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J Virol. 1994 August; 68(8): 4759-4767

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

Unité de Recherche Associée 1443 du Centre National de la Recherche Scientifique, Institut Curie, Centre Universitaire, Orsay, France.

ABSTRACT

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.

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