FUNCTIONAL AND BIOLOGICAL PROPERTIES OF AN AVIAN VARIANT LONG TERMINAL REPEAT CONTAINING MULTIPLE A-CONVERSION TO G-CONVERSION IN THE U3 SEQUENCE

We previously reported that infection of chicken embryonic neuroretina cells with Rous-associated virus type 1 leads to the frequent occurre nce of spliced readthrough transcripts containing viral and cellular s equences. Generation of such chimeric transcripts constitutes a very e arly step in oncogene transduction. We report, here, the isolation of a c-mil transducing retrovirus, designated IC4, which contains a highl y mutated U3 sequence in which 48% of A is converted to G. Functional analysis of this variant U3 indicated that these mutations do not impa ir viral transcription and replication; however, they abolish function ing of its polyadenylation signal, thus allowing readthrough transcrip tion of downstream cellular sequences. On the basis of these results, we designed a nonreplicative retroviral vector, pIC4Neo, expressing th e 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 occasional ly resulted in sustained cell proliferation. Two independent G418-resi stant proliferating cultures were found to express hybrid RNAs contain ing viral and cellular sequences. These sequences were characterized b y reverse transcription-PCR and were identified in both cultures, sugg esting that proliferation was correlated with a common integration loc us. These results indicate that IC4Neo virus functions as a useful ins ertional mutagen and may allow identification of genes potentially inv olved in regulation of cell division.