Mutation of polyadenylation signals generates murine retroviruses that produce fused virus-cell RNA transcripts at high frequency.

Retroviruses act as insertional mutagens and can also capture cellular sequences through a mechanism which initially requires the generation of RNA transcripts which fail to cleave and polyadenylate correctly. The correct termination of retroviral transcripts at the 3' LTR R/U5 junction is primarily dependent on the canonical AAUAAA polyadenylation signal, so we have analyzed the effect of mutating the polyadenylation signal sequences on the properties of a selectable murine retroviral vector. Mutation of consensus polyadenylation signal sequences in the 5' and/or 3' proviral LTRs demonstrated that a UA to GG change generated larger sized virus-specific RNA, consistent with loss of normal polyadenylation. Cell clones infected with viruses generated by proviral constructs containing this mutation in the 5' LTR express either normal-length or elongated viral RNA. Fused transcripts contained the mutant polyadenylation signal, while sequence analysis was consistent with the hypothesis that premature 5' to 3' primer strand transfer was responsible for the high frequency (80%) of wild-type polyadenylation. Cells infected by viruses from constructs mutated in both 5' and 3' proviral LTRs expressed poly(A)+ viral RNA between 0.3 and 3 kb larger than normal virus in 100% of infected clones, and sequence analysis of clones derived from either infected rodent or human cells confirmed that these transcripts contained both viral and adjacent cellular sequences. While mutant virus exhibits no increased ability to alter cell phenotypes, the read-through transcripts contain both unique and repetitive cell-derived sequences and can easily be recovered using PCR techniques, suggesting that these viruses may serve as effective tools for rapidly cloning cellular sequences and generating random genomic markers for gene mapping.

Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos.

The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways.

Phenotypic changes induced by wild type and variant c-src genes carrying C-terminal sequence alterations.

We previously reported the isolation of PR2257, a novel avian sarcoma retrovirus which transduced the c-src protooncogene. The v-src gene of PR2257 differs from the c-src gene by a sequence change after amino acid 525, resulting in the replacement of tyrosine 527 by a valine, and an extension of the open reading frame into the non coding region of c-src. We investigated the respective roles of Tyr527 mutation and of the C-terminal extension in activating the oncogenic properties of c-src. Therefore we overexpressed the wild type c-src gene and c-src variants, carrying either a substitution of tyrosine 527 or an extension of the C-terminus or both modifications in combination, in chicken embryo fibroblasts and post mitotic neuroretina (NR) cells, using replication defective retroviruses. We also used in vivo inoculation of plasmid DNA to assess the tumorigenicity of the various c-src genes. We report that, in contrast to previous results, overexpression of c-src is sufficient to induce NR cell division. While mutation of tyrosine 527 alone significantly activates c-src transforming and tumorigenic properties, its combination with the C-terminal extension of PR2257 confers to this gene full oncogenic properties and increased metastatic potential as compared to the v-src of Rous sarcoma virus strains.

Two novel variants of the v-src oncogene isolated from low and high metastatic RSV-transformed hamster cells.

Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-src protein in cell lines was not correlated with their metastatic potential in vivo. Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-src isoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and vsrcLM: 62 kDA. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-src sequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the of the oncoprotein. Both v-src variants and chimeric v-src with mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretinal cells were associated with the mutations in the carboxy-terminal region of the v-src oncogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.

Origin and evolution of the c-src-transducing avian sarcoma virus PR2257.

Avian sarcoma virus PR2257 transduced de novo the c-src gene and about 900 bp of 3' non-coding sequences belonging to the src locus. This virus contains only one mutation in the c-src coding sequence causing a reading frame shift after Pro-525. The molecular clone studied was derived from a cell line of transformed quail fibroblasts, C7. It contains endogenous virus (ev) derived sequences in the U5 and 3' non-coding regions, indicating that multiple recombination occurred with endogenous virus. Here we investigated the possible evolution of PR2257 when the original tumour was repeatedly passaged in vivo. After 16 passages a new virus, designated PR2257/16, appeared with a tenfold higher titre. The sequence of PR2257/16 was determined and showed that PR2257/16 resulted from recombination of PR2257 with the env gene of the helper virus (td daPR-C). This recombination expanded the env gene content in PR2257/16 and, in addition, five point mutations occurred in its genome. Because we thought that an endogenous virus might be involved in the mechanism of c-src transduction, we also reinvestigated the presence of ev sequences in PR2257 proviruses from several early passages of the original tumour. We found that in contrast with the first isolate from the C7 cell line, the provirus in these tumours did not contain such sequences. These results do not support the hypothesis that endogenous sequences were involved in the transduction process.

Functional and biological properties of an avian variant long terminal repeat containing multiple A to G conversions in the U3 sequence.

We previously reported that infection of chicken embryonic neuroretina cells with Rous-associated virus type 1 leads to the frequent occurrence of spliced readthrough transcripts containing viral and cellular sequences. Generation of such chimeric transcripts constitutes a very early step in oncogene transduction. We report, here, the isolation of a c-mil transducing retrovirus, designated IC4, which contains a highly mutated U3 sequence in which 48% of A is converted to G. Functional analysis of this variant U3 indicated that these mutations do not impair viral transcription and replication; however, they abolish functioning of its polyadenylation signal, thus allowing readthrough transcription of downstream cellular sequences. On the basis of these results, we designed a nonreplicative retroviral vector, pIC4Neo, expressing the neomycin resistance (Neo(r)) gene under the control of the IC4 long terminal repeat. Infection of nondividing neuroretina cells with virus produced by a packaging cell line transfected with pIC4Neo occasionally resulted in sustained cell proliferation. Two independent G418-resistant proliferating cultures were found to express hybrid RNAs containing viral and cellular sequences. These sequences were characterized by reverse transcription-PCR and were identified in both cultures, suggesting that proliferation was correlated with a common integration locus. These results indicate that IC4Neo virus functions as a useful insertional mutagen and may allow identification of genes potentially involved in regulation of cell division.

The 3' region of c-src gene mRNA is entirely included in exon 12 and does not encode another protein.

We previously reported the isolation of PR2257, a novel replication defective avian sarcoma virus which transduced the entire c-src coding region together with about 900 bp of c-src 3' non coding sequences. This virus originated from a chicken sarcoma induced by inoculation of a transformation-defective Rous sarcoma virus carrying only replicative genes. The 5' end of PR2257 was formed by a splice junction between viral leader sequences and the first exon of c-src. To understand the mechanism of 3' recombination between viral and cellular sequences, we analyzed the genomic organization of the 3' region in chicken and quail src DNA. We report that this sequence is colinear with that of a chicken src cDNA. Therefore, exon 12, which encodes the last 68 amino acids of c-src, also contains all 3' non coding sequences present in c-src mRNA and consequently, appears to be the last and largest (about 2 kbp) exon of c-src. We also show that the 3' regions of chicken and quail c-src genes does not contain the additional open reading frame (orf) which was previously reported (Dorai et al. (1991) Mol. Cell. Biol. 11, 4165-4176), and that no other significant conserved open reading frames could be found in this region for both species. Therefore, this region of src does not code for another protein. Taken together, our results suggest that PR2257 was generated by recombination at the RNA level. However, because of the absence of introns in this region of c-src, we cannot formally rule out the possibility that this recombination took place at the DNA level.

New case of c-src gene transduction: the generation of virus PR2257.

PR2257 is a new replication-defective avian sarcoma virus which harbours in addition to the spliced version of the c-src gene also about 950 bp of no-coding cellular sequences located downstream from the c-src stop codon (Geryk et al., 1989). Comparison of the 950 bp region transduced by PR2257 with the chicken c-src cDNA (Dorai et al., 1991) and genomic sequences of the c-src 3' non-coding region from chicken and quail has shown that there are no additional introns. The c-src 3' non-coding region represents the largest c-src exon (No. 12) comprising about 2 kb. Absence of conserved open reading frames within this region in chicken and quail genomic DNAs excludes the possibility for coding a protein by these sequences. Also, the possibility was excluded that numerous endogenous virus-derived sequences identified in molecularly cloned PR2257 provirus played a role in the c-src transduction. After serial passaging of PR2257 virus in vivo a variant PR2257/16 was isolated. In PR2257/16, the size of the env gene was increased due to homologous recombination with a helper virus. In addition to mutations in the viral leader and the v-src coding region, a large deletion in transduced c-src 3' non-coding sequences was found in the PR2257/16 genome. The significance of genome modifications for selective advantage of this viral variant in vivo is discussed.

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Virus-specific nucleotide sequences in duck cells transformed by chicken and duck-adapted Rous sarcoma virus.

Adaptation of PR-RSV-C on duck cells results in successful and efficient replication of the adapted virus in duck cells. The adapted variant, daPR-RSV-C, was compared with the parental chicken-cell derived PR-RSV-C. No differences in the efficiency of integration and in the number of integrated proviral copies in duck cells were found. However, the structure of proviral DNA of the adapted virus was different. Whereas EcoRI and HindIII digestion showed no differences between chicken-cell derived PR-RSV-C and the daPR-RSV-C, a new restriction site was found for BamHI endonuclease, which is probably located at the 3' end of the env gene.