For Northern blot analysis of viral RNA in denaturing conditions, RNA extracted as described above was resuspended in H2O after ethanol precipita-.
JOURNAL OF VIROLOGY, Feb. 1995, p. 1079–1084 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 69, No. 2
The Dimerization/Packaging Sequence Is Dispensable for both the Formation of High-Molecular-Weight RNA Complexes within Retroviral Particles and the Synthesis of Proviruses of Normal Structure ´ NIO THIERRY TCHE
AND
THIERRY HEIDMANN*
Unite´ de Physicochimie et Pharmacologie des Macromole´cules Biologiques, Centre National de la Recherche Scientifique URA 147, Institut Gustave Roussy, 94805 Villejuif Cedex, France Received 28 July 1994/Accepted 20 October 1994
Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the C packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and C packaging sequences—and even an RNA lacking the first 6,537 nucleotides of MoMLV— can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/C-deleted viral RNAs. These observations demonstrate that the DLS and C packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration. precluding the possible involvement of other viral sequences in intermolecular RNA linkage (19). In these studies, the DLS has been localized near the 59 end of the viral RNA and shown to be part of the C packaging sequence. In agreement with these observations, it has been shown that short regions encompassing the C packaging sequence can promote the efficient dimerization of RNAs transcribed in vitro (27, 38). However, the precise relationships between the DLS and C sequences are still unknown, and furthermore, the requirement for the DLS/C sequence to promote RNA dimerization in vivo within the retroviral particles has not been investigated. In this study, we have investigated in vivo the role of the DLS/C sequence in the dimerization of the viral RNA within the retroviral particles and in the reverse transcription process. An unexpected result is that no sequence within the first 6,500 nt of MoMLV, which include the DLS/C sequence, is critically required in cis for the occurrence of high-molecular-weight, slow-migrating RNA complexes within the retroviral particles and furthermore that the deletion encompassing the DLS/C sequence does not prevent the accurate reverse transcription of the viral genome and the formation of proviruses of normal structure.
Retroviruses are a family of RNA viruses which replicate through reverse transcription of their viral RNA and integration of the cDNA copy into the host cellular DNA. Extracellular viral particles usually contain two identical genomic RNA molecules which are packaged into virions by a highly efficient and selective process. Sequences that are strongly required in cis for viral RNA packaging have been localized within a short region of less than 400 nucleotides (nt) near the 59 end of the viral RNA, which is designated the encapsidation or packaging sequence, abbreviated E or C (16, 21, 39, 40). In the case of Moloney murine leukemia virus (MoMLV), deletion of the C sequence, mapped between nt 212 and 563 (21)—and in fact between nt 212 and 400 (30)—results in a decrease in viral RNA packaging efficiency by at least 1,000-fold (21, 35). Conversely, the C sequence of the Moloney sarcoma virus, and to a lesser extent that of MoMLV (1), is sufficient in cis to enhance packaging of nonretroviral RNAs into virions. In the case of MoMLV, secondary sequences localized within the first 1,100 bases of the viral RNA may also be involved in that process (3, 4, 24). The two genomic viral RNA molecules packaged into the virions are assembled as a dimer (5–7, 18, 19, 32). The two RNA monomers are linked by weak noncovalent bonds, since the dimer structure can be dissociated into monomers under relatively mild conditions. RNA dimerization initiates early in the course of retroviral particle formation, and the RNA dimer undergoes additional maturation processes subsequent to virion release from the cell (11, 32). Analyses of partially denatured dimers by electron microscopy have suggested that a sequence, designated the dimer linkage sequence (DLS), is involved in intermolecular RNA linkage (5, 6, 18, 25), without
MATERIALS AND METHODS Plasmids. All plasmids were constructed by standard cloning procedures; numbers refer to the distance from the viral MoMLV cap site. The pMLVC1SVtkneo plasmid (gift from C. Roy) was derived from pMLVSVtkneo (29) by extending the viral C packaging sequence to nt 1560 (instead of nt 563 in pMLV-SVtkneo; see Fig. 1). To do so, we replaced the ClaI (unique site in pBR322 upstream of the 59 long terminal repeat [LTR])-SalI (unique site just upstream of the simian virus early and herpesvirus thymidine kinase promoters in tandem [SVtk]) Klenow-treated fragment from pMLV-SVtkneo with a ClaI (unique site in pBR322) -XhoI (unique site in the gag gene at nt 1560) Klenowtreated fragment from a wild-type MoMLV provirus (pMov3 [12] recloned in pMLV-SVtkneo by homologous substitution of the proviral domains after enzyme restriction at unique sites in the LTRs). pMLVC2SVtkneo was derived from pMLVC1SVtkneo by substitution of the XbaI (nt 2151)-BstEII (nt 725) viral fragment from pMLVC1SVtkneo with the homologous fragment from pMovC2 (21), which carries the C sequence deletion (from nt 212 to 563)
* Corresponding author. Mailing address: Unite´ de Physicochimie et Pharmacologie des macromole´cules biologiques, CNRS URA 147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France. Phone: 33 / 1 45 59 49 70. Fax: 33 / 1 46 78 41 20. 1079
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FIG. 1. Structure of the transfected proviruses and cellular and virion RNAs. Northern blot analysis, under denaturing conditions, of cellular and virion RNAs isolated from C2-transfected cells. Polyadenylated cellular RNAs (0.5 mg) and virion RNAs were fractionated by electrophoresis through a 0.8% agarose– formaldehyde gel, transferred to a Hybond N membrane, and analyzed with a neo riboprobe. Lanes C1, C2, and none correspond, respectively, to C2 cells transfected with pMLVC1SVtkneo, pMLVC2SVtkneo, and no plasmid. The structure of the proviruses are shown below, with the C deletion, the internal SVtkneo reporter gene inserted between nt 1560 and 6535 of MoMLV, and the expected genomic and subgenomic transcripts. The amounts of virion RNA correspond to 1.5 ml of supernatant for C1 and 150 ml of supernatant for C2 and the control; exposure times were 16 h without intensifying screen for cellular RNAs and for C1 virion RNA and 10 days with an intensifying screen for C2 and control virion RNAs. r, positions of the 28S and 18S rRNAs.
indicated in Fig. 1. This plasmid was constructed by a three-fragment ligation, including an XbaI (nt 2151)-BstEII (nt 725) fragment derived from pMovC2, a BstEII (nt 725)-ClaI (unique site in pBR322), and a ClaI (in pBR322)-XbaI (nt 2151) fragment from pMLVC1SVtkneo. Plasmid pT7neo, used for riboprobe synthesis, was provided by S. Jensen. It was constructed by insertion of the neo open reading frame (ORF) fragment at the HindIII site of Bluescript M132 in the antisense orientation relative to that of the T7 promoter and after making the recessed ends blunt by Klenow treatment. For RNA transcription, pT7neo was linearized with NcoI, which cuts once in the neo ORF. Viruses and viral RNA analysis. For Northern (RNA blot) analysis of the RNA from extracellular virions in nondenaturing conditions, supernatants of G418r C2 cells were harvested three times every 10 to 14 h and filtered through 0.45-mm filters. Pooled collections (540 ml) were stored at 48C. The viral particles were first pelleted by centrifugation in a JA-14 rotor at 12,000 rpm at 48C for 12 h and then suspended in 8 ml of TNE (10 mM Tris-HCl [pH 7.2], 150 mM NaCl,
J. VIROL. 1 mM EDTA) and pelleted through a 20% sucrose cushion for 2 h in an SW41 rotor at 35,000 rpm and 48C. The pelleted virions were lysed for 30 min at 378C in 400 ml of lysis buffer (TNE plus 0.5% sodium dodecyl sulfate [SDS], 200 mg of proteinase K per ml, and 5 mM dithiothreitol) containing 40 mg of yeast tRNA. The lysate was gently extracted once with phenol containing 0.1% 8-hydroxyquinoline and saturated with 2 mM EDTA–400 mM NaCl–50 mM Tris-HCl (pH 7.4) and once with phenol-chloroform (1:1) containing 0.05% 8-hydroxyquinoline and saturated with 1 mM EDTA–400 mM NaCl–50 mM Tris-HCl (pH 7.4), and then RNA was precipitated by addition of sodium acetate to 0.2 M (final concentration) and 2.5 volumes of ethanol. Precipitated RNA was dissolved in buffer E (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 50 mM sodium citrate [pH 7.0], 5 mM b-mercaptoethanol) and heated at the indicated temperature in a calibrated thermal reactor for 10 min. We have verified that identical results were obtained upon heating for shorter times (3 min). After heating, RNA was chilled on ice and then size-fractionated by electrophoresis at 48C at about 8 V/cm through a 1% agarose gel in 0.53 TBE buffer (45 mM Tris-borate [pH 8.3], 1 mM EDTA). RNA was transferred to a Hybond N membrane by capillary blotting in 150 mM ammonium acetate after treatment of the agarose gel twice with 50 mM NaOH and 10 mM NaCl for 15 min and twice with 150 mM ammonium acetate for 15 min, and then fixed by UV irradiation of the wetted membrane for 5 min with the germicidal lamp of a cell culture hood. The nylon membranes were prehybridized for 6 h at 558C in 50% formamide–1M NaCl–103 Denhardt’s solution–50 mM Tris-HCl (pH 7.4)–0.1% pyrophosphate (Na4P2O7)–1% SDS–10% sulfate dextran and hybridized in the same buffer with an a-32P-labelled RNA probe for 20 h. Then, the nylon membranes were washed twice for 20 min with buffer A (50% formamide, 0.5 M NaCl, 1% SDS, 50 mM Tris HCl [pH 7.0], 1 mM EDTA) at 608C, four times with 0.53 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 608C for 10 min, once at 378C with 23 SSC plus RNAse A at a final concentration of 8 mg/ml to remove riboprobe molecules which are not annealed to complementary sequences, rinsed twice with 0.53 SSC–0.5% SDS, and washed for 20 min with buffer A at 608C. For Northern blot analysis of viral RNA in denaturing conditions, RNA extracted as described above was resuspended in H2O after ethanol precipitation, size-fractionated through a 1% agarose gel in formaldehyde as described previously (20), blotted to a Hybond N membrane, and hybridized with an a-32P-labelled RNA probe as described above. For the infectivity tests, the supernatants were harvested from cell cultures close to confluency, filtered through 0.45-mm filters, and immediately tested. Serial dilutions of the supernatants in growth medium plus Polybrene at a final concentration of 8 mg/ml were left in contact with murine WOP test cells (5 3 104 cells plated in a 6-cm-diameter culture dish the day before) for 24 h; cells were maintained in culture for another day in fresh medium and then selected for G418 resistance. Reverse transcriptase activity was determined as described previously (36). Cells and cellular nucleic acid analysis. Murine 3T3 cells transformed by the polyomavirus T antigen (WOP cells [8]) and C2 cells as well as their derivatives were grown in Dulbecco’s modified Eagle’s medium supplemented with 8% fetal calf serum (GIBCO) at 378C in 6% CO2. Transfections of C2 cells were achieved by the calcium phosphate method, and G418 selections were conducted in growth medium supplemented with 700 mg of G418 (GIBCO) per ml. For Northern blot analysis of cellular RNA in nondenaturing conditions, cells (about 4 3 106) were washed twice with isotonic phosphate-buffered saline (PBS), trypsinized, and harvested in 5 ml of PBS. Cells were pelleted by centrifugation at 250 3 g for 3 min, resuspended in 360 ml of TNE which was supplemented with 40 mg of yeast tRNA, 5 mM dithiothreitol, 0.4% Triton X-100 and, when indicated, concentrated virions, at 48C. The lysate was centrifuged for 8 min at 48C and 4,500 3 g to pellet the nuclei on 400 ml of a 20% sucrose cushion, and the supernatant was harvested and processed exactly as described above for virion RNA extraction. Briefly, the supernatant was supplemented with 0.5% SDS–200 mg of proteinase K per ml, incubated at 378C for 30 min, and extracted with phenol and phenol-chloroform, and RNA was precipitated with ethanol. RNA was dissolved in buffer E, heated when indicated, size-fractionated, and transferred to Hybond N membranes as for virion RNA. The blots were prehybridized in 0.5 M sodium phosphate (pH 6.8)–7% SDS–1 mM EDTA–5 mg of bovine serum albumin per ml for 2 h at 658C and then hybridized in the same buffer with a-32P-labelled DNA probes for 20 h. Blots were then washed twice at 658C in 0.13 SSC–0.1% SDS for 15 min. For Northern blot analysis in denaturing conditions, polyadenylated cellular RNAs were extracted and size-fractionated through 1% agarose gels in formaldehyde as described previously (20). Southern blot analyses were done as described previously (20). Hybridizations with a-32P-labelled DNA probes were done as described above.
RESULTS Deletion of the DLS/C sequence decreases RNA packaging into virions but does not prevent the accurate reverse transcription of retroviral RNA. Plasmids carrying the MLVC1SV tkneo and the DLS/C-deleted MLVC2SVtkneo proviruses, both of which contain a selectable marker conferring G418
VOL. 69, 1995
resistance (see Materials and Methods and Fig. 1), were introduced by transfection into C2 helper cells (21), and two independent G418r cell populations were isolated for each construct. Cells were expanded, and the culture supernatants were collected to recover the viral particles for further analysis. Northern blot analysis under denaturing conditions of the polyadenylated cellular RNAs (see Fig. 1) disclosed two major transcripts, as expected, one corresponding to the genomic RNA (gRNA; either C1 or C2) and one to the internal transcript initiated within the SVtk promoter (and not hybridizing to a 59 probe; not shown). Northern blot analysis under denaturing conditions of the RNA within the virions disclosed only the genomic transcript for the C1 construct, whereas for the C2 construct, both the genomic and the internal transcript could be detected; it should be emphasized that, in this latter case, the amount of packaged gRNA was 500- to 2,000-fold lower than for the C1 provirus (as determined by dot and Northern blot analyses [Fig. 1]; see amount of cell supernatant and time of exposure in the legend), and it cannot be excluded that similar amounts of the subgenomic SVtk-initiated transcripts are also packaged into virions produced by the MLVC1SVtkneo-transfected C2 cells. To test for the infectivity of the C2 virions, murine test cells were infected by serial dilutions of cell supernatant and then selected for G418r as described in Materials and Methods. G418-resistant cells were recovered at a low yield (25 6 4 U/ml), a value 2,500 6 500 times lower than that measured under identical conditions with the MLVC1SVtkneo-transfected C2 cells (after normalization of supernatant concentrations by their reverse transcriptase activity, less than 40% variation, and for the provirus transcription levels). As previously reported (21, 35), the decrease in RNA packaging efficiency by approximately 1,000-fold (see above) should therefore be the main factor accounting for the decrease in virus titer associated with the DLS/C deletion. The structures of the neo-marked retroviruses which have integrated into the cellular DNA of the test cells upon infection were further analyzed by Southern blots (Fig. 2). Interestingly, despite the deletion of the DLS/C sequence, we found that almost all the G418r cells contained a provirus of normal structure: actually, Southern blot analyses using a series of restriction enzymes, indicated in Fig. 2C, demonstrated that 20 of 24 and 18 of 19 G418r clones resulting from infection of test cells with supernatants from two independent C2-transfected C2 helper cell populations were of normal structure, with no detectable alterations (Fig. 2); moreover, in all cases the HindIII site located in place of the DLS/C deletion in the C2 construct was systematically recovered, thus clearly demonstrating the absence of recombination between the MLVC2 SVtkneo provirus and some possibly C-containing endogenous structures. Clearly then, removal of the DLS/C sequence, although it severely decreased the packaging efficiency of the viral RNA, still did not prevent the formation of integrated proviruses of normal structure. MLVC1SVtkneo and MLVC2SVtkneo transcripts are both extracted from virion particles as temperature-sensitive, highmolecular-weight, slow-migrating RNA complexes. Since previous studies have implicated the DLS/C sequence in the dimerization of the viral gRNA, we have comparatively analyzed the structure of MLVC1SVtkneo and MLVC2SVtkneo RNAs within retroviral particles by Northern blot in nondenaturing conditions. For each construct, the supernatants of the transfected G418r C2 helper cell populations were collected every 10 to 14 h for 36 h and then immediately processed to extract and analyze the viral RNAs under native conditions (according to a procedure derived from that described before
ROLE OF THE DLS/C SEQUENCE IN RETROVIRUSES
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FIG. 2. Deletion of the DLS/C packaging sequence does not prevent the formation of proviruses of normal structure. Southern blot analysis and restriction site mapping of proviruses within test cells infected with transfected C2 cell supernatants. Genomic DNAs extracted from C2 producer cells transfected with pMLVC1SVtkneo (lane A) or pMLVC2SVtkneo (lane B) as well as from the derived G418r cell clones generated upon infection of test cells with the corresponding cell supernatants (lanes A.1 and A.2 and B.1 to B.5) were analyzed by Southern blot with a 32P-labelled neo DNA probe after restriction with NheI (A), KpnI (B), or the enzymes indicated (C); the positions of the fragments expected for MLVC1SVtkneo and MLVC2SVtkneo proviruses are indicated (in kilobases). (C) Restriction site mapping of MLVC1SVtkneo and MLVC2SVtkneo proviruses generated upon infection; the occurrence and position of all the indicated enzymatic restriction sites were tested by Southern blots, using 32Plabelled neo and SV DNA probes. A, AflII; H, HindIII; K, KpnI; Nc, NcoI; N, NheI; X, XbaI.
[17]; see Materials and Methods). Northern blot analysis under these conditions disclosed, as previously reported for other wild-type retroviral transcripts, that the MLVC1SVtkneo transcripts are extracted from viral particles as heat-sensitive, slow-migrating RNA complexes (Fig. 3A); increasing the temperature of the RNA samples before gel electrophoresis resulted in the dissociation of the RNA complexes and the generation of a band of reduced molecular weight corresponding to the viral RNA monomers. For the MLVC2SVtkneo construct, since the RNA content of the virions was at least 1,000fold lower—see previous section and the legend to Fig. 3—a large amount of viral particles had to be processed to detect specific neo-hybridizing RNAs (see the negative control in Fig. 3 for a related amount of cell supernatant from nontransfected C2 helper cells). Rather unexpectedly, the neo-hybridizing
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FIG. 3. Evidence for high-molecular-weight, heat-sensitive, slow-migrating RNA complexes within retroviral particles. (A) Analysis of virion RNAs by Northern blot in nondenaturing conditions. RNAs extracted under native conditions from virions produced by C2 cells transfected with pMLVC1SVtkneo, pMLVC2SVtkneo, or no plasmid were treated at the indicated temperatures before analysis on nondenaturing Northern blots using a neo riboprobe, as described in Materials and Methods. The high-molecular-weight, slow-migrating, heat-sensitive RNA complexes are indicated by solid triangles, and the bands obtained after heat denaturation are indicated by open triangles. Amounts of virion RNAs deposited in each lane correspond to 2, 150, and 200 ml of C2 cell supernatants, respectively. Exposure times were 16 h for MLVC1SVtkneo RNAs and 14 days for the others. (B) Thermal stability of the neo-marked RNA complexes extracted from the virions. Values have been measured by scanning autoradiograms derived from two independent Northern blot analyses in nondenaturing conditions as in panel A.
RNAs also migrated as high-molecular-weight complexes; however, they disclosed a more diffuse migration pattern than the wild-type C1 transcripts (Fig. 3A), and their thermal stability was found to be slightly lower (68C; see Fig. 3B for two independent experiments). Finally, dissociation of the MLVC2SVtkneo complexes resulted in two RNA species, one almost comigrating with the MLVC1SVtkneo transcripts and therefore most likely corresponding to the genomic MLVC2 SVtkneo transcript, and a shorter one most likely associated with the internal transcript (see also Fig. 1). Two series of experiments suggest that the slow-migrating complexes isolated from the virions are not an artifact of the RNA extraction and analysis procedures. First, after complete dissociation of the RNA complex upon heating at 858C, no treatment related to those used for RNA extraction, such as ethanol precipitation or incubation at 378C in TNE buffer, could restore a slow-migrating RNA complex (data not shown). Second, nondenaturing Northern blot analysis of cellular RNAs extracted from MLVC2SVtkneo-transfected C2 cells using a procedure closely related to that for the virion RNAs (see below and Materials and Methods) did not reveal any slow-migrating, heat-sensitive RNA complexes (Fig. 4), even with an overexposure of the blot, but only two heatinsensitive bands, corresponding to the genomic and subgenomic transcripts. The procedure used in this experiment for the isolation, under native conditions, of the cellular RNAs was validated by demonstrating that slow-migrating viral RNA complexes could still be detected in the same nondenaturing Northern blot when concentrated virions were mixed with the C2 cells prior to their lysis (see Materials and Methods and Fig. 4). These results also indicate, in agreement with previous studies for MoMLV and Rous sarcoma virus (9, 23), that viral RNA can be found as high-molecular-weight complexes essentially in the virions and not in detectable amounts within the cells.
FIG. 4. Neo-marked viral RNAs in C2 cells are not isolated by nondenaturing extraction as high-molecular-weight, slow-migrating, heat-sensitive complexes. Northern blot analysis, under nondenaturing conditions, of cellular RNAs isolated from C2-transfected cells. RNAs from two different pMLVC2SVtkneotransfected C2 cell populations (lanes c1 and c2) and from pMLVC2SVtkneotransfected C2 cells with concentrated virions added to the cells before RNA isolation as a control (lane c2 1 v) were extracted as described in Materials and Methods, heated or not at 858C for 5 min, and analyzed by Northern blot in nondenaturing conditions as described in the legend to Fig. 3. The same amount of concentrated virion (corresponding to 75 ml of pMLVC1SVtkneo-transfected C2 cell supernatant) has been deposited alone in lane v. The high-molecularweight, slow-migrating, heat-sensitive RNA complexes are indicated by solid triangles, and heat-insensitive bands are indicated by open triangles.
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ROLE OF THE DLS/C SEQUENCE IN RETROVIRUSES
DISCUSSION The retroviral DLS/C sequence has been found to be involved in RNA packaging (16, 21, 39, 40), RNA dimerization (see references 5, 6, 18, and 25 for electron microscopy analyses and reference 27 and references in 38 for in vitro studies), and, to some extent, viral RNA processing, as it has been previously suggested that deletion of the DLS/C may affect the structure of the packaged RNA complex within the virion and perturb reverse transcription (34, 35). To investigate the relative contribution of the DLS/C sequence in these various processes, an SVtkneo-marked C1 MoMLV proviral structure (MLVC1SVtkneo) and one derivative with a deletion between nt 212 and 567, which includes the DLS and C packaging sequences (21, 25, 27), were introduced by transfection into C2 helper cells, and the properties of the resulting virions comparatively analyzed. As previously reported, we observed that the DLS/C deletion resulted in a large decrease in viral titer, approximately 2,500-fold, which can be essentially accounted for by a decrease in viral RNA packaging efficiency of 500- to 2,000-fold. However, an unexpected result was that almost all the proviruses generated upon infection (38 of 43 proviruses analyzed for two independent MLVC2SVtkneo C2 producer cell populations tested) disclosed no structural alteration, as determined by refined restriction mapping. This indicates that viral particles which contain a DLS/C-deleted retroviral genomic transcript can achieve the accurate reverse transcription of this RNA and therefore that the DLS/C sequence is essential only for RNA packaging and dispensable for reverse transcription and provirus formation. These results contrast somewhat with those from a previous study in which deletion of the C packaging sequence was reported to result in numerous alterations in the proviral structures generated upon infection (35); although the reasons for these discrepancies are unclear, it can be noted that in the latter case, the C-deleted RNAs were likely to be copackaged with C1 RNAs because of the use of a nondefective helper virus, which could greatly enhance recombination during reverse transcription. As the DLS/C sequence has also been reported to be involved in the dimerization of the viral RNAs, we have analyzed by Northern blots, under nondenaturing conditions, the structure of the viral RNAs packaged within the virions. An unexpected result was the demonstration that deletion of the DLS/C sequence does not prevent the formation of heat-sensitive, slow-migrating, high-molecular-weight viral RNA complexes within the retroviral particles produced by MLVC2SV tkneo-transfected C2 cells. These complexes dissociate at a temperature only a few degrees lower than that for the control MLVC1SVtkneo RNA complexes isolated from the corresponding virions but still display a more heterogeneous structure, as evidenced by a diffuse pattern of migration. The dissociation temperature for the MLVC1SVtkneo RNA complexes was close to 608C, as found by others for the dissociation into monomers of the packaged viral RNA dimers (5); the different value reported before (11) may be attributed to differences in the buffer used. We also found that the major neo-marked RNA in the slow-migrating complex within the virions produced by the MLVC2SVtkneo-transfected C2 cells is the SVtk-promoted subgenomic viral RNA, which lacks the first 6,537 nt of MoMLV. All together, these results clearly demonstrate that at least part of the intermolecular RNA bonds within the retroviral particles do not rely on sequences in cis within the 59 MoMLV RNA, a conclusion in agreement with previous observations by electron microscopy disclosing
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that viral RNA molecules are held together at many locations within the dimer structure (19). It can therefore be proposed that the intermolecular RNA linkage within the virion is not sequence specific, as not depending on stringently defined sequences in cis, but rather results simply from the conditions encountered during retroviral particle formation. For instance, the local concentration and condensation of the viral RNA at the budding membrane during the packaging process and/or its high local concentration within the virions could promote the spontaneous and DLS-independent annealing of the RNAs, also possibly catalyzed by the high local concentration of the NC p10 nucleocapsid viral protein (2, 27, 28). Accordingly, one would expect that the viral RNAs within the MLVC2SVtkneo virions are complexed with various RNA species, depending on their relative abundance, into the retroviral particles. Dimers of MLVC2SVtkneo-encoded RNAs could exist, but most likely a large fraction of the MLVC2SVtkneo-encoded RNAs are linked to heterologous RNAs, such as endogenous MLV-related or VL30 RNAs (22, 31, 33, 38), the latter displaying a high packaging efficiency into the MoMLV particles (38). Heterologous complexes would result in a low sequence identity between the MLVC2SVtkneo transcripts and the copackaged RNAs, which could account for the absence—or at least a very low rate—of recombination during reverse transcription (41). It could further imply that the complete reverse transcription of the genomic MLVC2SVtkneo RNA into cDNA requires only one RNA molecule, a conclusion in agreement with previous studies suggesting that one RNA molecule should be sufficient to generate a complete provirus (13, 15, 26). Accordingly, what should be the role of the DLS sequence per se? This sequence has been previously identified as a sequence involved in RNA dimerization by electron microscopy and in vitro studies, and the colocalization of the DLS and the C sequence has suggested a close relationship between the RNA packaging process and the formation of RNA dimers. Accordingly, it has been previously hypothesized that a cellular DLS-mediated annealing of the RNAs, and the resulting dimeric structural assembly, is required for the packaging process and that this requisite can be overcome only with a very low efficiency by monomeric RNAs; although attractive, definite evidence in favor of this hypothis is still lacking (see Discussion in reference 11), and another, nonexclusive, hypothesis could be proposed on the following basis. The highmolecular-weight complexes extracted from the MLVC2SV tkneo virions are somehow related to the ‘‘immature’’ RNA dimers previously characterized in nascent or protease-defective retroviral particles (10, 11); in all cases, the observed high-molecular-weight viral RNA complexes display an altered electrophoresis mobility and a lower thermal stability than mature C1 RNA dimers. Then, it could be hypothesized that ‘‘immature’’ RNA complexes are spontaneously formed during and/or immediately after packaging (possibly in a DLS-independent manner; see preceding section) and that maturation of these complexes within the retroviral particles requires the DLS sequence in cis as well as some cleavage products of the gag and pol polyproteins in trans (11, 23). Transformation of an ‘‘immature’’ complex into the ‘‘mature’’ RNA dimer within retroviral particles would therefore involve the intermolecular linkage of the DLS/C sequences, possibly catalyzed by the NC p10gag cleavage product (23). Although we found that the DLS/C sequence was dispensable for accurate reverse transcription, it actually remains possible that the DLS sequence, by imposing an ordered annealing of the retroviral RNA molecules within the viral particle, promotes efficient and accurate interstrand jumps of the reverse transcriptase during provirus
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