Activation of cryptic splice sites in murine sarcoma virus-124 mutants.

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing.     

S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus

The structures of murine sarcoma virus (MuSV) ts110 viral RNA and intracellular RNA present in MuSV ts110-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV ts110 viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV ts110 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 delta 1) 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 p10 coding sequences. The 3' border of the delta 1 deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5-kb MuSV ts110 RNA, the 5' border of the deleted central region (termed delta 2) 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 ts110 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 ts110 RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33 degrees C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39 degrees C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33 degrees C permits the reappearance of the 3.5-kb RNA at its original level.     

Temperature-sensitive splicing defect of ts110 Moloney murine sarcoma virus is virus encoded.

ts110 Moloney murine sarcoma virus (Mo-MuSV)-nonproductively infected cells (6m2) have a transformed phenotype at 28 to 33 degrees C and a normal phenotype at 39 degrees C. At temperatures permissive for transformation, 6m2 cells contain P58gag produced from the 4.0-kilobase (kb) viral RNA genome and P85gag-mos translated from a 3.5-kb spliced mRNA. At 39 degrees C, only the 4.0-kb RNA and its product P58gag are detected. Two temperature-sensitive defects have been observed in ts110-infected 6m2 cells: (i) the splicing of the 4.0-kb RNA to the 3.5-kb RNA; and (ii) the thermolability of P85gag-mos and its kinase activity relative to the wild-type revertant protein, termed P100gag-mos (R.B. Arlinghaus, J. Gen. Virol. 66:1845-1853, 1985). In the present study, we examined the mos gene products of two cell lines (204-2F6 and 204-2F8) obtained by infection of normal rat kidney cells with ts110 Mo-MuSV as a simian sarcoma-associated virus pseudotype to see whether the temperature-sensitive splicing defect could be transferred by viral infection. Southern blot analysis of these two cell lines showed that viral DNAs containing restriction fragments from cellular DNA are different from those in 6m2 cells, indicating that 204-2F6 and 204-2F8 cells have different ts110 provirus integration sites from those of 6m2 cells. Northern blots, S1 mapping analyses, and immunoprecipitation experiments showed unequivocally that the splicing defect of ts110 Mo-MuSV is virus encoded and is independent of host cell factors.     

Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells.

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 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees 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-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 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 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees 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 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.     

Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line

Carcinogenic metal compounds, with the exception of chromium(VI), have been found to be poorly mutagenic in both prokaryotic and mammalian cell mutagenesis assays, yet they are clearly clastogenic (Hansen and Stern, 1984). Thus, the role of metals as initiators in carcinogenesis has been difficult to delineate. In an effort to develop a model system capable of assaying DNA damage caused by carcinogenic metals, we have investigated the role of NiCl2, CdCl2, Na2CrO4, and NMU in a murine sarcoma virus-infected mammalian cell line in which expression of the retroviral v-mos gene is growth-temperature regulated. This cell line, designated 6m2, contains a single-copy, stably integrated, mutant Moloney murine sarcoma virus DNA (designated MuSVts110) and is temperature sensitive for morphological transformation due to a conditionally defective viral RNA-splicing event that in turn regulates expression of the viral transforming gene. Mutations affecting the viral DNA in 6m2 cells can be detected if these alterations lead to changes in the structure or expression of the transforming protein encoded by the MuSVts110 v-mos gene. Analysis of the viral proteins from 6m2 'revertant' cell lines (as defined by reversion to the transformed phenotype at all growth temperatures) selected after treatment with the above agents showed that NiCl2, NMU, and Na2CrO4 each induced a different yet specific type of mutation. NiCl2 and NMU each altered the temperature sensitivity of viral RNA splicing, possibly due to base substitution mutations, but did so to distinctly different extents. Na2CrO4 affected the structure of the viral proteins by inducing what appear to be short frameshift mutations that resulted in the temperature-dependent translation of a novel virus-encoded transforming protein, P100gag-mos. CdCl2 also induced frameshift mutations but, in one case, induced a mutation which may result from a deletion of about 300 bases within the MuSVts110 DNA.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Branchpoint and polypyrimidine tract mutations mediating the loss and partial recovery of the Moloney murine sarcoma virus MuSVts110 thermosensitive splicing phenotype.

Balanced splicing of retroviral RNAs is mediated by weak signals at the 3' splice site (ss) acting in concert with other cis elements. Moloney murine sarcoma virus MuSVts110 shows a similar balance between unspliced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N-nitroso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicing was virtually complete at all temperatures and have investigated the molecular basis of this reversion on the assumption that the findings would reveal cis-acting elements controlling MuSVts110 splicing thermosensitivity. In a representative revertant (NMU-20), we found that complete splicing was conferred by a G-to-A substitution generating a consensus branchpoint (BP) signal (-CCCUGGC- to -CCCUGAC- [termed G(-25)A]) at -25 relative to the 3' ss. Weakening this BP to -CCCGAC- [G(-25)A,U(-27)C] moderately reduced splicing at the permissive temperature and sharply inhibited splicing at the originally nonpermissive temperature, arguing that MuSVts110 splicing thermosensitivity depends on a suboptimal BP-U2 small nuclear RNA interaction. This conclusion was supported by results indicating that lengthening the short MuSVts110 polypyrimidine tract and altering its uridine content doubled splicing efficiency at permissive temperatures and nearly abrogated splicing thermosensitivity. In vitro splicing experiments showed that MuSVts110 G(-25)A RNA intermediates were far more efficiently ligated than RNAs carrying the wild-type BP, the G(-25)A,U (-27)C BP, or the extended polypyrimidine tract. The efficiency of ligation in vitro roughly paralleled splicing efficiency in vivo [G(-25)A BP > extended polypyrimidine tract > G(-25)A,U(-27)C BP > wild-type BP]. These results suggest that MuSVts110 RNA splicing is balanced by cis elements similar to those operating in other retroviruses and, in addition, that its splicing thermosensitivity is a response to the presence of multiple suboptimal splicing signals.     

Mapping the encapsidation determinants of feline immunodeficiency virus

Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis-acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans. When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5' leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.     

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.