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J Virol. 2009 December; 83(23): 12526–12534.
Published online 2009 September 23. doi:  10.1128/JVI.01219-09
PMCID: PMC2786734

Packaging of Host mY RNAs by Murine Leukemia Virus May Occur Early in Y RNA Biogenesis[down-pointing small open triangle]


Moloney murine leukemia virus (MLV) selectively encapsidates host mY1 and mY3 RNAs. These noncoding RNA polymerase III transcripts are normally complexed with the Ro60 and La proteins, which are autoantigens associated with rheumatic disease that function in RNA biogenesis and quality control. Here, MLV replication and mY RNA packaging were analyzed using Ro60 knockout embryonic fibroblasts, which contain only ~3% as much mY RNA as wild-type cells. Virus spread at the same rate in wild-type and Ro knockout cells. Surprisingly, MLV virions shed by Ro60 knockout cells continued to package high levels of mY1 and mY3 (about two copies of each) like those from wild-type cells, even though mY RNAs were barely detectable within producer cells. As a result, for MLV produced in Ro60 knockout cells, encapsidation selectivity from among all cell RNAs was even higher for mY RNAs than for the viral genome. Whereas mY RNAs are largely cytoplasmic in wild-type cells, fractionation of knockout cells revealed that the residual mY RNAs were relatively abundant in nuclei, likely reflecting the fact that most mY RNAs were degraded shortly after transcription in the absence of Ro60. Together, these data suggest that these small, labile host RNAs may be recruited at a very early stage of their biogenesis and may indicate an intersection of retroviral assembly and RNA quality control pathways.

Retroviruses are ribonucleoprotein (RNP) complexes that assemble at host cell plasma membranes. Their predominant RNA component is the unspliced retroviral genome, but virions also contain a number of host cell noncoding RNAs (3, 38, 45). Subsets of these RNAs are overrepresented in virions relative to their abundance in host cells (38). Other than the primer tRNA, which anneals to the primer binding site on viral genomic RNA (gRNA) and initiates minus-strand DNA synthesis, the manner of recruitment of these RNAs and whether they function in retrovirus biology are unknown (5, 29, 30). Virion assembly does not require viral gRNA, but RNA of some sort is required for assembly, thus suggesting that host RNA can play this role (27, 33). Although prevailing notions suggest nonspecific RNA interactions drive retrovirus assembly (20), the concentration within virions of particular subsets of host cell noncoding RNAs suggests that their recruitment may have functional significance for viral assembly or other replication processes.

Among the most highly recruited noncoding cellular RNAs in Moloney murine leukemia virus (MLV) are mY1 and mY3 (38). These RNAs are enriched in MLV particles to a degree similar to that of the highly packaged 7SL RNA (also called 7S or SRP RNA) (38). Packaging of 7SL RNA, the scaffolding RNA of host cell signal recognition particles, is observed for a number of retroviruses, including Rous sarcoma virus (4), MLV (11, 38, 41), and human immunodeficiency virus type 1 (HIV-1) (39). In MLV, 7SL is present at three- to fourfold molar excess to gRNA and therefore is packaged at approximately six to eight copies per virion (38). HIV-1 particles contain roughly 10 to 14 molecules of 7SL per gRNA dimer (39). Like MLV, HIV-1 also packages at least some Y RNAs, albeit at a lower level of enrichment than 7SL (1, 25, 57).

mY1 and mY3 are host RNA polymerase III transcripts of ~100 nucleotides (nt). Both of these mouse RNAs fold into similar structures consisting of 5′ and 3′ ends joined in a base-paired stem surrounding an internal, largely single-stranded loop (8). Within cells, most mY RNAs are complexed with the host cell protein Ro60, which appears to function in quality control of misfolded noncoding RNAs (7, 36, 44). Structural analysis suggests that Y RNA binding may inhibit Ro60 access to misfolded RNAs, as Ro60 binding sites for Y RNAs partially overlap those for misfolded RNAs (18, 54, 62). In agreement with this hypothesis, a bacterial Y RNA inhibits the function of its Ro orthologue in 23S rRNA maturation (9). It has been suggested that Y RNA binding might sequester Ro60 in the cytoplasm, thereby preventing Ro60's interaction with nascent nuclear transcripts (36, 42). Consistent with this view, the mouse Ro protein was recently shown to contain a signal for nuclear accumulation that is masked by Y RNA binding (50).

Here, we demonstrate that mY1 and mY3 are recruited into MLV virions without the cellular protein Ro60. Ro60 is necessary for the cellular stability of mY1 and mY3, and therefore, Ro60 knockout cells contain only very low levels of these RNAs. Surprisingly, MLV packaged high levels of mY1 and mY3 when it replicated in Ro60 knockout cells. These findings demonstrate a remarkable degree of selectivity for mY RNA encapsidation into MLV particles and suggest mY RNAs are recruited for MLV packaging from a very early stage in their biogenesis.


Cells and virus.

NIH 3T3 and derivative cell lines were maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% bovine serum (Invitrogen). Wild-type mouse embryonic fibroblasts (MEFs) and Ro60−/− MEFs were maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini). Wild-type and Ro60−/− MEFs were prepared by backcrossing 129/Sv × C57BL/6 Ro−/− mice (63) with C57BL/6 mice for six successive generations (50). Embryonic fibroblasts were prepared and immortalized by repeated passages (58). Wild-type MLV particles were obtained by collecting supernatants from 70% to 100% confluent NIH 3T3 cells, wild-type MEFs, and Ro60−/− MEFs chronically infected with wild-type MLV at 8- to 16-h intervals.


All riboprobe templates were derivatives of pBSII SK(+) (Stratagene). pEG604-1 was constructed with synthetic oligonucleotides for 95 nt of mY1 (nt 1 to 95) with SalI and EcoRI sticky ends and for 65 nt of mouse 7SL RNA (nt 125 to 189) with PstI and NotI sticky ends. The insert in pEG467-10, which was generated by PCR and subcloned into the EcoRV site, included portions complementary to both the MLV 5′ untranslated region (nt 55 to 214) and 100 nt of 7SL RNA (38).

Viral- and cellular-RNA extraction.

All supernatants were filtered using 0.2-μm MCE syringe filters (Fisher Scientific) and stored at −70°C prior to their use. Virus was concentrated at 4°C by centrifugation at 25,000 rpm for 90 min using the AH629 rotor in a Sorvall discovery ultracentrifuge. Viral pellets were resuspended in TRIzol (Invitrogen), and RNA was extracted following the manufacturer's instructions. RNA from chronically infected cells was also extracted with TRIzol. Samples were resuspended in either diethyl pyrocarbonate-treated double-distilled H2O or TENS (10 mM Tris [pH 8.0], 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 100 mM NaCl).

Northern blots.

Northern blots were used both to visualize RNAs and as controls to normalize protein samples for Western blotting. The hybridization probes were oligonucleotides complementary to the RNAs of interest, 5′ end labeled using [γ-32P]ATP (Perkin-Elmer) and T4 polynucleotide kinase (NEB). Labeled oligonucleotides were separated from unincorporated nucleotides on Sephadex G-25 columns (Roche). The oligonucleotides included 5′-TGCTCCGTTTCCGACCTGGGCCGGTTCACCCCTCCTT-3′ for 7SL, 5′-CTGACTGTGAACAATCAATTGAGATAACTCACTAC-3′ for mY1, 5′-TAACTGGTTGTGATCAATTAGTTGTAAACACCACTACTC-3′ for mY3, 5′-CGTGTCATCCTTGCGCAGGGGCCATGCTAATCTTCTCTGT-3′ for U6, 5′-GAGTCCCACGCTCTACCAACTGAGCTAGCTG-3′ for tRNALys1, and 5′-TGCTCCGTTTCCGACCTGGGAAAAACTGACTGTGAACAATCAATTGAGATAACTCACTAC-3′ as the chimeric 7SL-mY1 probe. Viral and cellular RNAs were separated by 5% polyacrylamide-8 M urea gel electrophoresis in 1× Tris-borate-EDTA (TBE). For high-resolution denaturing Northern blots, viral and cellular RNAs were separated on 8% polyacrylamide-8 M urea gels (0.4-mm thick) in 1× TBE using glass plates pretreated with Sigmacote (Sigma) to facilitate separation from the glass plates prior to transfer. RNAs were subsequently transferred by electroblotting them to Zeta-probe GT nylon membranes (Bio-Rad) in 0.5× TBE. The membranes were air dried, UV cross-linked (Stratalinker; Stratagene), and prehybridized at 45°C in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5× Denhardt's solution-0.5% SDS-0.025 M sodium phosphate-625 μg/ml denatured salmon sperm DNA. Oligonucleotide probes were denatured at 85°C for 5 min before being added to the membranes, and hybridization was at 45°C. Blots were washed under the following conditions: (i) the blots were washed first in 2× SSC-0.1% SDS at 25°C and then in 0.5× SSC-0.1% SDS at 25°C (see Fig. 2A and B); (ii) the blots were washed first in 2× SSC-0.1% SDS at 25°C and then in 0.33× SSC-0.1% SDS at 55°C (see Fig. Fig.3A);3A); (iii) the blots were washed first in 2× SSC-0.1% SDS at 52°C and then in 0.33× SSC-0.1% SDS at 52°C (see Fig. 4A and B and 5A and B). The damp blots were wrapped in plastic wrap and exposed to phosphorimager screens and/or film. For reprobing, the blots were stripped by at least two washes in 0.1% SDS at 80°C and then prehybridized and probed as described above.

FIG. 2.
mY1 and mY3 RNA recruitment in the absence of Ro60. Northern blots were coprobed for 7SL and either mY1 (A) or mY3 (B). The left half of each blot shows cellular-RNA samples from wild-type Ro60+/+ MEFs (lanes 1), Ro60−/− ...
FIG. 3.
Stoichiometric analysis of mY1 RNA packaging. (A) Northern blot of MLV RNA from Ro60 wild-type and knockout MEFs probed with a chimeric oligonucleotide complementary to both 7SL RNA and mY1. (B) RPA of cellular and supernatant/viral RNA for mY1 and 7SL ...
FIG. 4.
mY1 RNA redistribution in Ro60−/− cells. (A) Northern blot of RNA from total (T), nuclear (N), and cytoplasmic (C) fractions of both wild-type Ro60+/+ MEFs and knockout Ro60−/− MEFs probed for mY1. (B) Northern ...
FIG. 5.
mY1 RNA processing intermediates in cell and virus samples. (A) Northern blot of RNA from total cell and viral samples from wild-type Ro60+/+ MEFs and knockout Ro60−/− MEFs separated on an 8% acrylamide, 8 M urea ...


To detect mY1 and 7SL, pEG604-1 was linearized with XhoI and transcribed with T3 RNA polymerase (Promega) and [α-32P]CTP to create a 217-nt transcript that protected 95 nt of mY1 and 65 nt of 7SL RNA. To detect 7SL and MLV gRNA, pEG467-10 was linearized with HindIII and transcribed with T3 RNA polymerase to create a 364-nt transcript that protected 160 nt of MLV gRNA and 100 nt of 7SL RNA. Previously described RNase protection assay (RPA) approaches (38) were modified by extending hybridization times to 16 h and by digestion with a fivefold excess of RNase T1 (Applied Biosystems). Bands were quantified by phosphorimager analysis using a Typhoon PhosphorImager for detection and ImageQuant TL for analysis. Bands were adjusted for the number of radiolabeled Cs incorporated. In one case (see Fig. Fig.3),3), product bands in two separate RPAs (see Fig. Fig.3B)3B) were corrected for the 18 Cs in the mY1-protected fragment and 25 for 7SL and then multiplied by 7.75 7SL RNAs per virion, as determined by two separate RPAs (see Fig. Fig.3C).3C). The product bands were corrected for 53 labeled C residues in the fragment protected by MLV gRNA and the 35 Cs in the 7SL-protected fragment (see Fig. Fig.3C).3C). These data were consistent with direct quantification of the amount of MLV gRNA per mY1 by RPAs performed in triplicate (data not shown).

Exogenous RT assay.

Media supernatants were harvested, filtered through 0.2-μm MCE syringe filters (Fisher Scientific), and stored at −70°C. Reverse transcriptase (RT) assays were based on a protocol of Goff et al. (21) as described previously (56). Briefly, 3 μl of viral supernatant was incubated with 12 μl of a 1.2× solution {60 mM Tris (pH 8.3), 24 mM dithiothreitol, 0.7 mM MnCl2, 75 mM NaCl, 0.06% NP-40, 6 μg/ml oligo(dT), 12 μg/ml poly(rA), 10 μCi/ml [α-32P]TTP at 3 Ci/mmol} at 37°C for 2 h. Following incubation, 3 μl of the reaction mixture was spotted onto DEAE paper, dried, washed with 2× SSC followed by 95% ethanol, and once again dried. The spots were quantified by phosphorimager analysis.

Cellular fractionation.

Wild-type and Ro60−/− MEFs were fractionated using a modification of the procedure of Siomi et al. (53). Briefly, nuclear and cytoplasmic fractions were obtained by washing adherent cells twice with ice-cold 1× phosphate-buffered saline, followed by one wash with buffer RSB100 (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, and 100 mM NaCl). Adherent cells were permeabilized by incubation with 4 ml RSB100 supplemented with 0.01% (wt/vol) digitonin (Sigma) on ice for 5 min. The RSB100 buffer-digitonin mixture was removed from the adherent cells and spun at 200 × g for 5 min to pellet residual cell debris. The RSB100 buffer supernatant served as the cytoplasmic fraction, and RNA was extracted from this aqueous mixture with TRIzol LS (Invitrogen) according to the manufacturer's recommended protocol. The remaining nuclei were washed once with ice-cold phosphate-buffered saline before the addition of 1 ml of TRIzol (Invitrogen) and RNA extraction according to the manufacturer's protocol.


MLV replication is unaltered in Ro60−/− cells.

Ro RNP RNAs mY1 and mY3 are among the most highly enriched host RNAs in MLV particles (38). Within cells, most Y RNAs exist in RNPs containing the Ro60 cellular protein, and most Ro60 protein is associated with Y RNAs (40, 61). When Y1 RNA levels were used to normalize parallel viral- and cellular-protein extracts, the Ro60 protein in virus was below the limit of detection by Western blotting (37). To test the inference that Y RNAs were not recruited as parts of Ro RNPs, the kinetics of viral spread and Y RNA recruitment were analyzed in Ro60 knockout cells.

Ro60 binding is necessary for the stable accumulation of Y RNAs (6, 28, 63). Accordingly, embryonic fibroblasts prepared from Ro knockout mice contain only very low levels of mY1 and mY3 (50). Thus, studying MLV infectivity in Ro60 knockout cells allowed an examination of the effects of limiting mY RNA availability on viral replication, as well as possible roles of Ro60 itself. Ro60−/− MEFs and isogenic wild-type cells were infected with MLV, and infected and control uninfected cells were serially passaged. Culture media were collected and assayed for RT activity at various time points postinfection to monitor virus spread (Fig. (Fig.1).1). Control uninfected cells retained only background levels of RT activity throughout the time course. For medium samples from wild-type- and Ro60−/−-infected MEFs, the levels of RT activity rose above background on the same day (day 7) (Fig. (Fig.1).1). Thereafter, the kinetics of virus spread in infected wild-type and Ro60−/− cells remained approximately equal, with infected Ro60−/− supernatants containing levels of RT activity that differed by less than twofold from those of infected wild-type MEFs (data not shown). Thus, Ro60 was dispensable for the assembly and infectivity of infectious MLV particles.

FIG. 1.
Time course of MLV spread in Ro60−/− cells and wild-type MEFs. Wild-type (+/+) and Ro knockout (−/−) cells were infected with identical amounts of MLV, and virus spread was monitored by assaying for RT activity ...

MLV virions from both wild-type and Ro60−/− MEFs package high levels of mY1 and mY3.

Because MLV replicated normally in cells containing very low levels of mY RNAs, it initially seemed likely that virions shed from Ro knockout cells would contain correspondingly low levels of mY RNAs. To address directly whether reduced mY RNA levels in Ro60 knockout cells led to diminished mY RNA recruitment by MLV, mY1 and mY3 in viral supernatants shed by wild-type and Ro60−/− MEFs were compared to their intracellular levels by Northern blotting (Fig. (Fig.2).2). Quantifying virus by RT assay and Northern blotting confirmed previous findings that the host cell noncoding RNA 7SL is enriched in MLV in proportion to viral proteins (38). Thus, 7SL served as a loading control. Consistent with previous studies (7, 50), both infected and uninfected Ro60−/− MEFs contained 30-fold less mY1 and mY3 than wild-type cells (Fig. 2A and B, lanes 1 to 4). Surprisingly, despite the very low levels of mY RNAs in Ro60−/− cells, MLV virions produced from these cells continued to package high levels of mY RNAs (Fig. 2A and B, lanes 7 and 8). When normalized to the copackaged 7SL RNA, which is undiminished in Ro60−/− cells, virus produced from the Ro60−/− cells contained nearly the same amount of mY RNA per virion as virus from wild-type cells (Fig. 2A and B, lanes 7 and 8).

Because the Y/7SL RNA ratios in virus versus those in cells were the same for both the mY1 and the mY3 blots in Fig. Fig.2,2, this suggests that each MLV virion encapsidates the same number of mY1 molecules as mY3 molecules. However, because the blots shown in Fig. Fig.22 resulted from coprobing with two separate oligonucleotide probes—one for mY RNA and the other for 7SL—the absolute number of Y RNAs per virion could not be addressed by the radioactive signals observed in Fig. Fig.2.2. Thus, to ensure uniformity in probe-specific activities and to estimate the number of mY1 RNAs packaged per virion, virus and cell RNA samples were subsequently probed with a single radiolabeled oligonucleotide complementary to both 7SL and mY1 (Fig. (Fig.3A).3A). The results indicated that approximately twofold less mY1 than 7SL RNA was encapsidated into MLV produced by wild-type cells (Fig. (Fig.3A,3A, lane 1) and slightly less mY1 was packaged into MLV produced from Ro60 knockouts (Fig. (Fig.3A,3A, lane 2). RPA for mY1 and 7SL RNA confirmed this quantification and revealed a nearly 2:1 ratio of 7SL to mY1 in virus from wild-type cells (Fig. (Fig.3B,3B, lane 7). When normalized to 7SL, a detectable but less than twofold decrease in mY1 packaging was observed in virus from Ro60 knockouts (Fig. (Fig.3B,3B, lane 8). The gRNA-to-7SL ratios in virus from both cell types (Fig. (Fig.3C,3C, lanes 3 and 4) were consistent with previously established levels of 7SL packaging (three- to fourfold molar excess to gRNA) (38). Taken together, these results indicated that each MLV virion from wild-type MEFs contained four or five copies of both mY1 and mY3 and that virions from Ro60 knockouts contained approximately two copies each of mY1 and mY3.

The subcellular distribution of mY1 RNA is altered in Ro60−/− cells.

Ordinarily, most Y RNA localizes to the cytoplasm, where it is bound to Ro60 in Ro RNPs (35, 40). The reduced levels of mY RNAs in Ro60−/− cells are believed to reflect their decreased intracellular stability when their cognate RNP is not present (6, 28, 63). Thus, a reduction in mY RNA stability in Ro60−/− cells would likely be accompanied by a more severe deficit of mY RNA in the cytoplasm than in the nucleus.

To determine the subcellular distribution of the residual mY1 RNA in Ro60−/− cells, RNA was extracted from total, nuclear, and cytoplasmic fractions of knockout and isogenic wild-type MEFs (Fig. (Fig.4).4). Northern blots were probed for mY1 or coprobed for nuclear U6 snRNA and cytoplasmic tRNALys1 to control for fractionation efficiency. The results indicated that the cytoplasm of wild-type cells contained at least fourfold more mY1 RNA than their nuclei (Fig. (Fig.4A,4A, lanes 3 and 5). In contrast, nuclear and cytoplasmic fractions of Ro−/− MEFs contained similar amounts of mY RNA (Fig. (Fig.4A,4A, lanes 4 and 6: the nuclear-fraction lane contains 10% more Y1 than the cytoplasmic-fraction lane). Compared to wild-type cells, knockout cell cytoplasmic amounts of mY1 were reduced about 20-fold, and nuclear amounts were reduced about 4-fold. Because the nuclear levels of the control RNA U6 are not reduced in knockout cells, the fourfold decrease of nuclear Y1 in the knockouts suggests at least some of the RNA degradation associated with the absence of Ro60 may initiate in the nucleus. These findings confirm a marked alteration in the subcellular location of mY RNAs, from their cytoplasmic prominence in wild-type cells to a far greater depletion from the cytoplasm than from the nucleus for the residual mY RNAs in Ro60 knockout cells.

The results described here showed mY RNAs were recruited at wild-type levels from Ro60 knockout cells, despite 20- to 30-fold reductions in mY RNA intracellular levels. When mY-to-gRNA ratios in wild-type cells and virus were compared, mY RNAs were selected for packaging three- to fourfold less well than MLV gRNAs (38). Thus, whereas virions produced by wild-type cells package mY RNAs slightly less effectively than viral gRNAs, when MLV replicated in Ro knockout cells, mY1 and mY3 displayed a roughly 5- to 10-fold-higher packaging selectivity than gRNAs.

mY RNA packaging is independent of the Y RNA-processing step.

The biosynthesis of host RNP complexes involves a series of RNA-processing steps and alternate protein associations. Significant uncertainty remains about both the temporal and spatial aspects of these steps in Ro RNP assembly. Nonetheless, it is clear that transcription of Y RNAs, as is the case for all RNA polymerase III transcripts, terminates in a run of uridines and that the resulting 3′ ends are bound by the La protein, which recognizes RNAs ending in three or more uridines. Subsequent end trimming by exonucleases removes some of the terminal uridines, resulting in a population of RNAs slightly shorter than the initial transcription products; some of these no longer possess La binding sites. Neither the identity of the exonuclease nor its subcellular location is known. Ro travels to the nucleus prior to Ro RNP assembly and binds the bulged Y RNA stem region (22, 51). Although La has been observed to shuttle between the nucleus and the cytoplasm (15), La binding retards nuclear export of Y RNAs (23, 52). Thus, Y RNA 3′-end shortening and elimination of the La binding site may occur prior to nuclear export (24, 46). Ro and La can bind a single Y RNA simultaneously. In unstressed wild-type cells, most Y RNAs exist in the cytoplasm in a shorter, matured form in an RNP complex containing Ro but not La (shorter mY1 RNAs lack the 3′ La binding site) (Fig. (Fig.4A)4A) (31, 60).

To address whether mY RNA encapsidation into MLV represented diversion of the RNAs from a specific stage in their maturation pathway, RNAs from virions produced by wild-type and Ro60−/− cells were compared to those in cells for signatures of Y RNA processing intermediates. Consistent with the Y RNAs in the knockout MEFs representing newly synthesized RNAs, the residual Y RNAs in Ro60−/− cells migrated slightly more slowly than the majority of the Y RNAs in wild-type cells and thus exhibited the pattern observed for nascent La-bound Y RNAs (Fig. (Fig.4A)4A) (7).

The lengths of Y RNA processing intermediates were also examined using high-resolution denaturing acrylamide gels (Fig. (Fig.5A).5A). The results confirmed that the spectra of mY RNAs in knockout cells were longer than those in wild-type cells. The short lengths of mY RNAs in virions from wild-type cells suggested that La binding was not necessary for mY RNA packaging. However, the encapsidated RNAs from knockout cells were longer than those from wild-type cells. In both cell types, the RNA length distribution in virus was found to resemble that in the producer cells (Fig. (Fig.5A),5A), suggesting that packaging did not require mature 3′-end formation.

To further address possible packaging of specific Y RNA subsets, high-resolution gels were used to examine mY1 processing species distribution in cytoplasmic and nuclear fractions (Fig. (Fig.5B).5B). Under the fractionation conditions used, the spectra of RNAs in knockout cells' cytoplasmic and nuclear fractions were indistinguishable (Fig. (Fig.5B,5B, lanes 5 and 6). A slight subtle but reproducible bias toward more completely processed products was observed both in wild-type cells' cytoplasmic fractions (Fig. (Fig.5B,5B, lane 3) and in virions (Fig. (Fig.5A,5A, lane 6) compared to these cells' nuclear RNA spectrum (Fig. (Fig.5B,5B, lane 2).


Retroviruses like MLV encapsidate distinct subsets of cellular noncoding RNAs (3, 38). The data presented here demonstrated that mY1 and mY3 RNAs were recruited into budding MLV at four or five copies apiece from wild-type MEFs and about two copies apiece from Ro60 knockouts. Although most mY RNA in cells resides in Ro RNPs, these RNAs were recruited into MLV without their cognate RNP, Ro60.

Similar observations of host protein-independent packaging of RNP RNA have been made for the SRP RNA, 7SL, which is packaged without the 54-kDa SRP protein in both MLV and HIV-1 (references 25 and 39 and unpublished data). Because of its essential role in signal recognition particles, direct knockdown of 7SL RNA is not readily achievable (reference 1 and unpublished data). Facilitating our analysis of MLV mY RNA packaging, viable Ro60 knockout mice have been generated. Their cells lack Ro RNPs and display vastly reduced levels of mY RNAs (63).

We therefore used Ro60−/− cells to examine the intersection of mY RNA biogenesis with MLV replication. The normal spread of MLV in Ro60−/− embryonic fibroblasts demonstrated that the Ro60 protein is not necessary for virus replication. Because mY1 and mY3 are highly labile in the absence of Ro60, knockout of Ro60 leads to a 30-fold reduction in these RNAs (63). Strikingly, this intracellular reduction was not accompanied by a proportional reduction in mY RNA packaging. Instead, mY1 and mY3 were so highly enriched that MLV's selectivity for mY RNAs, from among all RNAs in Ro60 knockout cells, was 5- to 10-fold higher than selectivity for its own genome.

MLV's high level of selectivity for mY RNAs led to our model, in which Ro RNP and MLV assembly pathways intersect at an early step in Ro RNP biogenesis (Fig. (Fig.6).6). In this model, for the encapsidation of mY RNAs without Ro60, the pathways of mY RNA biogenesis and MLV assembly intersect, and a virion-specifying factor diverts Ro60-free mY RNAs from host cell RNA degradation machinery toward assembly sites on the plasma membrane (Fig. (Fig.66).

FIG. 6.
Ro RNP biogenesis and late stages of MLV replication. A speculative model for their intersection was based on observations reported here and Ro RNP assembly properties, as described in the text. The light-gray oval represents the nucleus; the dark-gray ...

The similar Y RNA packaging observed in virions produced by cells with either high or very low intracellular Y RNA levels suggests that these RNAs are not recruited from Ro RNPs but from a separate intracellular pool of Ro60-free RNAs. In an attempt to localize this intracellular pool, cell fractionation suggested MLV recruitment from an early pool of nascent RNAs. Consistent with the role of Ro60 in stabilizing mY RNAs (6, 28, 63), as well as the likely importance of Ro60 binding to Y RNA nuclear export (52), cytoplasmic pools of mY RNAs decreased more than the residual pool of nuclear mY RNAs in Ro60 knockout cells. Because this redistribution did not result in a corresponding decrease in Y RNA recruitment by MLV, it suggests that recruitment occurs at an unaffected, and possibly earlier, step in Y RNA biogenesis.

Precisely where this occurs was not resolved by monitoring 3′-end modifications that accompany Y RNA maturation. In virus from wild-type cells, encapsidated Y RNAs resembled the biased pattern of mature Y RNAs in the cytoplasm. However, encapsidated RNAs from Ro60 knockout cells, like the Y RNAs in these cells, resembled nascent RNAs that had not undergone 3′-end maturation. Thus, although we cannot rule out a cytoplasmic point of recruitment from a small pool of immature cytoplasmic Y RNA, these data suggest that recruitment into particles is independent of 3′-end maturation, which likely occurs prior to cytoplasmic entry for the bulk of Y RNA.

Assuming that mY RNAs are recruited at the same step of their biosynthesis in both cell types, these results suggest that recruitment for packaging occurs before 3′-end maturation is completed but does not preclude subsequent 3′-end maturation. Both La and Ro recognize and act on RNA motifs in the Y RNA stem and 3′ tail region, while other factors, such as nucleolin and hnRNP I, are known to interact with some Y RNAs via the internal loop (12, 16, 22). Thus, these findings may indicate that recruitment of Y RNAs occurs via interactions with the loop region that prevent degradation of Ro-deficient RNAs but do not preclude 3′-end maturation (Fig. (Fig.6).6). Speculatively, the slower migration of residual Y RNAs in knockout cells may be suggestive of a role for Ro60 in exposing Y RNA 3′ ends for completing their exonucleolytic processing by an as yet unknown mechanism.

Because they appear to be recruited early, one possibility is that mY RNAs may be selected for encapsidation into MLV from near their site of transcription in the nucleus (Fig. (Fig.6).6). The possibility of nuclear recruitment is plausible, considering that known pools of protein-free Y RNAs localize to perinucleolar sites of early RNP assembly (32). Although assembly of MLV, as for all retroviruses classically described as type C, is first visualized at the plasma membrane (17), the notion that retroviral late replication phases may include a nuclear step is not unprecedented, as it has been shown that a portion of avian sarcoma virus Gag molecules transit through the nucleus prior to assembly (47-49). Although one study reported that 18% of MLV-infected cell-associated Gag immunoprecipitated from nuclear fractions, it remains controversial whether retroviruses other than avian sarcoma virus share this step (34).

If mY RNAs are recruited in the nucleus, they may join a complex that includes MLV gRNA. The hypothesis that MLV gRNAs transit directly from the nucleus to sites of assembly is supported by the propensity of sibling MLV gRNAs, but not those of HIV-1, to self-associate for packaging (13). When sibling gRNAs are expressed from a single nuclear locus or proximal integration sites, they associate randomly (14, 26, 43). The impact of nuclear distance on gRNA dimer partner selection argues for an early association of gRNA siblings and an early formation of a subviral RNP destined for encapsidation at the plasma membrane. If mY RNAs are recruited in the nucleus, they may join this hypothetical subviral RNP and accompany it to the plasma membrane, with the possibility that mY RNA binding may modulate the RNP's intracellular trafficking, as it does for Ro RNPs (Fig. (Fig.6)6) (50).

These findings of highly specific recruitment and enrichment of host RNP RNAs into MLV particles, even when the particles are produced by cells in which the RNAs are barely detectable, add to growing evidence that the pathway of retroviral assembly—from nuclear provirus to plasma membrane-released virion—may be less linear than previously believed (55). Whether the apparent intersection of host and viral RNP biogenesis pathways is a fortuitous convergence, represents an evolved viral reliance on host RNP biosynthetic machinery, or is indicative of an abortive attempt of the host RNA quality control circuitry to thwart viral attack is not clear. The comparable packaging of similar subsets of noncoding RNAs in several different retroviral species does not immediately differentiate between these possibilities (1, 25, 38, 57), although the argument for chance interactions may be weakened if some retroviruses include a nuclear preassembly step while others do not (10, 13). Accumulating evidence suggests that some viruses coopt protein quality control machinery to aid their replication (59); the work here adds to evidence for a similar intersection between retrovirus assembly and cellular machinery associated with RNA quality control (2, 19).


This work was supported by NIH grants R21 AI080276 to A.T. and R01GM073863 to S.L.W., as well as by a Gates Grand Challenges Exploration Grant to A.T. S.S. was supported by a postdoctoral fellowship from the Arthritis Foundation.

We thank Michael Malim for constructive comments on the manuscript.


[down-pointing small open triangle]Published ahead of print on 23 September 2009.


1. Bach, D., S. Peddi, B. Mangeat, A. Lakkaraju, K. Strub, and D. Trono. 2008. Characterization of APOBEC3G binding to 7SL RNA. Retrovirology 5:54. [PMC free article] [PubMed]
2. Beliakova-Bethell, N., C. Beckham, T. H. Giddings, Jr., M. Winey, R. Parker, and S. Sandmeyer. 2006. Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components. RNA 12:94-101. [PubMed]
3. Berkowitz, R., J. Fisher, and S. P. Goff. 1996. RNA packaging. Curr. Top. Microbiol. Immunol. 214:177-218. [PubMed]
4. Bishop, J. M., W. E. Levinson, N. Quintrell, D. Sullivan, L. Fanshier, and J. Jackson. 1970. The low molecular weight RNAs of Rous sarcoma virus. I. The 4 S RNA. Virology 42:182-195. [PubMed]
5. Cen, S., H. Javanbakht, S. Kim, K. Shiba, R. Craven, A. Rein, K. Ewalt, P. Schimmel, K. Musier-Forsyth, and L. Kleiman. 2002. Retrovirus-specific packaging of aminoacyl-tRNA synthetases with cognate primer tRNAs. J. Virol. 76:13111-13115. [PMC free article] [PubMed]
6. Chen, X., A. M. Quinn, and S. L. Wolin. 2000. Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation. Genes Dev. 14:777-782. [PubMed]
7. Chen, X., J. D. Smith, H. Shi, D. D. Yang, R. A. Flavell, and S. L. Wolin. 2003. The Ro autoantigen binds misfolded U2 small nuclear RNAs and assists mammalian cell survival after UV irradiation. Curr. Biol. 13:2206-2211. [PubMed]
8. Chen, X., and S. L. Wolin. 2004. The Ro 60 kDa autoantigen: insights into cellular function and role in autoimmunity. J. Mol. Med. 82:232-239. [PubMed]
9. Chen, X., E. J. Wurtmann, J. Van Batavia, B. Zybailov, M. P. Washburn, and S. L. Wolin. 2007. An ortholog of the Ro autoantigen functions in 23S rRNA maturation in D. radiodurans. Genes Dev. 21:1328-1339. [PubMed]
10. Dorman, N., and A. Lever. 2000. Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus. J. Virol. 74:11413-11417. [PMC free article] [PubMed]
11. Duesberg, P. H., and W. S. Robinson. 1966. Nucleic acid and proteins isolated from the Rauscher mouse leukemia virus (MLV). Proc. Natl. Acad. Sci. USA 55:219-227. [PubMed]
12. Fabini, G., R. Raijmakers, S. Hayer, M. A. Fouraux, G. J. Pruijn, and G. Steiner. 2001. The heterogeneous nuclear ribonucleoproteins I and K interact with a subset of the ro ribonucleoprotein-associated Y RNAs in vitro and in vivo. J. Biol. Chem. 276:20711-20718. [PubMed]
13. Flynn, J. A., W. An, S. R. King, and A. Telesnitsky. 2004. Nonrandom dimerization of murine leukemia virus genomic RNAs. J. Virol. 78:12129-12139. [PMC free article] [PubMed]
14. Flynn, J. A., and A. Telesnitsky. 2006. Two distinct Moloney murine leukemia virus RNAs produced from a single locus dimerize at random. Virology 344:391-400. [PMC free article] [PubMed]
15. Fok, V., K. Friend, and J. A. Steitz. 2006. Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling. J. Cell Biol. 173:319-325. [PMC free article] [PubMed]
16. Fouraux, M. A., P. Bouvet, S. Verkaart, W. J. van Venrooij, and G. J. Pruijn. 2002. Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes. J. Mol. Biol. 320:475-488. [PubMed]
17. Freed, E. O. 1998. HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251:1-15. [PubMed]
18. Fuchs, G., A. J. Stein, C. Fu, K. M. Reinisch, and S. L. Wolin. 2006. Structural and biochemical basis for misfolded RNA recognition by the Ro autoantigen. Nat. Struct. Mol. Biol. 13:1002-1009. [PubMed]
19. Gallois-Montbrun, S., R. K. Holmes, C. M. Swanson, M. Fernandez-Ocana, H. L. Byers, M. A. Ward, and M. H. Malim. 2008. Comparison of cellular ribonucleoprotein complexes associated with the APOBEC3F and APOBEC3G antiviral proteins. J. Virol. 82:5636-5642. [PMC free article] [PubMed]
20. Ganser-Pornillos, B. K., M. Yeager, and W. I. Sundquist. 2008. The structural biology of HIV assembly. Curr. Opin. Struct. Biol. 18:203-217. [PMC free article] [PubMed]
21. Goff, S. P., P. Traktman, and D. Baltimore. 1981. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38:239-248. [PMC free article] [PubMed]
22. Green, C. D., K. S. Long, H. Shi, and S. L. Wolin. 1998. Binding of the 60-kDa Ro autoantigen to Y RNAs: evidence for recognition in the major groove of a conserved helix. RNA 4:750-765. [PubMed]
23. Grimm, C., E. Lund, and J. E. Dahlberg. 1997. In vivo selection of RNAs that localize in the nucleus. EMBO J. 16:793-806. [PubMed]
24. Gwizdek, C., B. Ossareh-Nazari, A. M. Brownawell, A. Doglio, E. Bertrand, I. G. Macara, and C. Dargemont. 2003. Exportin-5 mediates nuclear export of minihelix-containing RNAs. J. Biol. Chem. 278:5505-5508. [PubMed]
25. Khan, M. A., R. Goila-Gaur, S. Opi, E. Miyagi, H. Takeuchi, S. Kao, and K. Strebel. 2007. Analysis of the contribution of cellular and viral RNA to the packaging of APOBEC3G into HIV-1 virions. Retrovirology 4:48. [PMC free article] [PubMed]
26. Kharytonchyk, S. A., A. I. Kireyeva, A. B. Osipovich, and I. K. Fomin. 2005. Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site. Retrovirology 2:3. [PMC free article] [PubMed]
27. Klein, K. C., J. C. Reed, and J. R. Lingappa. 2007. Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag. AIDS Rev. 9:150-161. [PubMed]
28. Labbe, J. C., S. Hekimi, and L. A. Rokeach. 1999. The levels of the RoRNP-associated Y RNA are dependent upon the presence of ROP-1, the Caenorhabditis elegans Ro60 protein. Genetics 151:143-150. [PubMed]
29. Levin, J. G., and J. G. Seidman. 1981. Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. J. Virol. 38:403-408. [PMC free article] [PubMed]
30. Levin, J. G., and J. G. Seidman. 1979. Selective packaging of host tRNAs by murine leukemia virus particles does not require genomic RNA. J. Virol. 29:328-335. [PMC free article] [PubMed]
31. Maraia, R. J., and R. V. Intine. 2001. Recognition of nascent RNA by the human La antigen: conserved and divergent features of structure and function. Mol. Cell. Biol. 21:367-379. [PMC free article] [PubMed]
32. Matera, A. G., M. R. Frey, K. Margelot, and S. L. Wolin. 1995. A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. J. Cell Biol. 129:1181-1193. [PMC free article] [PubMed]
33. Muriaux, D., J. Mirro, D. Harvin, and A. Rein. 2001. RNA is a structural element in retrovirus particles. Proc. Natl. Acad. Sci. USA 98:5246-5251. [PubMed]
34. Nash, M. A., M. K. Meyer, G. L. Decker, and R. B. Arlinghaus. 1993. A subset of Pr65gag is nucleus associated in murine leukemia virus-infected cells. J. Virol. 67:1350-1356. [PMC free article] [PubMed]
35. O'Brien, C. A., K. Margelot, and S. L. Wolin. 1993. Xenopus Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonucleoproteins. Proc. Natl. Acad. Sci. USA 90:7250-7254. [PubMed]
36. O'Brien, C. A., and S. L. Wolin. 1994. A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors. Genes Dev. 8:2891-2903. [PubMed]
37. Onafuwa-Nuga, A. A. 2007. Host cell RNA packaging in retroviruses. Ph.D. thesis. University of Michigan, Ann Arbor.
38. Onafuwa-Nuga, A. A., S. R. King, and A. Telesnitsky. 2005. Nonrandom packaging of host RNAs in Moloney murine leukemia virus. J. Virol. 79:13528-13537. [PMC free article] [PubMed]
39. Onafuwa-Nuga, A. A., A. Telesnitsky, and S. R. King. 2006. 7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles. RNA 12:542-546. [PubMed]
40. Peek, R., G. J. Pruijn, A. J. van der Kemp, and W. J. van Venrooij. 1993. Subcellular distribution of Ro ribonucleoprotein complexes and their constituents. J. Cell Sci. 106:929-935. [PubMed]
41. Peters, G., F. Harada, J. E. Dahlberg, A. Panet, W. A. Haseltine, and D. Baltimore. 1977. Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis. J. Virol. 21:1031-1041. [PMC free article] [PubMed]
42. Pruijn, G. J., F. H. Simons, and W. J. van Venrooij. 1997. Intracellular localization and nucleocytoplasmic transport of Ro RNP components. Eur. J. Cell Biol. 74:123-132. [PubMed]
43. Rasmussen, S. V., and F. S. Pedersen. 2006. Co-localization of gammaretroviral RNAs at their transcription site favours co-packaging. J. Gen. Virol. 87:2279-2289. [PubMed]
44. Reinisch, K. M., and S. L. Wolin. 2007. Emerging themes in non-coding RNA quality control. Curr. Opin. Struct. Biol. 17:209-214. [PubMed]
45. Rulli, S. J., Jr., C. S. Hibbert, J. Mirro, T. Pederson, S. Biswal, and A. Rein. 2007. Selective and nonselective packaging of cellular RNAs in retrovirus particles. J. Virol. 81:6623-6631. [PMC free article] [PubMed]
46. Rutjes, S. A., E. Lund, A. van der Heijden, C. Grimm, W. J. van Venrooij, and G. J. Pruijn. 2001. Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs. RNA 7:741-752. [PubMed]
47. Scheifele, L. Z., R. A. Garbitt, J. D. Rhoads, and L. J. Parent. 2002. Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein. Proc. Natl. Acad. Sci. USA 99:3944-3949. [PubMed]
48. Scheifele, L. Z., S. P. Kenney, T. M. Cairns, R. C. Craven, and L. J. Parent. 2007. Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology. J. Virol. 81:10718-10728. [PMC free article] [PubMed]
49. Scheifele, L. Z., E. P. Ryan, and L. J. Parent. 2005. Detailed mapping of the nuclear export signal in the Rous sarcoma virus Gag protein. J. Virol. 79:8732-8741. [PMC free article] [PubMed]
50. Sim, S., D. E. Weinberg, G. Fuchs, K. Choi, J. Chung, and S. L. Wolin. 2009. The subcellular distribution of an RNA quality control protein, the Ro autoantigen, is regulated by noncoding Y RNA binding. Mol. Biol. Cell 20:1555-1564. [PMC free article] [PubMed]
51. Simons, F. H., G. J. Pruijn, and W. J. van Venrooij. 1994. Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes. J. Cell Biol. 125:981-988. [PMC free article] [PubMed]
52. Simons, F. H., S. A. Rutjes, W. J. van Venrooij, and G. J. Pruijn. 1996. The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA. RNA 2:264-273. [PubMed]
53. Siomi, M. C., P. S. Eder, N. Kataoka, L. Wan, Q. Liu, and G. Dreyfuss. 1997. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J. Cell Biol. 138:1181-1192. [PMC free article] [PubMed]
54. Stein, A. J., G. Fuchs, C. Fu, S. L. Wolin, and K. M. Reinisch. 2005. Structural insights into RNA quality control: the Ro autoantigen binds misfolded RNAs via its central cavity. Cell 121:529-539. [PMC free article] [PubMed]
55. Swanson, C. M., and M. H. Malim. 2006. Retrovirus RNA trafficking: from chromatin to invasive genomes. Traffic 7:1440-1450. [PubMed]
56. Telesnitsky, A., S. Blain, and S. P. Goff. 1995. Assays for retroviral reverse transcriptase. Methods Enzymol. 262:347-362. [PubMed]
57. Tian, C., T. Wang, W. Zhang, and X. F. Yu. 2007. Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles. Nucleic Acids Res. 35:7288-7302. [PMC free article] [PubMed]
58. Todaro, G. J., and H. Green. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17:299-313. [PMC free article] [PubMed]
59. Wileman, T. 2007. Aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design? Annu. Rev. Microbiol. 61:149-167. [PubMed]
60. Wolin, S. L., and T. Cedervall. 2002. The La protein. Annu. Rev. Biochem. 71:375-403. [PubMed]
61. Wolin, S. L., and J. A. Steitz. 1983. Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome. Cell 32:735-744. [PubMed]
62. Wolin, S. L., and J. A. Steitz. 1984. The Ro small cytoplasmic ribonucleoproteins: identification of the antigenic protein and its binding site on the Ro RNAs. Proc. Natl. Acad. Sci. USA 81:1996-2000. [PubMed]
63. Xue, D., H. Shi, J. D. Smith, X. Chen, D. A. Noe, T. Cedervall, D. D. Yang, E. Eynon, D. E. Brash, M. Kashgarian, R. A. Flavell, and S. L. Wolin. 2003. A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen. Proc. Natl. Acad. Sci. USA 100:7503-7508. [PubMed]

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