Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a-1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39°C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33°C, however, MuSVtsllO-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent SI nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33°C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-ms. In the present study, DNA primers hybridizing to the MuSVts11O 4-anid 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVtsll0 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVtsllO 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVtsllO 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVtsllO 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed. As a complement to the above sequence data, blot hybridization studies showed unequivocally that MuSVtsll0-infected nonproducer 6m2 cells contain a single, approximately 4.4-kb MuSVtsll0-related viral genome. The restriction map of this provirus was consistent with a relationship to wild-type MuSV-349 viral DNA by way of a 1.5-kb deletion between the gag and mos genes. No 3.5-kb provirus could be detected in 6m2 cells, necessitating that the 3.5-kb RNA be derived from the transcription product of the 4.4-kb genome. In MuSVtsllO producer 206-21C cells chronically superinfected with Moloney murine leukemia virus, however, an integrated 3.9-kb MuSVtsll0-related genome was readily apparent. From its restriction map, this 206-2...

The structures of murine sarcoma virus (MuSV) tsllO viral RNA and intracellular RNA present in MuSV tsllO-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV tsllO viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV tsllO RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed Al) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and plO coding sequences. The 3' border of the Al deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5kb MuSV tsllO RNA, the 5' border of the deleted central region (termed A2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV tsllO RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV tsllO RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33°C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39°C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33°C permits the reappearance of the 3.5-kb RNA at its original level.

We searched for the presence of common RNA structural motifs in mammalian type C retroviruses related to murine leukemia viruses and the closely related avian spleen necrosis virus. A novel motif consisting of a pair of hairpins, called hairpin pair motif, was detected in the 5' untranslated regions of the genomes of these retroviruses. A combination of computational analyses that included the assessment of phylogenetic sequence conservation by multiple alignment, the search for regions with unusual RNA folding properties, and the analysis of RNA secondary structure by suboptimal free-energy calculations highlighted the significance of this hairpin pair motif. The hairpin pair motif encompasses 70 to 80 nucleotides between the splice donor site and the gag translational initiation codon of these viruses. The motif is composed of two adjacent hairpins both with a perfectly conserved GACG tetraloop. We propose that the novel GACG-hairpin pair motif described here constitutes an essential component of the regulatory machinery in these type C retroviruses.

We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39°C and a transformed phenotype at 28 or 33°C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33°C, whereas only P58gag is expressed at 39°C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-m0s. Using both Northern blot and Si nuclease analyses, we show here that the 3.5-kb RNA iF the predominant viral RNA species in 6m2 cells grown at 28°C, whereas only the 4.0-kb RNA is detected at 39°C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39°C and is detected again after a shift back from 39 to 28°C. By Southern blot analysis, we have detected only one tsllO proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39°C indicates that the splicing reaction is thermosensitive. The splicing defect of the tsllO Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb tslO RNA was detected in acutely superinfected 6m2 cells maintained at 39°C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39°C, suggesting that the lack of tsllO viral RNA splicing at 39°C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39°C as well as at 28°C and contain proviral DNAs corresponding to the two viral RNA species. In these cells, the splicing of the 4.0-kb RNA appears to remain thermosensitive; however, additional 3.5-kb RNA can be independently transcribed from its own proviral DNA at either permissive or nonpermissive temperatures, thus bypassing the usual splicing requirement.

Nuclei of cells infected with Moloney murine leukemia virus (MoMuLV) were examined for the presence of gag proteins. This analysis was performed in conjunction with other studies suggesting a possible role for gag proteins in regulating nuclear events relating to processing and/or transport of viral genomic RNA. We detected Pr659'9 and a p30.related protein in a nuclear fraction of infected cells. We also found evidence that a highly conserved amino acid sequence, which is shared by p30 and Ul small nuclear ribonucleoprotein 70-kDa protein, is a component of the nuclear targeting sequence for Pr659'9. Immunoelectron microscopy studies with a monoclonal anti-p12 antibody established that approximately 18% ofgag-containing proteins of MoMuLV are located in the nucleus. Such gag-containing proteins from a mutant MoMuLV that lacks N-terminal myristic acid had greater affinity for the nucleus, suggesting that fatty acid acylation of Pr659ar plays a role in overcoming the proposed nuclear transport signal. The possible roles that nuclear gag proteins may play in retroviral replication are discussed.