Functional and biological properties of an avian variant long terminal repeat containing multiple A to G conversions in the U3 sequence.
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.
Felder
M P
MP
Unité de Recherche Associée 1443 du Centre National de la Recherche Scientifique, Institut Curie, Centre Universitaire, Orsay, France.
Laugier
D
D
Yatsula
B
B
Dezélée
P
P
Calothy
G
G
Marx
M
M
D000225:Adenine; D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D002642:Chick Embryo; D004279:DNA, Viral / Q000235:genetics; D005822:Genetic Vectors; D006147:Guanine; D008969:Molecular Sequence Data; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf; D012326:RNA Splicing; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014161:Transduction, Genetic
D004279:DNA, Viral; D011518:Proto-Oncogene Proteins; D006147:Guanine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D000225:Adenine