Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells

Abstract

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

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

“…Therefore, constitution of 5' ends by a splice process could represent a general mechanism in retrovirus transduction of proto-oncogenes. We previously proposed that a precursor of these early virus forms could be an alternatively spliced U5leader-Ac-onc RNA (9). Obtaining a complete retrovirus requires the acquisition of 3' sequences including a U3 region.…”

“…IC2 and IC3 were isolated during early passages of RAV-1 on NR cells, whereas ICI was isolated at later passages of culture supernatants during the same experiment. We were able to demonstrate a generation relationship between these earlyand late-passage viruses on the basis of structural analyses of their genomes (9).…”

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.…”