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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.

International audience; We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

We previously reported that serial passaging of Rous-associated virus type 1 in nondividing chicken embryo neuroretina cells leads to reproducible generation of acutely mitogenic retroviruses that transduced the catalytic domain of c-mil/c-raf or c-Rmil/B-raf. On the basis of structural analysis of several retroviruses, we proposed that the early step of oncogene transduction is the constitution of alternatively spliced leader-delta onc-poly(A) transcripts. Here, we show that neuroretina cells do synthesize hybrid leader-delta mil and leader-delta Rmil RNAs and that these RNAs exhibit mitogenic properties and serve as templates for the generation of transducing retorviruses.