Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1

Abstract

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the millraf 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 ICll, from NR cells infected at the third virus passage of the same experiment. Both IC1l 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. ICll 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-l-Rmil junction of ICll 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 ICll in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that ICll 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.

Raf‐1 and B‐Raf proteins have similar regional distributions but differential subcellular localization in adult rat brain

“…Therefore, all B‐Raf isoforms contain this peptide and are recognized by the IS11 (as an antiexon 11 serum). The IS11 serum was previously used in several studies to immunoprecipitate B‐Raf proteins from neuroretina infected cells (Felder et al . 1991), Cos1‐transfected cells (Eychène et al .…”

Occurrence of alternatively spliced leader-delta onc-poly(A) transcripts in chicken neuroretina cells infected with Rous-associated virus type 1: implication in transduction of the c-mil/c-raf and c-Rmil/B-raf oncogenes

“…This experimental cell system also proved useful in establishing a precursor-product relationship between viruses generated successively during milIRmil transduction. Specifically, we showed that late virus forms were generated by further recombination involving sequence homologies between oncogenes and the RAV-1 gag gene (5,6). Therefore, the proposed model may account for the generation of the vast majority of transforming viruses which contain a 5' junction between gag and oncogene exonic sequences (22).…”

“…We reported that serial in vitro passaging of Rous-associated virus type 1 , an avian retrovirus that does not carry an oncogene, in chicken embryo neuroretina (NR) cells results in reproducible transduction of the catalytic domain of two related serine/ threonine protein kinases (15,16), c-mil/c-raf (13,14,18) and c-Rmil/B-raf (10). By comparing the genetic organization of several viruses isolated after few or multiple passages of culture supernatants on NR cells, we were able to establish a precursor-product relationship between viruses generated at different stages of transduction (5,6). We showed that the 5' end of early-passage viruses was generated by a splice mechanism between the donor site of RAV-1 leader sequences and acceptor sites of the activated exons.…”

“…Late-passage isolates acquired additional viral sequences (gag) by further recombination between the early virus and RAV-1 genomes. We proposed that the precursor of early virus forms could be an alternatively spliced leader-Aonc-poly(A) RNA (5,6). Here we present evidence that NR cells infected with RAV-1 do synthesize chimeric transcripts containing the RAV-1 leader sequence spliced to the catalytic domains of c-mil and c-Rmil oncogenes.…”

A model system for nonhomologous recombination between retroviral and cellular RNA

“…Homologous recombination in retroviruses, also proposed to occur during reverse transcription (15,19,20,40), occurs at a very high frequency, with markers as close as 1 kb apart segregating independently, suggesting that RT jumps frequently between copackaged genomic RNA (29). In the case of nonhomologous recombination, it is not known whether certain sequences or short stretches of homology promote RT jumps, since examples of recombination breakpoints at areas of either patchy homology (11,23,45) or no homology (1,26,38,43) are found in naturally occurring transforming viruses. Several systems have been described to study nonhomologous recombination during reverse transcription.…”