 32P-labeled tracer in each band. (c) Dimerization as a function of KCl concentration in the absence of magnesium. After treatment with 50 mM EDTA, monomeric RNA (0.02 mg/ml) was incubated at 37  C for one hour in the presence of varying concentrations of KCl. In the presence of magnesium, the amount of dimer formed was not dependent on KCl concentration (data not shown). (d) Kinetics of dimerization as a function of MgCl2 concentration. Monomeric RNA (0.02 mg/ml) was incubated at standard dimerization conditions for varied times with 0.5 mM ( & ), 2 mM (*), 10 mM (~) and 50 mM (^) MgCl2.

Monomeric TR(28-258) was mixed with varying concentrations of KCl, and dimerization was assessed after a 60 minutes incubation under standard conditions. The dependence of dimerization on salt concentration indicates that classical Watson-Crick interactions play an important role in dimer formation and/or stabilization. When the concentration of monovalent cation was varied in the presence of 5 mM MgCl2, however, no signi®cant effect on dimerization was seen (data not shown).

Magnesium ion concentration had a pronounced effect on the dimerization of RNAs containing DLS204-228. For these experiments, monomeric RNA was ®rst stripped of bound magnesium ions by heating to 98  C in the presence of 50 mM EDTA. If treatment with EDTA was omitted, enough Mg2‡ remained bound to the RNA in the denatured state to mask the response to change in Mg2‡ concentration (data not shown). Monomeric RNA (0.02 mg/ml) was incubated at standard dimerization conditions with varying concen-

Moloney Murine Leukemia Virus RNA Dimerization

trations of MgCl2 (Figure 7(d)). Dimerization reactions were stopped by transferring samples to ice. Zero time-points were kept on ice in identical solution conditions throughout the experiment. The effect of Mg2‡ was detected in the rate of the ®rst ``fast'' phase of dimer formation. The rate of dimerization in the ``slow'' phase was essentially the same at different concentrations of Mg2‡. These results suggest that Mg2‡ is involved in the process of forming important dimer contacts but that it plays a minor role, at most, in structural rearrangements of the various monomeric conformations of the RNA.

Discussion We have identi®ed and characterized a novel DLS (dimer linkage structure) within the region 199-243 of MoMLV gRNA. This region is at the 50 end of the Psi domain, which is involved in encapsidation (Mann et al., 1983; Mann & Baltimore, 1985; Mougel & Barklis, 1997; Adam & Miller, 1988) as well as in dimerization (Roy et al., 1990) and recombination (Mikkelsen et al., 1998) of the viral genome. Secondary structure prediction analysis for the 199-243 region of MoMLV shows the presence of a ten base palindrome (210-219) that is predicted to be part of a hairpin-loop structure at positions 204-228 (Figure 3). The previously discovered DLS278-303 from MoMLV contains a 16 residue palindrome that is predicted to fold into a hairpinloop structure (Prats et al., 1990; Girard et al., 1996). Recently, two additional stem-loops, H2 and H3, which are adjacent to DLS278-303,were shown to contribute to dimerization (De Tapia et al., 1998). In addition, the sequence-related retrotransposon

609 VL30 contains similar hairpin sequences (Torrent et al, 1994). Such hairpin-loops are thought to be involved in genome dimerization (for review, see Paillart et al., 1996), as well as recombination (Mikkelsen et al., 1998) of retroviruses through a ``kissing-loop'' mechanism (Girard et al., 1995; Laughrea & Jette 1996; Paillart et al., 1996). Overall similarities discussed here suggest that dimerization driven by DLS204-228 also occurs through a kissing-loop interaction. Recently, the kissing-loop model received additional support from NMR studies on the HIV-1 DLS (Mujeeb et al., 1998), which demonstrated that formation of a kissingloop complex can occur via direct base-pairing between unpaired bases in the loops. The kissingloop structure is thought to be only an intermediate that is subsequently transformed into mature dimer through melting of the hairpin part of the complex and extension of base-pairing (Girard et al., 1995). We used RNA secondary structure prediction (GENETYX v.8.0 , Software Development Corp.) to model interactions between two copies of the 199243 region of MoMLV gRNA (Figure 8). Although we used a variety of restraints and simulated different conditions in these predictions, this pair of RNA molecules was always predicted to form a stable dimer between positions 210 and 219. Moreover, sequences at both the 50 and 30 ends of DLS204-228 were predicted to form additional intermolecular interactions, and thus, could be involved in the overall stabilization of the dimer. A region important to dimerization or to other critical viral functions such as recombination should be conserved in related viruses. Sequence alignments of viral 50 untranslated regions show

Figure 8. Proposed model for the progression of the initial ``kissing-loop'' interaction between 204-228 hairpins to the formation of a matured, dimerized state. The model is based on secondary structure prediction using the program Genetyx 8.0 (Software Delevopment Corp.). (a) Hairpin-loop structures exist in each of the monomeric RNAs. (b) A ``kissing-loop'' complex involving nucleotides in the unpaired loops forms between the two RNAs. (c) Additional interactions between sequences ¯anking the palindrome stabilize the ``matured'' dimer.

610

Figure 9. Conservation of DLS structures in various C-type retroviruses. Genomes of many C-type retroviruses (Cof®n et al., 1997) were compared for the presence and integrity of palindrome sequences contained in DLS204-228 and DLS278-303 of MoMLV. Comparisons were made using GCG software (Genetics Computer Group, Madison, WI). A dash indicates sequence identity. Variable space between the two palindromes is indicated by a number in parentheses; in cases where the second palindrome is not found, that result is indicated. Lines a-e represent groups of isolates with the given sequence identity. The viruses represented by each group are listed along with their EMBL accession number: a) Moloney murine leukemia virus (J02255); Moloney murine sarcoma virus (J02263); Abelson murine leukemia virus (V01541); (b) AKV murine leukemia virus (J01998); FBR murine osteosarcoma virus (K02712); FBJ murine osteosarcoma virus (J02084); Friend spleen focus-forming virus (K00021); radiation murine leukemia virus (K03363); Kirsten murine leukemia virus (X00984); MCF1233 murine leukemia virus (U13766); (c) Friend murine leukemia virus (M93134); Cas-Ba-E murine leukemia virus (X57540); Rauscher murine leukemia virus (U94692); (d) Gibbon ape leukemia virus (M26927); X-strain gibbon leukemia virus (U60065); simian sarcoma virus (J02394); (e) (SM-FeLV) feline sarcoma virus (M23024); GA-strain feline leukemia virus subgroup B (K01209); feline leukemia virus subgroup A (FeLV-FAIDS) (M18247).

that this is the case (Figure 9). The sequence of the palindrome (210-219) in DLS204-228 is conserved throughout C-type retroviruses isolated from murine hosts. In some less closely related C-type retroviruses, the palindrome sequence is also conserved, and an additional group retains the palindrome with a single mutation that does not disrupt base-pairing. Moreover, some viruses that completely lack the palindrome of DLS278-303 still have the DLS204-228 palindrome present in their genome. More distantly related C-type retroviruses have neither palindrome sequence, but carry similar size palindromes of unrelated sequence in approximately the same position (data not shown). This conservation underlines the probable importance of these sequences in the viral life-cycle. These palindromes also have another important function. Recent data shows that sites of recombination of retroviruses coincide with predicted sites of dimerization of gRNAs (Mikkelsen et al., 1998). One of the most active recombination sites in Akv MLV gRNA was found at the SIWIII locus (position 199-219). This site contains a ten base palindrome (209-218). The hairpin and palindrome align with DLS204-228 and its palindrome at 210-219. The only site that was more active in recombination in

Moloney Murine Leukemia Virus RNA Dimerization

this system included a 16 base palindrome sequence that aligns with DLS278-303 of MoMLV. Tounetki et al. (1992) showed that nucleotides around position 215 showed changes in reactivity following dimerization of MoMLV RNA. Perhaps because this area was at the extreme 50 end of the RNA fragment they studied, no suggestion was made linking the 204-228 region to dimer formation. In light of our data, that earlier study can be seen as evidence that nucleotides around position 215 change their conformation during dimer formation. Our data indicate that short RNAs containing DLS204-228 form dimers with dissociation constants similar to those previously reported for either gRNA or shorter sequences containing DLS278-303 (Figure 7(b)). At the physiological temperature of 37  C, the dimerization reaction seems to consist of a relatively ``fast'' phase followed by a ``slow'' phase (Figure 7(a)). This effect is very similar to the kinetics of dimer formation observed for DLS278-303 (Girard et al., 1995) or whole MoMLV gRNA (Roy et al., 1990). The two phases have been attributed to the presence of multiple conformational isomers in the initial pool of monomeric RNA. Only some of these conformations are competent to form dimers. The fast phase can be modeled as the dimerization of the competent subpopulation. The slower phase requires a slow conformational change in the initially incompetent subpopulation. In our results, a residual 20 % of the monomeric TR(28-258) RNA could not be shifted into dimeric form even after prolonged incubation. After heating and slow cooling, however, this residual population was capable of dimer formation. More support for this model comes from the optimum temperature for dimer formation, which we found to be near 55  C. An optimum in dimer formation at approximately 55  C with reduction in dimer formation at lower temperatures has been seen with RNAs comprising the entire 50 untranslated region (755 nucleotides) (Roy et al., 1990). Ehresmann and colleagues (Roy et al., 1990) attributed the temperature-dependence of dimer formation to the necessity of overcoming an energy barrier. This barrier separates folded states of the monomeric form that are incapable of dimerization from forms that can dimerize. Increased temperature can destabilize RNA's secondary and/or tertiary structure, accelerating the transition to forms capable of dimerization. As one might expect, the optimum is slightly below the calculated melting temperature of 63  C for the hairpin loop. Catalysis of these slow conformational changes in vivo may be a major role for the nucleocapsid protein (p10), which is thought to have nucleic acid chaperonin activity (Prats et al., 1988, 1990; Girard et al., 1996) and participates in dimer formation (Bieth et al., 1990; De Roquigny et al., 1992; Lapadat-Talposky et al., 1995). The ability of elevated concentrations of monovalent salts to promote dimerization of DLS-containing RNAs suggests that classical Watson-Crick

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Moloney Murine Leukemia Virus RNA Dimerization

interactions play a signi®cant role in some step in dimer formation (Roy et al., 1990). Our data con®rms this observation for DLS204-228 (Figure 7(c)). Magnesium ions promote dimerization of gRNAs and fragments containing DLS structures (Roy et al., 1990). Signi®cantly, we found that RNAs containing DLS204-228 followed this pattern. In fact, the presence of a residual magnesium ion concentration carried over during puri®cation was enough to mask the effect of monovalent ions on dimerization of TR(28-258) unless the RNA was treated with EDTA. The most prominent effect of Mg2‡ was detected in the ®rst (``fast'') phase of dimer formation (Figure 7(d)). Moreover, our results from experiments with heat and formamide denaturation indicate that Mg2‡ promotes dimerization of MoMLV gRNA and stabilizes dimers against denaturation (Figure 6(a)). Our data show that the effect of Mg2‡ has a weakly cooperative character (Figure 6(b)). Cooperativity suggests multiple sites of binding for Mg2‡ on the RNA and their mutual contribution to dimer stabilization. Some of these magnesium ions undoubtedly mediate tertiary contacts. Sequence swapping experiments (e.g. see Yang & Temin, 1994) suggest that many dimerization contacts are not dependent on primary sequence. On the other hand, the rate of dimerization in the ``late'' phase of dimer formation (Figure 7(d)) was essentially the same at different concentrations of Mg2‡, suggesting that Mg2‡ plays a minor if any role in structural rearrangements of the various monomeric conformations of the RNA. Magnesium ions have emerged as an important element in the three-dimensional structure of RNAs. Recent crystal structures have revealed Mg2‡ in the folded core of RNAs (Correll et al., 1997; Cate et al., 1997). Although an NMR structure for an HIV-1 kissing-loop structure has been solved (Mujeeb et al., 1998), limitations of the NMR method prevented authors from addressing the structural role of magnesium. As we gain additional information about the boundaries of regions involved in RNA dimerization, it begins to seem possible to use X-ray crystallographic methods to address these questions. A combination of biochemistry and structural biology will reveal the three-dimensional structure of the C-type retrovirus genomic dimer.

Materials and Methods Standard practices (Sambrook et al., 1989) were used for all molecular biology procedures not speci®cally described. Construction of plasmids The pNCA plasmid (Colicelli & Goff, 1988), carrying a complete MoMLV proviral DNA, was used as a template for high-®delity PCR ampli®cation (Pwo thermostable DNA polymerase, Boehringer Mannheim), to create cDNAs for selected portions of MoMLV gRNA. Restric-

tion sites for BamHI and EcoRI were simultaneously incorporated into these PCR products at the 50 and 30 ends, respectively, by PCR primer-directed mutagenesis (Higuchi, 1990). The pGEM-4 plasmid (Promega Corp.) was used as a general-purpose vector for subcloning of these cDNAs under control of phage T7 and SP6 RNA polymerase promoters at 50 and 30 ends, respectively. pGEM-4 was cleaved with BamHI and EcoRI endonucleases, and PCR-generated cDNAs were inserted with phage T4 DNA ligase. To minimize the number of nucleotides not originating from MoMLV in the ®nal RNA, these intermediate plasmids were cleaved with HindIII and BamHI, blunt ends were created by ®lling in with Klenow, and the plasmid DNAs were religated. Plasmids were designated pT(x ÿ y), where x and y are the ®rst and last nucleotides of the MoMLV proviral sequence inserted. In vitro transcription and purification of viral RNAs To produce RNA, DNA templates were ®rst ampli®ed from pT plasmids by high-®delity PCR with a T7/SP6 primer set. The templates were digested with EcoRI to create a 50 end containing only virally derived sequence with the exception of ®ve terminal nucleotides derived from the EcoRI restriction site. Before transcription, the templates were puri®ed by agarose gel electrophoresis using low melting point agarose. The DNA templates were transcribed in vitro by T7 RNA polymerase in 100 ml reactions containing 5 pmol of DNA template, 20 mg of BSA, 100 mM Tris-HCl (pH 7.6), 3.2 mg of PEG 8000, 20 mM MgCl2, 2 mM spermidine (pH 8.0), 4 mM of each NTP (pH 7.5), 2 mM DTT, and 200 units of T7 RNA polymerase. Reactions were incubated for 6-16 hours at 37  C, treated with RNAse-free DNAse for one hour at 37  C, and then extracted with phenol/chloroform. The RNA from each reaction was ethanol-precipitated, air-dried and resuspended in 0.5 volume of diethylpyrocarbonate (DEPC)treated water. After denaturation at 96  C for ®ve minutes, the RNA was chilled on ice. The full-length RNA was then puri®ed by electrophoresis in a 2.5 % low melting point agarose gel. Purity and integrity of the RNAs were analyzed by electrophoresis in 6 % or 12 % polyacrylamide, 8 M urea, 40 % formamide gels. RNA was visualized by staining with methylene blue (0.2 mg/ml) for one hour followed by destaining in water. The concentration of each RNA was determined by measuring absorbance at 260 nm and assuming that an absorbance of 1.0 (1 cm path length) is equivalent to 40 mg/ml RNA. RNA was stored at about 1 mg/ml at ÿ70  C in DEPCtreated water. All RNAs were designated TR(n-m), where n and m are the ®rst and last nucleotides of the included MoMLV sequence (Figure 1). Every RNA had 20 extra nucleotides on its 50 end and ®ve extra nucleotides on its 30 end that originated in the multicloning site of pGEM-4. 50 -end labeling of TR(28-258) RNA Gel-puri®ed RNA was labeled for two hours at 37  C in a 10 ml reaction containing 35-100 mCi of [32P]ATP, 1 ml of T4 polynucleotide kinase, 1 ml of T4 polynucleotide kinase buffer and 1 ml of RNAse inhibitor (AmBion). The labeled RNA was ethanol-precipitated after addition of 0.1 volume of 3 M sodium acetate (pH 5.2). After airdrying, the pellet was resuspended in 50 ml of DEPC treated water.

612 Dimerization of RNAs Unless speci®cally noted, dimerization reactions contained 0.2 mg of RNA incubated in 10 ml of dimerization buffer (30 mM bistrispropane (pH 7.6), 100 mM KCl, 10 mM MgCl2) for one hour at 37  C. Dimerization was then stopped by placing reaction tubes on ice. These conditions are designed to closely mimic the dimerization conditions determined previously for MoMLV gRNA and RNAs from the PSI region in order to allow comparison with these other results. We call these ``standard dimerization conditions.`` Sterile glycerol (1 ml) was added to each stopped reaction before electrophoresis to facilitate loading onto the gel. Quantitation of RNA dimerization After dimeric and monomeric forms of RNA were separated by electrophoresis in native 2.5 % agarose or 4 % polyacrylamide gels, the RNA bands were stained with SYBR Gold ¯uorescent stain (Molecular Probes), used at 1:10,000 dilution. Staining was in 10 mM Tris (pH 8.0), 1 mM EDTA, for 40 minutes with constant agitation. Bands were scanned on a Storm PhosphorImager MD840 (Molecular Dynamics) in blue ¯uorescence mode. The intensity of ¯uorescence from each of the RNA bands was integrated and the ratio of dimeric and monomeric forms for each sample was calculated. Gels with 32 P-labeled RNA were exposed to phosphor storage screens and images were read and integrated using the phosphorimager in phosphor screen mode. Estimation of dissociation constants Dissociation constants for dimerization of RNAs were estimated by the method of Paillart et al. (1996). Nonlinear curve-®tting was performed with DeltaGraph software (version 4.0, DeltaPoint Inc.) Estimation of tm for RNA dimers Preformed dimer was puri®ed on 2.5 % low melting point agarose gels. RNA dimer eluted from the gel was extracted with phenol/chloroform, precipitated with 2.5 volumes of ethanol, washed four times with 70 % ethanol, air-dried and resuspended in DEPC-treated water. RNAs were then mixed with 1 ml of 10 buffer (300 mM bistrispropane (pH 7.6), 1 M KCl) in a total volume of 10 ml. Reactions were placed in a thermal cycler and the temperature was elevated from 44  C to 100  C in 8 deg. C steps with a duration of seven minutes per step. At the end of each step, a set of samples were quickly transferred and stored on ice. Finally, samples (5 ml aliquots) from each temperature were mixed with 1 ml of sterile glycerol and immediately separated on a 2.5 % agarose gels. Melting temperature was de®ned as the temperature at which 50 % of the RNA dimer converted back to the monomeric form.

Acknowledgements We thank Glen Lindwall for helpful comments on this manuscript and Maura Jess for help in preparing illustrations. The plasmid pNCA was kindly supplied by S. Goff and A. Telesnitsky. This work was supported by the Universitywide AIDS research program grant #R95-

Moloney Murine Leukemia Virus RNA Dimerization SB-155 and by National Science Foundation grant #MCB9505977. L.A.K. is an American Cancer Society Junior Faculty Fellow (JFRA-572).

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Edited by D. E. Draper (Received 15 March 1999; received in revised form 28 June 1999; accepted 29 June 1999)