Modulation of Homo- and Heterodimerization of Harvey Sarcoma Virus RNA by GACG Tetraloops and Point Mutations in Palindromic Sequences

doi:10.1016/S0022-2836(02)00966-X available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 323, 613–628

Modulation of Homo- and Heterodimerization of Harvey Sarcoma Virus RNA by GACG Tetraloops and Point Mutations in Palindromic Sequences Søren Vestergaard Rasmussen1, Jacob Giehm Mikkelsen1 and Finn Skou Pedersen1,2* 1

Department of Molecular Biology, University of Aarhus C.F. Mollers Alle, Bldg 130 DK-8000 Aarhus C, Denmark 2 Department of Medical Microbiology and Immunology University of Aarhus, DK-8000 Aarhus C, Denmark

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV). By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers. A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of g-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: retrovirus; RNA dimerization; recombination; kissing-loop

Introduction Present address: J. G. Mikkelsen, Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305, USA. Abbreviations used: DLS, dimer linkage structure; HaSV, Harvey sarcoma virus; HIV-1, human immunodeficiency virus type 1; MLV, murine leukemia virus; Mo-MLV, Moloney murine leukemia virus; Pal, palindrome; PBS, primer binding site; SL, stem-loop. E-mail address of the corresponding author: [email protected]

A retroviral particle contains two plus-strand copies of viral RNA. Studies of homodimers formed in vivo and in vitro show non-covalent linkage of the two RNAs at the dimer linkage structure (DLS) close to the 50 end.1 – 3 RNA isolated from freshly budded virus particles is dimeric, indicating that dimerization is a prerequisite for encapsidation of RNA into the virus particles.4,5 Dimerization of the retroviral genome and its packaging into virus particles is mediated by overlapping RNA elements, suggesting a linkage

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

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Homo- and Heterodimerization of HaSV RNA

Figure 1. Motifs implicated in homodimerization of MLV and HaSV RNAs. Key motifs in the leader region RNA of MLV involved in dimerization and packaging are Pal I,28,29 Pal II22,23 and the GACG stem-loops,7,9,33,46 here designated SL1MLV and SL2MLV. The origin of HaSV RNA leader sequences from VL30 (white) and Mo-MLV (dark grey) is shown. A black box indicates spacer sequence. The spacer sequence harbours the Pal I palindrome implicated in dimerization.36,37 Pal I nucleotide positions that differ between Akv/Mo-MLV are underlined in the HaSV sequence. Thin broken lines indicate sequences not contained in HaSV. SL4 and SL5 represent the GACG stem-loops also implicated in dimer formation.7,33

between the two processes.6 – 9 Co-packaging of two distinct RNAs is a prerequisite for recombination between retroviral genomes by template switching of reverse transcriptase.10,11 While recombination between closely related viruses or between different mutants of the same viral strain is frequent, recombinants between less closely related viruses also arise.12 – 15 It may be hypothesized that formation of heterodimers is needed for reverse transcriptase-mediated retroviral recombination to take place. In support of this, we have previously shown that introduction of mutations into a suspected dimerization signal affects recombination specificity in vivo.16 However, no direct evidence for formation of heterodimers between different retroviruses in vivo has been provided. Studies of dimer formation between in vitrotranscribed RNAs have led to the identification of autocomplementary regions in the 50 leader of human immunodeficiency virus type 1 (HIV-1),17,18 avian sarcoma leukosis virus (ASLV)19,20 and murine leukemia viruses (MLVs)7,21 – 23 that are important for in vitro dimerization. These sequences are thought to fold into a stem-loop

(SL) structure exposing a palindromic loop sequence, known as a kissing-loop. A loop-loop contact between two kissing-loops initiates dimerization, possibly leading to the formation of an antiparallel extended duplex with Watson – Crick base-pairing between the two strands.19,22,24,25 The mechanisms involved in heterodimerization of RNA from different viruses have not been given much attention and, to our knowledge, only one report exists. This in vitro study showed that HIV-1 could form heterodimers with Mo-MLV and Rous sarcoma virus RNA.26 The formation of heterodimers was suggested to involve purinerich sequences; however, no genetic evidence for this interaction has been provided.27 In Mo-MLV, a 16 nucleotide palindromic sequence (Pal II) known as the DLS within the packaging signal22 and a ten nucleotide sequence (Pal I) upstream of Pal II are thought to contribute equally to in vitro formation of dimers (Figure 1).28,29 Additionally, two stem-loop structures (SL1MLV, SL2MLV) constituting the core packaging signal have been shown to support the formation of dimers generated in vitro.7 Both SL1MLV and

Figure 2. Enzymatic probing of RNA secondary. (a) Structural data obtained from 50 end-labelled RNA in monomer conformation (i) position 203– 238 (iii), 239– 280, (iv) 280– 353 and (ii) dimeric conditions conformation position 203– 238. The cleavage products were resolved on 7 – 12% (w/v) polyacrylamide sequencing gels. Above each lane, the enzyme used is indicated (RNase A, T2, T1 and V1). An alkaline RNA degradation and a G-specific sequencing with RNase T1 (G-seq) is included. In the control lane, no RNases were included in order to identify potential unspecific RNA degradation. (b) Summary of structural probing data of 50 (data shown in (a)) and 30 end-labelled (data not shown) RNA and the proposed secondary structure of the HaSV packaging signal. The digestion products of 30 endlabelled RNA gave rise to “double bands” due to non-template RNA synthesis; for simplicity, these data are not shown. This phenomenon was not observed with 50 end-labelled RNA. RNase V1 differs from the single-strand-specific RNases, in leaving a 50 phosphate group; this gives rise to more slowly migrating digestion products for the 50 endlabelled RNA and faster migrating products in the 30 labelled RNA. This was especially evident for the shorter cleavage products (,50 nt); however, by combining data from 30 and 50 end-labelled RNA this change of mobility could be adjusted. The probing data were provided for the mfold v. 3.0 program; thus the suggested structure is on the basis of both probing data and thermodynamic folding.39,40

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Homo- and Heterodimerization of HaSV RNA

Figure 2 (legend opposite)

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SL2MLV expose a GACG tetraloop and are perhaps the most conserved structures among the g-retroviruses.9,30 – 32 Recent NMR studies suggested that two SL2MLV structures interact through a kissing-loop mechanism involving two interstrand base-pairs stabilised by unusually strong electrostatic interactions around the base-pairs and by intensive stacking with the surrounding bases.33 In addition, motifs downstream of the GACG loops have been suggested to support dimer formation in Mo-MLV23 and in the closely related Moloney murine sarcoma virus.34 Harvey sarcoma virus (HaSV), believed to originate through recombination between Mo-MLV and rat retrotransposon virus-like 30 S RNA (ratVL30)35 can be packaged and replicated by the Mo-MLV machinery. HaSV RNA is nearly identical with Mo-MLV in the upstream part of the leader and identical with ratVL30 sequence in the downstream part (see Figure 1). The VL30 part of the 50 leader region involved in packaging exhibits 57% sequence identity with Mo-MLV as well as with Akv-MLV, while the latter two exhibit 73% sequence identity in the same window. The Mo-MLV sequence and the ratVL30 sequence are spaced by a region showing homology to Mo-MLV sequences. This sequence coincides with the Pal I region in Mo-MLV, the sequence involved in RNA dimerization in vitro.29 HaSV RNA has been shown to homodimerize in vitro involving this spacer sequence36,37 and the downstream ratVL30-derived sequence.37 Autocomplementarity in the spacer sequence has been reported to mediate dimerization, probably through kissingloop interaction.37 Others suggest that a G-rich sequence in the same region mediates dimerization through co-ordination of a metal ion.36 Furthermore, the downstream ratVL30 sequence was shown to dimerize independently of the autocomplementary region.37 Two stem-loop structures (SL4 and SL5) in the downstream part of HaSV RNA both expose a GACG tetraloop (Figure 1). Here, we first study the mechanism of in vitro homodimerization of HaSV RNA and show that autocomplementarity of the spacer sequence can direct homodimerization independently of the G-rich region. Moreover, we show that it is possible to design alternative non-palindromic sequences that specifically direct heterodimerization with complementary sequences. We find that the GACG tetraloops of SL4 and SL5 contribute to formation of these dimers, and that the GACG motif is critical for this enhancement. Heterodimerization between HaSV and the two murine exogenous viruses Mo-MLV and Akv-MLV is enhanced by the GACG tetraloops. Heterodimer formation could be modulated by mutation of the spacer sequence of HaSV to create perfect complementarity to Pal I of MLV. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of

Homo- and Heterodimerization of HaSV RNA

g-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.

Results Secondary structure probing In HaSV, the packaging signal has been mapped to position 205– 380,38 this sequence is sufficient and necessary to drive effective packaging of heterologous RNA into Mo-MLV particles. Seemingly, this region is involved in in vitro RNA dimerization.37,38 The secondary structure of this region is not known; however, it contains the conserved GACG motifs of the g-retroviruses, which have been shown by biochemical experiments and phylogenetic comparisions to fold into a stable hairpin structure with the GACG sequence exposed in a tetraloop.21 To confirm the potential existence of these structures and determine other secondary structures possibly involved in dimerization, the secondary structure of the packaging signal was probed enzymatically. Probing of the secondary structure would assist the design of point mutations affecting specific structures. For the experiments, an either 30 or 50 end labelled RNA spanning the entire packaging signal from position 205 to 380 was employed (HaSV(205 – 380)wt)). The RNA was probed under both monomer and dimer conditions. Monomer conditions were obtained with a low concentration of RNA (< 2 nM) and short incubation time (five minutes), dimer conditions were obtained with a high concentration of RNA (< 250 nM) and long incubation time (30 minutes). Under monomer conditions, less than 2% of the RNA was dimeric; under dimeric conditions, RNA was approximately 95% dimeric. Formation of monomers and dimers was conducted by use of the standard dimerization procedure as employed in all the following dimerization experiments. After incubation under monomer or dimer conditions, the reactions were stopped by transferring to ice. Immediately, the RNA was supplemented with either single-strandspecific RNase T1 (G specific), RNase A (U and C specific)or RNase T2 (preferentially A), or doublestrand-specific RNase V1 (no sequence specificity). Digestion products were analysed by denaturing PAGE. By employing both 50 and 30 end-labelled RNA under various electrophoretic conditions, we were able to acquire structural data for both monomers and dimers (Figure 2(a)). The best-defined probing data were used as constraints in the thermodynamic structure modelling using the RNA folding program mfold.39,40 Of the structures predicted by mfold, the structure in best agreement with the

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Homo- and Heterodimerization of HaSV RNA

remaining probing data is presented in Figure 2(b). The predicted structure in Figure 2(b) has a calculated free energy DG8 of 2 57.0 kcal mol21 compared to a calculated minimum DG8 of 2 65.5 kcal mol21 without constraints from probing data (1 cal ¼ 4.184 J). Especially the SL-4 and SL-5 are well documented both by thermodynamic predictions and enzymatic probing. In the 50 end of the RNA, the structural data are more complicated and more than one conformation is possible. However, only one band was observed by PAGE under native conditions (data not shown), arguing against large variations in structure. Comparing the monomer and dimer cleavage patterns revealed two regions that showed a change in reactivity. The first region is mapped to the potential loop sequence of SL1 which, under monomer conditions, is cut strongly at position 216 with RNase V1, whereas under dimer conditions both positions 215 and 216 are cleaved strongly by RNase V1 (Figure 2(a)). However, the change in reactivity is suggesting an intermolecular interaction involving formation of Watson – Crick base-pairing in this region. The other region affected by dimerization is positioned in 233 –238, which is cut weakly by single-strand-specific RNases under monomer conditions and cut strongly under dimer conditions. This may be due to overall structure change in the dimer resulting in increased accessibility of RNases toward RNA in this region. Several regions are involved in HaSV homodimer formation in vitro To map regions responsible for dimerization, we introduced deletions into the full-length HaSV packaging signal. In an effort to avoid deleting part of stem-loop structures, the deletions were designed on the basis of the putative secondary structure (Figure 2(b)) and the position of the 50 and 30 end for each construct is indicated with a subscript. The HaSV(205 – 271) construct was included, however, to confirm results obtained.38 All dimerization experiments were done by titrating a fixed amount of radioactively labelled RNA with increasing amounts of non-labelled RNA. Under the conditions used, the full-length RNA HaSV(205 – 380) shows a high level of dimerization at low concentrations of RNA (Figure 3), as do all RNAs harbouring SL1 (HaSV(205 – 380), HaSV(205 – 324), HaSV(205 – 271)). In all cases, deletion of SL1 resulted in strong reduction in the amount of dimers

Figure 3. Homodimer formation. (a) Formation of homodimers of RNAs spanning different regions of the HaSV packaging signal. RNAs were incubated under standard dimerization conditions (see Materials and

Methods). The total concentration of RNA is noted above each lane. Monomer and dimer bands are marked with M and D, respectively. Bands of lower mobility likely represent formation of trimers, tetramers, etc. (b) The graph shows the molar ratio (Yc) of dimers of the different RNAs as a function of total RNA concentration (on a log scale).

618

Homo- and Heterodimerization of HaSV RNA

Figure 4. Introduction of alternative SL1 sequences in HaSV. (a) The alternative SL1 sequences introduced in HaSV and their putative secondary RNA structure. Palindromic and non-palindromic sequences are marked with þ and 2 , respectively. (b) The HaSV(205 – 324)SL1 mut RNAs were titrated with HaSV(205 – 380)SL1 mut harbouring an identical SL1 sequence, and the relative amount of radioactivity located in the heterodimer bands (heterodimeric RNA ratio) is plotted as a function of HaSV(205 – 380)SL1 mut RNA concentration. (c) Radioactively labelled HaSV(205 – 380) SL1 mut RNAs were titrated with increasing amounts of non-labelled HaSV(205 – 380)SL1 mut RNAs containing an identical SL1 sequence. The molar ratio (Yc) is plotted as a function of total RNA concentration.

formed at a given concentration. We observed that HaSV(205 – 324) and HaSV(227 – 324) did not dimerize as well as their counterparts HaSV(205 – 380) and HaSV(227 – 380). These constructs differ by the presence or absence of SL5. Altogether, these results show that the SL1 region is a key mediator of homodimerization, and that the SL5 region supports dimerization both in the presence and in the absence of SL1. Further deletions of the SL4 and SL3 regions had no effect in this assay. However, our results do not allow us to conclude if SL4 and SL5 act in concert. Stem-loop 1 functions as a kissing-loop To analyse the function of the SL1 region, we focused on the palindrome (Pal) of nucleotides 210 –219 (Figure 2(b)). Mutations (Figure 4(a)) were at first introduced into the HaSV(205 – 324) constructs and dimerization was analysed with a HaSV(205 – 380) RNA harbouring the same mutations (Figure 4(b)). Formation of a complex between the labelled mutated RNA (HaSV(205 – 324)SL1 mut)) and

the longer HaSV(205 – 380) mutated RNA would give rise to a shift of the homodimer band to a heterodimer band with lower mobility (Figure 4(b)). Most mutations conserved the ten nucleotide palindrome and the composition of the loop with two cytosine bases and two guanine bases as in the wild-type sequence. With the exception of the CGCG mutation, all mutations conserving the palindromic sequence gave rise to efficient dimerization comparable to the wild-type level, indicating the importance of autocomplementarity. The mutation of G217 to A, in a HaSV(205 – 271) construct, was previously shown to inhibit dimerization strongly.36 G217 was proposed to participate in the formation of a purine tetrade consisting only of the 50 G-rich sequence in position 205 –220 (Figure 2(c)) in HaSV.36 However, G217 is part of the palindrome constituting the putative SL1,37 and a kissing-loop mechanism is likely to be affected by this mutation. Consistent with these previous results, we found the mutation to impair heterodimer formation strongly between HaSV(205 – 324)SL1 CGGCCA and HaSV(205 – 380)SL1 CGGCCA

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Homo- and Heterodimerization of HaSV RNA

RNA (Figure 4(b)). To examine whether this effect was due to destruction of the palindromic sequence or the G-rich sequence, a complementary mutation was introduced to restore the palindrome. This changed the putative closing basepair from wild-type C212-G217 to U212-A217 (Figure 4(a)). As the only construct harbouring a wildtype sequence of the putative tetraloop, HaSV(205 – 324)SL1 UGGCCA was found to dimerize to almost wild-type level, reaching a higher level of heterodimerization than the wild-type construct and only a 50% increase in CH50% (the concentration of unlabelled RNA giving rise to 50% of labelled RNA bound in heterodimers). These results demonstrate the requirement of loop complementarity rather than a G-rich sequence for efficient dimerization (Figure 4(b)). Equivalent to the above experiments, the palindromic mutations were introduced into the longer HaSV(205 – 380) constructs and tested in a homodimerization assay. These results again showed the importance of autocomplementarity for efficient dimerization (Figure 4(c)). However, by employing the longer constructs, the specificity of dimerization was not as pronounced, possibly due to the contribution of sequences outside the HaSV(205 – 324) in the dimerization process. The GACG loop motif is important for homodimer formation From deletion and SL1-mutation experiments, it was obvious that SL1 was not the sole sequence responsible for dimerization. Deletion analysis suggested SL5 to be involved in dimerization and, interestingly, both SL4 and SL5 contain GACG motifs previously proposed to be important for dimer formation.7 Therefore, we wanted to specifically analyse the GACG nucleotides. Both structures expose a GACG loop sequence that was mutated to a UUCG sequence, thereby generating the constructs HaSV(205 – 380)SL4(UUCG), HaSV(205 – 380)SL5(UUCG) and HaSV(205 – 380)SL4þ5(UUCG). The UUCG sequence was chosen as it has been shown to adopt a very stable tetraloop structure,41 thus limiting the possibility that the mutation would change the secondary structure of SL4 and SL5. The mutated RNA constructs were labelled and the experiments were carried out as described previously by adding increasing amounts of unlabelled identical RNA (Figure 5). Mutation of one GACG had a crucial effect on dimerization activity, resulting in impairment of dimerization comparable with, or more drastic than, the deletion of SL1. Apparently, SL4 contributes the most to dimerization ðYCð500 nMÞ ¼ 0:62Þ; whereas mutation of SL5 has less effect ðYCð500 nMÞ ¼ 0:81Þ: Mutation of both GACG motifs in HaSV(205 – 380)SL4þ5(UUCG) reduced dimerization further ðYCð500 nMÞ ¼ 0:58Þ: This demonstrates clearly the involvement of the GACG tetraloop sequences in dimerization. Alternative tetraloop sequences were tested in SL4 (GAUA, GGCG,

Figure 5. Involvement of the SL4 and 2 5 GACG tetraloop in dimerization. Different HaSV derived RNA constructs harbouring mutations in SL4 (HaSV(205 – 380)SL4 UUCG), SL5 (HaSV205 – 380)SL5 UUCG) or both (HaSV(205 – 380)SL4þ5(UUCG)) were used in homodimerization experiment, as described in the text. The chart shows Yc as a function of the total concentration of RNA.

GGUA, AACG, GUCG); none of these was capable of mediating dimerization to the wild-type level (data not shown), indicating the existence of strong structural restrictions on GACG interactions. Formation of heterodimers between SL1modified HaSV RNAs To further test the need for complementarity of loop sequences for dimer formation, we wanted to explore the possible design of constructs capable of directing heterodimerization rather than homodimerization. Experiments were carried out as described above by titrating an HaSV construct having a non-autocomplementary sequence exposed in SL1 (HaSV(205 – 380)SL1 CGGC, HaSV(205 – 380)SL1 GCCG) with a construct that has a sequence complementary to this sequence (Figure 6(c)). For added focus on the dimerization effects of SL1, the experiments were carried out also in SL4 þ 5 mutated constructs HaSVSL1 GCCG SL4þ5(UUCG)) (HaSVSL1 CGGC SL4þ5(UUCG), (Figure 6(a) and (b)). The results showed a strong selectivity towards a construct harbouring a complementary sequence, this was especially evident when the background dimerization of the GACG stem-loops were eliminated in the HaSVSL1 mut SL4þ5(UUCG) constructs. This once again emphasises the complementarity of SL1 as a determinant of the selection of interaction partner in HaSV dimerization. Although the three dimerization signals located in SL1, SL4 and SL5 were mutated, relatively large amounts of homodimers were found, especially at concentrations of RNA above 25 nM. Strikingly, these homodimers

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Homo- and Heterodimerization of HaSV RNA

Figure 6. Dimerization of RNAs harbouring non-autocomplementary SL1 sequences. (a) Labelled HaSV(205 – 380)SL1 CGGC SL4þ5(UUCG) and HaSV(205 – 380)SL1 GCCG SL4þ5(UUCG) were titrated with their SL1 complementary or noncomplementary HaSV(205 – 380)SL1mut SL4þ5(UUCG) RNA. Above each gel the labelled construct is marked with an asterisk ( p ) and in each lane the RNA concentration of the non-labelled construct is noted. The mobility of monomer (M) and dimer (D) is marked. (b) The chart shows a summary of data from (a), with the relative amount of radioactivity located in dimer bands (dimeric RNA ratio) as a function of RNA concentration (on a log scale) of the non-labelled construct. The labelled construct is marked with an asterisk (p ). (c) The chart shows hetero- and homodimerization of HaSV(205 – 380)SL1 mut constructs. The dimeric RNA ratio is plotted as a function of RNA concentration of non-labelled construct. (d) The chart shows a time-course experiment of hetero- and homodimerization of HaSV(205 – 380)SL1 mut SL4þ5(UUCG) constructs. The dimeric RNA ratio is plotted as a function of incubation time at 45 8C.

showed lower mobility than heterodimers in which intermolecular SL1 base-pairing was possible, indicating that homo- and heterodimers have different conformations. It is unclear which RNA motive is responsible for homodimer formation in these constructs or if it is involved in formation of heterodimers. A time-course experiment revealed that

SL1 in this set-up supports dimerization both kinetically (on rate) and thermodynamically (Kd) (Figure 6(d)). Altogether, these results point to complementarity of the ten nucleotides palindrome of the SL1 motif as an important determinant for dimer formation. However, other regions are strongly

Homo- and Heterodimerization of HaSV RNA

involved in dimerization, as lack of complementarity and the GACG stem-loops do not abolish dimerization. Formation of heterodimers between HaSV and MLV RNAs Previous studies of in vitro transcribed RNA from the leader region of Mo-MLV have identified several regions capable of mediating dimerization, all localised either in the packaging signal or in close proximity to the packaging signal. To avoid exclusion of any of these elements from our constructs, a long segment including the packaging signal and the adjacent sequences was initially applied. All of the constructs were on the basis of the secondary structure suggested for Mo-MLV21,42 including nucleotides 177– 620 in Mo-MLV and 176– 638 in the closely related Akv-MLV. These RNA constructs thereby included all the described dimerization elements, among which we possibly also would find elements mediating heterodimerization. The tested HaSV RNAs were all labelled and titrated with increasing amounts of either Akv MLV or Mo-MLV derived RNAs (Figure 7). As it can be seen, addition of Akv-MLV/Mo-MLV RNA gave rise to several bands with lower mobility than the HaSV homodimer band. By running a DNA marker next to the lanes, these bands were identified as Akv-MLV or Mo-MLV monomers and dimers that are capable of interacting with HaSV (Figure 7(d)). The use of high concentrations of RNA (500 nM) and long exposure revealed a number of low-mobility complexes, which are likely to be trimers and tetramers of MLV RNA associated with a number of HaSV RNAs. The exact composition of these hetero-complexes is not known. PhosphorImager analysis of total intensity per lane indicated that loading variation was within 5% except for the highest concentration of RNA at 500 nM, where intensity was reduced by 10 – 15% (data not shown). We cannot exclude the possibility that this may result from selective loss due to RNA aggregation, but note that such minor variation will have no significant influence on overall conclusions. Akv RNA in particular has a high affinity for HaSV RNA, showing a CH50% of only 20 nM, equivalent to that of HaSV(205 – 380) itself. Mo-MLV was not as active and showed a CH50% of 400 nM (Figure 7(b) and (c)). This difference in activity of Akv and Mo-MLV RNA was observed with several different RNA preparations. Stem-loop 1 and GACG loops are important for formation of stable heterodimers between HaSV and MLV RNAs As shown above, homodimerization of HaSVderived RNA is mediated through SL1 consisting of a ten nucleotide palindrome. The ten nucleotide palindrome differs from the Pal I sequence of Mo-MLV and Akv-MLV at only two positions.

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Therefore, the intensive heterodimerization observed between HaSV and Akv/Mo-MLV could be mediated through a kissing-loop interaction involving either a loop-loop interaction or possibly an extended duplex with incomplete base-pairing. To investigate this, dimer formation experiments were carried out with the SL1 deleted construct HaSV(227 – 380) (Figure 7). Furthermore, an Akv-MLV and a Mo-MLV construct in which the ten nucleotide palindrome was deleted (Akv-MLV(219 – 638) and Mo-MLV(221 – 625)) was included in these experiments (data not shown). The results showed a modest reduction in heterodimerization of the HaSV(227 – 380) RNA compared to wild-type; however, due to the deletion, it is difficult to do a direct comparison with the wild-type construct. Thus, the reduction may not be significant, and it is clear that SL1 is not the main mediator of heterodimerization. An additional experiment including the GACG mutated constructs HaSV(205 – 380)SL4 UUCG, HaSV(205 – 380)SL5 UUCG and HaSV(205 – 380)SL4þ5(UUCG) revealed a dramatic reduction in heterodimerization, clearly showing an important function of the GACG stem-loop structures in heterodimerization (Figure 7). Analogous to experiments with HaSV, SL4 was most important for heterodimerization, whereas SL5 mutation showed less effect. It was observed that heterodimers formed between SL4 and SL5 mutated constructs and Akv/Mo-MLV, gave rise to mainly heterodimers with low mobility, whereas the wild-type constructs gave rise to both high and low-mobility heterodimers (Figure 7). The explanation for this observation is probably that at the concentration of RNA at which Akv/Mo-MLV gives rise to heterodimers with SL4 and SL5 mutated HaSV constructs, the Akv/Mo-MLV RNA is found to be mainly dimeric; thus, the interaction will occur primarily between HaSV monomers and Akv/Mo-MLV dimers. An explanation of the lack of dependence upon palindromic sequences in heterodimerization between HaSV and MLV RNAs could be that the palindromic sequences do not make an exact match. This was overcome by carrying out the same experiments with the construct HaSV(205 – 380)SL1 UGGCCA. This construct restores a perfect match between HaSV stem-loop I and the MLV palindrome. The experiments showed that the affinity of HaSV-derived constructs towards Mo-MLV could be modulated strongly by introduction of two point mutations (Figure 7(c)). The affinity of HaSV(205 – 380)SL1 UGGCCA for Mo-MLV was enhanced strongly, as CH50% was reduced sevenfold from 400 nM to 60 nM, whereas the mutation showed little effect on heterodimerization with Akv-MLV. To further characterise heterodimerization between HaSV and Mo-MLV, the thermal stability of heterodimers was tested. The heterodimers were formed at 45 8C for 30 minutes as described. Following standard incubation the dimerization buffer was changed to a low ionic strength buffer using spin columns (see Materials and Methods).

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Homo- and Heterodimerization of HaSV RNA

Figure 7. Heterodimerization between HaSV and Akv/Mo-MLV RNAs. (a) The gels show heterodimerization between labelled HaSV-derived constructs and non-labelled Akv-MLV RNA constructs. The concentration of Akv RNA is noted above each lane and the monomer (M), dimer (D) and heterodimer bands (HD1/HD2) are marked. Equivalent experiments were carried out with Mo-MLV RNA (the results are summarized in (c)). (b) Heterodimerization of HaSV with AkvMLV. HaSV constructs were titrated with increasing amounts of Akv-MLV RNA. The relative amount of HaSV RNA bound in complexes with Akv-MLV RNA (heterodimeric RNA ratio) is plotted as a function of Akv-MLV RNA concentration. (c) Heterodimerization of HaSV with Mo-MLV. HaSV constructs were titrated with increasing amounts of Mo-MLV RNA. The heterodimeric RNA ratio of HaSV RNA is plotted as a function of Mo-MLV RNA concentration. (d) Identification of heterodimers. Lane 1, labelled HaSV(205 – 380) RNA (10 nM) þ unlabelled Akv(176 – 638) RNA (100 nM); M marks the HaSV monomeric RNA; D marks the HaSV homodimers; HD1 marks the high-mobility heterodimer; and HD2 marks the low-mobility heterodimer. Lane 2, labelled Akv(176 – 638) at 100 nM RNA conc, M (monomer), D (dimer). Lane 3, labelled HaSV denatured at 95 8C in 7 M urea. Lane 4, labelled Akv(176 – 638) denatured at 95 8C in 7 M urea. Lane 5, DNA marker.

Homo- and Heterodimerization of HaSV RNA

Figure 8. Stability of heterodimers between HaSV and Akv/Mo-MLV. For each dimerization experiment, the relative amount of heterodimers at 45 8C prior to denaturation is set as 1. The chart shows the amount of heterodimers relative to the amount of heterodimers measured at 45 8C as a function of denaturation temperature.

The samples were incubated at the indicated temperatures for ten minutes immediately prior to loading on a native gel. The amount of heterodimers after incubation at the indicated temperature relative to the amount of heterodimers at the standard temperature of 45 8C was plotted as a function of denaturing temperature (Figure 8). Heterodimers between HaSV(205 – 380) harbouring wild-type SL1 and Akv/Mo-MLV all melted between 35 8C and 50 8C. However, in case of the SL1-modulated construct, HaSV(205 – 380)SL1 UGGCCA, the shape of the melting graph differs from the theoretical sigmoid curve of a homogeneous population. This correlates with the increase in heterodimerization between HaSV(205 – 380)SL1 UGGCCA and Akv/Mo-MLV compared to constructs harbouring the wild-type SL1. Seemingly, one population of heterodimers between HaSV(205 – 380)SL1 UGGCCA and Akv/Mo-MLV exhibits a greater heat-resistance (Figure 8). Interestingly, it could be observed on the gels that heterocomplexes consisting of one HaSV RNA linked to one Akv/Mo-MLV RNA, analogous to the indicated heterodimer (HD1) in Figure 7, melted at a temperature above 45 8C.

Discussion Our studies of determinants needed for in vitro dimerization of HaSV RNA are consistent with earlier studies by Torrent et al.36 and by Feng et al.37. Both of these studies identified the region around nucleotides 210 –220 as important for in vitro dimerization. Based upon mutation of G217 and metal ion specificity in thermal stability,

623

Torrent et al. proposed the involvement of noncanonical base-pairs and possibly purine –purine interactions in this region. Feng et al. proposed a secondary structure including SL1, and noted that such a structure might be involved in a kissingloop mechanism of dimerization through autocomplementary sequences. One common problem in RNA dimerization is the risk of unspecific RNA interactions. This has been observed when employing small RNAs that cover only a fragment of the 50 untranslated region (UTR) in Mo-MLV7 or the gag coding region in HIV-1.43 These apparently fold into different conformations from those in the “parental” RNA when the flanking sequences are present. In the experiments presented here, most RNAs cover an entire functional packaging signal and when inserted into a vector, the sequences mediate in vivo dimerization, packaging and transduction with great efficiency.38 Thus, it is fair to assume that RNA sequences employed in these experiments attend a biologically relevant conformation in vitro that is not influenced significantly by flanking sequences, since this is not the case in vivo. Furthermore, a previous study showed comparable dimerization activities of RNAs spanning either the 1 – 1088 or the 205 –378 region of HaSV.38 We performed enzymatic secondary structure probing on the packaging signal of HaSV and found that a kissing-loop structure (SL1) exposing the palindrome in a loop indeed is consistent with our data. In the kissing-loop structure, a large bulge of seven nucleotides is found adjacent to the palindromic sequence. It has been found that small bulges in the kissing-loop of the Cop A/T system are involved in the propagation from an initial loop-loop interaction to an extended duplex.44 In HIV-1, a similar internal loop seems to be involved in the formation of an extended duplex, also involving the binding of NCp to this sequence.45 Whether this is the case in HaSV has not been investigated; however, it has been found that HaSV RNA can form dimers of different stability,37 possibly corresponding to loop-loop interaction and extended duplex. Two probing experiments were done to identify regions with reactivity towards RNases; one on mainly monomeric RNA and the other on mainly dimeric RNA. The most pronounced effect was obtained from the potential kissing-loop structure in which the loop sequence reactivity towards double-stranded specific RNase is enhanced by dimerization. These changes in reactivity of the region suggest strongly that it is involved in dimer formation through Watson–Crick baseparing. Whether the dimer is stabilised by looploop interaction or possibly an extended duplex cannot be concluded from these experiments. Also a region in position 233– 238 showed a change in reactivity, by becoming more sensitive towards single-strand-specific RNases in the dimer conformation than in the monomer. This indicates that dimerization may involve an overall change in

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structure giving rise to a change in accessibility of the site. Our results add further support to the kissingloop hypothesis by showing that alternative palindromes of nucleotides 212– 217 supported homodimerization, while a single nucleotide disruption of the palindrome reduced dimerization. Moreover, our results show that non-palindromic tetraloop sequences can sustain efficient dimer formation in vitro if provided with mutant RNA with a complementary tetraloop. Hence, the presence of a palindrome is not required and our overall data show that it is possible to specifically modulate homo- and heterodimerization of HaSV mutant RNAs by introduction of matching or non-matching alternative palindromic and non-palindromic sequences in the tetraloop of SL1. In agreement with Feng et al.,37 we found that nucleotides downstream of position 271 contribute to dimer formation. We note here the presence of the putative stem-loops, SL4 and SL5 both having a tetraloop sequence GACG as recorded for a number of g-retroviruses.30 Recently, interaction between GACG-loops was proposed to involve Watson– Crick base-pairing of the 30 CG dinucleotide with stabilizing stacking and electrostatic interactions.33 Notably, the GACG tetraloop was sensitive to cleavage by single-stranded-specific RNases, under conditions of both monomeric and dimeric RNAs. This has been observed by chemical probing of the GACG structures in Moloney MLV.21 We show here that the GACG motif is sensitive to mutations and that in the present set-up SL4 seems to contribute the most to dimer formation. In vivo studies have shown that deletion of the GACG stem-loops reduces RNA encapsidation and replication in Mo-MLV9,31,46 and in spleen necrosis virus.32 Since RNA dimerization may be a prerequisite for encapsidation,4,47,48 these in vivo effects may be caused partly or solely by an effect on dimer formation as studied here. However, no in vivo study has addressed the effect of singlenucleotide alterations in the GACG tetraloops. Interestingly, the SL1 palindrome of HaSV located at the border between the MLV and the ratVL30 derived sequences has an eight out of ten nucleotides match to the Pal I palindrome recently proposed to be involved in the dimerization of Mo-MLV RNA28,29 in addition to the formerly identified Pal II and GACG stem-loops. Moreover, Pal I and SL1 seem to be located at equivalent sites of the two genomes, Mo-MLV and HaSV, respectively, as judged from the distances to the primer binding site (PBS) and the GACG motifs. We report here that heterodimer formation between HaSV and Mo-MLV or Akv-MLV is relatively insensitive to the presence or absence of SL1 or Pal I sequences in the respective RNAs, indicating that the SL1 and Pal I palindromes are of minor importance for heterodimerization. The GACG stem-loops are, however, responsible for heterodimerization, giving rise to large amounts of relatively unstable heterodimers. Noticeably,

Homo- and Heterodimerization of HaSV RNA

Akv RNA was found to be more reactive in heterodimerization with HaSV than Mo-MLV RNA, although they both carry the GACG stem-loops. This indicates that secondary structure is not the sole determinant of dimerization, and it may be that a compatible orientation of the stems in the tertiary structure is also a requirement for effective dimerization. As the GACG stem-loops are very conserved among the g-retroviruses, heterodimerization mediated through the GACG stem-loops could represent a general mechanism by which two distantly related viruses could interact, although they share no complementary palindromes. Although the wild-type SL1 palindrome does not participate in heterodimerization, it can, by the mutation of two nucleotides to a palindrome matching MLV Pal I, greatly facilitate heterodimerization. Hence, mutations of SL1 sequences allow control of dimerization with wild-type and mutant HaSV RNAs as well as with Mo-MLV and Akv MLV RNAs. We have no evidence for a role in heterodimerization of the Pal II motif previously found to be involved in kissing-loop interaction in the formation of MLV homodimers in vitro22 and in recombination in vivo.49 Interestingly, a search for palindromic sequences in HaSV revealed a 12 nucleotide long palindromic sequence in position 268– 279 downstream of the SL3. This palindrome shows no homology with Pal II of MLVs; however, it is located in approximately the same position relative to the PBS, Pal I and the GACG motifs. This “genetic organisation” of Pal I, Pal II and GACG motifs is found by database search in more distantly related murine viruses and in some simian viruses (e.g. gibbon ape leukemia virus) harbouring non-homologous palindromes in this position (data not shown). The conserved organisation of GACG motifs and palindromes indicates a similar function of these sequences, suggesting that the 268– 279 palindrome of HaSV may be involved in homodimerization equivalent to the Pal II of the MLVs. Recently, it has been experimentally demonstrated that homodimerization in Mo-MLV does involve the interaction of both Pal I and Pal II.28 The 268 –279 palindrome in HaSV is not predicted to attend a kissing-loop conformation by structural probing, and potential dimerization through this palindrome may involve the nucleic acid chaperone activity of the nucleocapsid protein. By pure speculation, this palindrome might be responsible for dimerization of constructs having non-complementary sequences in SL1 and UUCG substitutions in the loops of SL4 and SL5. The need for structural rearrangements prior to dimerization correlates with the slow dimerization kinetics observed for these constructs. Likewise, structural rearrangements appear to be necessary for dimerization of Mo-MLV RNA through Pal II.7,22 However, these issues have not been addressed experimentally in this study. In Mo-MLV, the dimerization through Pal II apparently results in the formation of an extended duplex, as the dissociation kinetics of

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Homo- and Heterodimerization of HaSV RNA

such dimers are very slow compared to what is expected of a loop-loop interaction.7 Possibly, the extended symmetry around the palindrome of SL1 may lead to formation of an extended duplex in HaSV RNA homodimers. In heterodimers formed between the SL1 UGGCCA mutant of HaSV RNA and Mo-MLV or Akv-MLV, sequence analysis indicates that a putative duplex may be less extended. There is some precedence that in vitro dimerization of retroviral RNA in the presence or absence of NC protein may reflect features relevant for the formation of dimers in vivo.27,50,51 The in vitro results presented here predict that in mixed stocks of HaSV and MLV from the same producer cells, the two types of homodimers may be favoured over heterodimers. Moreover, heterodimer formation with MLV may be increased in the SL1 (UGGCCA) mutant. Also, the possibility of specific heterodimerization by alternative non-palindromic sequences of SL1 is testable in vivo directly. Altogether, we propose that the dimerization region of g-retroviruses contains GACG stemloops as common determinants of dimerization and one or more autocomplementary sequences that modulate the specificity of interaction among viral RNAs. In all cases reported so far, these autocomplementary motifs in the 50 leader RNA are located upstream of the GACG motifs. Further studies may investigate if natural and engineered variations in such autocomplementary sequences have an effect on heterodimerization and recombination between different g-retroviruses.

Materials and Methods Template construction and RNA preparation Plasmid DNA carrying the leader region of Mo-MLV, Akv-MLV and HaSV, was used in a high-fidelity PCR amplification (mixture of Pfu (Promega) and Taq gold (Perkin – Elmer)), to generate the phage T7 templates. The forward primer included a T7 promoter and sitedirected mutations, the downstream primer was fully complementary to the template or harboured sitespecific mutations. The PCR products were extracted with phenol/chloroform and precipitated in sodium acetate/ethanol prior to T7 transcription. The RNA was transcribed using a T7 Megascript (Ambion), in reaction volumes of 10 ml. The reaction was added to an equal volume of formamide loading buffer and denatured for two minutes at 95 8C prior to loading on an 8 M urea/ polyacrylamide gel. The RNA was visualised by UVshadow paper, and the appropriately sized bands were excised and the RNA extracted into a 0.25 M sodium acetate (pH 6.0) and phenol solution. After extraction with phenol/chloroform, the RNA was precipitated in sodium acetate/ethanol, air-dried and finally dissolved in 20 ml of doubly distilled water. The concentration of each RNA was determined by measuring absorbance at 260 nm.

Dephosphorylation and 50 end-labelling Purified RNAs were dephosphorylated at their 50 ends with shrimp alkaline phosphatase (Amersham), extracted with phenol/chloroform, and precipitated in sodium acetate/ethanol. Dephosphorylated RNA was 50 end-labelled with [a-32P]ATP (100 mCi; 8000 Ci mmol21; ICN), by incubating with ten units of phage T4 polynucleotide kinase (USB). Full-length RNAs were purified by PAGE as described above. 30 End-labelling Cordycepin phosphate (Cp) was phosphorylated with [a-32P]ATP (100 mCi; 8000 Ci mmol21; ICN) by incubating with ten units of T4 polynucleotide kinase (USB) in a total volume of 10 ml. The labelled Cp was used in a ligation reaction of approximately 1 mg of RNA using a T4 RNA ligase (NEB) in a 20 ml reaction for three hours at room temperature. Full-length RNAs were purified by PAGE as described above. Enzymatic probing End-labelled RNA was probed using both monomeric and dimeric conditions. Under monomeric conditions, approximately 10 pmol of labelled RNA was diluted in 80 ml of water and denatured at 95 8C for two minutes and snap-cooled on ice for two minutes. Following snap-cooling, 20 ml of a 5 £ dimerization-buffer (see section Dimerization of RNAs) was added and incubated at 45 8C for five minutes. The RNA was less than 2% dimeric. Samples were transferred to ice and added to 100 ml of a 100 ng ml21 of tRNA solution. Each sample of 200 ml was incubated on ice for 15 minutes with 2 ml of RNase A (2 pg ml21) (Sigma), RNase T2 (200 units ml21) (In Vitrogen), RNase T1 (4 units ml21) (Sigma) and RNase V1 (100 units ml21) (Pierce). The reactions were stopped by addition of phenol, resulting in degradation of approximately 2 – 5% of total labelled RNA. After extraction with phenol/chloroform, the RNA was precipitated in sodium acetate/ethanol, air-dried and finally dissolved in 7 ml of formamide loading buffer. Under dimeric conditions, approximately 10 pmol of labelled RNA was added to non-labelled RNA and diluted to 80 ml, giving a total concentration of RNA of approximately 250 nM. RNA dimerization was carried out as described below with incubation at 45 8C for 30 minutes. The dimerization reaction was split into 10 ml aliquots and RNases were added to each aliquot as described above. The reactions were stopped as described above. Denaturing gel analysis and RNA sequencing Approximately 20 pmol of labelled RNA was added to 5 mg of tRNA and dried in a Speedvac. The RNA was dissolved in 10 ml of G-buffer (20 mM sodium citrate (pH 4.5), 7 M urea, 1 mM EDTA, 0.025% (w/v) xylene cyanol, 0.025% (w/v) bromophenol blue). After denaturation at 95 8C for two minutes, 1 ml of RNase T1 (100 units ml21) (Sigma) was added to the sequencing reaction and incubated at 55 8C for 15 minutes. The RNA degradation ladder was prepared by dissolving approximately 50 pmol of labelled RNA in 2 £ alkaline hydrolysis buffer (final concentration 50 mM NaHCO3/ Na2CO3 (pH 9.0), 1 mM EDTA, 250 ng ml21 of carrier tRNA) and incubating at 99.5 8C for 20 minutes. The reaction was stopped by transferring on ice and an

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equal amount of 90% formamide buffer added. RNAladder, RNA sequence and enzymatic digestion products were analysed using 7– 12% sequencing gels in TBE. Secondary structure modelling The best-defined probing data were used as constraints in the thermodynamic structure modelling using the RNA folding program mfold.39,40 Of the structures predicted by mfold the structure in best agreement with the remaining probing data is presented in Figure 2(b).

Homo- and Heterodimerization of HaSV RNA

by BioRad software, Quantity One. From the data collected, the molar ratio and the relative radioactivity located in the heterodimer band (heterodimeric RNA ratio) were calculated and plotted in graphs as a function of RNA concentration. In the reactions including only identical RNA, the molar ratio (Yc) was calculated and plotted as a function of total RNA concentration.

Acknowledgements Preparation of internally labelled RNA The T7 templates were transcribed using T7 Megascript, (Ambion), including about 15 mCi of [a-32P]UTP (800 Ci mmol21; Amersham) per nmol of UTP. The RNA was purified as described above and the concentration of the RNA was determined by scintillation counting. Integrity and purity of labelled and cold RNAs were analysed on denaturing 7 M urea/6 – 8% (w/v) polyacrylamide gel, and visualised by autoradiography and ethidium bromide, respectively. Dimerization of RNAs The following standard protocol for RNA dimerization was used unless anything else is noted. The labelled RNA was present at a fixed concentration of 5 nM and then titrated with increasing amounts of non-labelled RNA (5 – 500 nM). The dimerization reactions were mixed in thin-walled PCR tubes on ice, subsequently heated for two minutes at 95 8C and chilled on ice for two minutes. After addition of 2 ml of 5 £ dimerizationbuffer (final concentration, 50 mM sodium cacodylate (pH 7.5), 300 mM KCl, 5 mM MgCl2), the reactions were incubated for 30 minutes at 45 8C. Following incubation, the reactions were stopped on ice and 2 ml of 6 £ loading-buffer (80% (w/v) glycerol, 0.025% bromophenol blue, 1 mM EDTA) was added. Heat stability experiments Following standard RNA dimerization in 50 ml volume (see above), the reactions were put on ice and 500 ml of 20 mM Tris – HCl (pH 7.5), 50 mM NaCl was added. The total volume was transferred to a Centricon 30 spincolumn (Millipore) and spun at 4 8C, reducing the volume to approximately 20 ml. This step was repeated twice, keeping the samples on ice and finally diluted to a total volume of 100 ml with DE buffer. From this solution, aliquots of 10 ml were incubated at the temperatures noted for ten minutes. The reaction was stopped by placing the samples on ice prior to the addition of 2 ml of 6 £ loading-buffer. The samples were loaded immediately onto a native agarose gel. Gel electrophoresis and analysis The reactions were loaded onto 2.5% (w/v) agarose gels with TBE running buffer. The gels were run at 7 V cm21 at 4 8C for about four hours. After electrophoresis, the gels were dried in a BioRad gel vacuum drier at 60 8C. The dried gels were visualised on a PhosphorImager (BioRad Personnel FX, Kodak phosphorimager storage screens). Analysis and quantification were aided

We thank Lars Aagaard and Jørgen Kjems for critical reading of the manuscript. This work was supported by the Danish Research Agency, Bavarian Nordic Research Institute, the Danish Cancer Society, and the Karen Elise Jensen Foundation.

References 1. Kung, H. J., Hu, S., Bender, W., Bailey, J. M., Davidson, N., Nicolson, M. O. & McAllister, R. M. (1976). RD-114, baboon, and woolly monkey viral RNA’s compared in size and structure. Cell, 7, 609– 620. 2. Bender, W. & Davidson, N. (1976). Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules. Cell, 7, 595– 607. 3. Murti, K. G., Bondurant, M. & Tereba, A. (1981). Secondary structural features in the 70 S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy. J. Virol. 37, 411– 419. 4. Fu, W. & Rein, A. (1993). Maturation of dimeric viral RNA of Moloney murine leukemia virus. J. Virol. 67, 5443– 5449. 5. Fu, W., Gorelick, R. J. & Rein, A. (1994). Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions. J. Virol. 68, 5013– 5018. 6. Prats, A. C., Roy, C., Wang, P. A., Erard, M., Housset, V., Gabus, C. et al. (1990). cis elements and transacting factors involved in dimer formation of murine leukemia virus RNA. J. Virol. 64, 774–783. 7. De Tapia, M., Metzler, V., Mougel, M., Ehresmann, B. & Ehresmann, C. (1998). Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278 – 303) and new roles for stem-loops H2 (310– 352) and H3 (355 – 374). Biochemistry, 37, 6077– 6085. 8. Mann, R., Mulligan, R. C. & Baltimore, D. (1983). Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell, 33, 153– 159. 9. Mougel, M. & Barklis, E. (1997). A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions. J. Virol. 71, 8061– 8065. 10. Hu, W. S. & Temin, H. M. (1990). Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc. Natl Acad. Sci. USA, 87, 1556– 1560.

627

Homo- and Heterodimerization of HaSV RNA

11. Stuhlmann, H. & Berg, P. (1992). Homologous recombination of copackaged retrovirus RNAs during reverse transcription. J. Virol. 66, 2378– 2388. 12. Wolgamot, G., Bonham, L. & Miller, A. D. (1998). Sequence analysis of Mus dunni endogenous virus reveals a hybrid VL30/gibbon ape leukemia viruslike structure and a distinct envelope. J. Virol. 72, 7459–7466. 13. Mikkelsen, J. G., Lund, A. H., Duch, M. & Pedersen, F. S. (1999). Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses. J. Gen. Virol. 80, 2957– 2967. 14. Mikkelsen, J. G. & Pedersen, F. S. (2000). Genetic reassortment and patch repair by recombination in retroviruses. J. Biomed. Sci. 7, 77 –99. 15. Chong, H., Starkey, W. & Vile, R. G. (1998). A replication-competent retrovirus arising from a splitfunction packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences. J. Virol. 72, 2663– 2670. 16. Mikkelsen, J. G., Lund, A. H., Duch, M. & Pedersen, F. S. (2000). Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo. J. Virol. 74, 600–610. 17. Paillart, J. C., Marquet, R., Skripkin, E., Ehresmann, B. & Ehresmann, C. (1994). Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. J. Biol. Chem. 269, 27486– 27493. 18. Skripkin, E., Paillart, J. C., Marquet, R., Ehresmann, B. & Ehresmann, C. (1994). Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc. Natl Acad. Sci. USA, 91, 4945– 4949. 19. Polge, E., Darlix, J. L., Paoletti, J. & Fosse, P. (2000). Characterization of loose and tight dimer forms of avian leukosis virus RNA. J. Mol. Biol. 300, 41 – 56. 20. Fosse, P., Motte, N., Roumier, A., Gabus, C., Muriaux, D., Darlix, J. L. & Paoletti, J. (1996). A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization. Biochemistry, 35, 16601– 16609. 21. Tounekti, N., Mougel, M., Roy, C., Marquet, R., Darlix, J. L., Paoletti, J. et al. (1992). Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. J. Mol. Biol. 223, 205 –220. 22. Girard, P. M., Bonnet-Mathoniere, B., Muriaux, D. & Paoletti, J. (1995). A short autocomplementary sequence in the 50 leader region is responsible for dimerization of MoMuLV genomic RNA. Biochemistry, 34, 9785– 9794. 23. Girard, P. M., de Rocquigny, H., Roques, B. P. & Paoletti, J. (1996). A model of PSI dimerization: destabilization of the C278-G303 stem-loop by the nucleocapsid protein (NCp10) of MoMuLV. Biochemistry, 35, 8705– 8714. 24. Laughrea, M. & Jette, L. (1996). Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248– 271 are dispensable for dimer formation. Biochemistry, 35, 1589–1598. 25. Muriaux, D., De Rocquigny, H., Roques, B. P. & Paoletti, J. (1996). NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop

26.

27.

28. 29.

30.

31.

32.

33. 34.

35.

36.

37. 38.

39.

40.

41.

complex into a stable dimer. J. Biol. Chem. 271, 33686 – 33692. Marquet, R., Baudin, F., Gabus, C., Darlix, J. L., Mougel, M., Ehresmann, C. & Ehresmann, B. (1991). Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism. Nucl. Acids Res. 19, 2349– 2357. Haddrick, M., Lear, A. L., Cann, A. J. & Heaphy, S. (1996). Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1. J. Mol. Biol. 259, 58 – 68. Ly, H. & Parslow, T. G. (2002). Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus. J. Virol. 76, 3135– 3144. Oroudjev, E. M., Kang, P. C. & Kohlstaedt, L. A. (1999). An additional dimer linkage structure in Moloney murine leukemia virus RNA. J. Mol. Biol. 291, 603– 613. Konings, D. A., Nash, M. A., Maizel, J. V. & Arlinghaus, R. B. (1992). Novel GACG-hairpin pair motif in the 50 untranslated region of type C retroviruses related to murine leukemia virus. J. Virol. 66, 632 –640. Fisher, J. & Goff, S. P. (1998). Mutational analysis of stem-loops in the RNA packaging signal of the Moloney murine leukemia virus. Virology, 244, 133 –145. Yang, S. & Temin, H. M. (1994). A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA. EMBO J. 13, 713 –726. Kim, C. H. & Tinoco, I. (2000). A retroviral RNA kissing complex containing only two G.C base pairs. Proc. Natl Acad. Sci. USA, 97, 9396– 9401. Ly, H., Nierlich, D. P., Olsen, J. C. & Kaplan, A. H. (1999). Moloney murine sarcoma virus genomic RNAs dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines. J. Virol. 73, 7255 –7261. Soeda, E. & Yasuda, S. (1985). RNA Tumor Viruses; Molecular Biology of Tumor Viruses (Weiss, N., Teich, H., Varmus, H. & Coffin, J. M., eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Torrent, C., Bordet, T. & Darlix, J. L. (1994). Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus. J. Mol. Biol. 240, 434– 444. Feng, Y. X., Fu, W., Winter, A. J., Levin, J. G. & Rein, A. (1995). Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro. J. Virol. 69, 2486– 2490. Torrent, C., Gabus, C. & Darlix, J. L. (1994). A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virusVL30-derived vectors for gene transfer. J. Virol. 68, 661 –667. Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. RNA Biochemistry and Biotechnology NATO ASI Series (Barciszewski, J. & Clark, B. F. C., eds), pp. 11 – 43, Kluwer Academic Publishers, Dordrect. Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911 – 940. Tuerk, C., Gauss, P., Thermes, C., Groebe, D. R., Gayle, M., Guild, N. et al. (1988). CUUCGG hairpins: extraordinarily stable RNA secondary structures

628

42.

43.

44.

45.

46.

Homo- and Heterodimerization of HaSV RNA

associated with various biochemical processes. Proc. Natl Acad. Sci. USA, 85, 1364– 1368. Mougel, M., Tounekti, N., Darlix, J. L., Paoletti, J., Ehresmann, B. & Ehresmann, C. (1993). Conformational analysis of the 50 leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization. Nucl. Acids Res. 21, 4677– 4684. Sundquist, W. I. & Heaphy, S. (1993). Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc. Natl Acad. Sci. USA, 90, 3393– 3397. Kolb, F. A., Westhof, E., Ehresmann, C., Ehresmann, B., Wagner, E. G. & Romby, P. (2001). Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense – target RNA complex. Nucl. Acids Res. 29, 3145– 3153. Takahashi, K. I., Baba, S., Chattopadhyay, P., Koyanagi, Y., Yamamoto, N., Takaku, H. & Kawai, G. (2000). Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome. RNA, 6, 96 –102. Mougel, M., Zhang, Y. & Barklis, E. (1996). cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA. J. Virol. 70, 5043– 5050.

47. Levin, J. G., Grimley, P. M., Ramseur, J. M. & Berezesky, I. K. (1974). Deficiency of 60 to 70 S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J. Virol. 14, 152– 161. 48. Housset, V., De Rocquigny, H., Roques, B. P. & Darlix, J. L. (1993). Basic amino acids flanking the zinc finger of Moloney murine leukemia virus nucleocapsid protein NCp10 are critical for virus infectivity. J. Virol. 67, 2537– 2545. 49. Mikkelsen, J. G., Lund, A. H., Dybkaer, K., Duch, M. & Pedersen, F. S. (1998). Extended minus-strand DNA as template for R-U5-mediated second-strand transfer in recombinational rescue of primer binding site-modified retroviral vectors. J. Virol. 72, 2519– 2525. 50. Clever, J. L. & Parslow, T. G. (1997). Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation. J. Virol. 71, 3407– 3414. 51. Paillart, J. C., Berthoux, L., Ottmann, M., Darlix, J. L., Marquet, R., Ehresmann, B. & Ehresmann, C. (1996). A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis. J. Virol. 70, 8348–8354.

Edited by J. Karn (Received 27 May 2002; received in revised form 4 September 2002; accepted 5 September 2002)