Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells

Abstract

Avian erythroblastosis virus (AEV) causes erythroblastosis and sarcomas in birds and transforms erythroblasts and fibroblasts to neoplastic phenotypes in culture. The viral genetic locus required for oncogenesis by AEV is at present poorly defined; very little is known of the mechanism of tumorigenesis by the virus. To facilitate further analysis of these problems, molecular cloning was used to isolate the genome of AEV as recombinant DNA in a prokaryotic vector. The identity of the isolate DNA was verified by mapping with restriction endonucleases and by tests for biological activity. The circular form of unintegrated AEV DNA was purified from synchronously infected quail cells and cloned into the EcoRI site of .lambda.gtWES .cntdot. B. A restriction endonuclease cleavage map was established. By hybridization with complementary DNA probes representing specific parts of avian retrovirus genomes, the restriction map of the cloned AEV DNA was correlated with a genetic map. Nucleotide sequences unique to AEV comprise at least 50% of the genome and are located approximately in the middle of the AEV genome. The data confirm and extend previous descriptions of the AEV genome obtained by other procedures. Two recombinant clones containing AEV DNA were studied; the topography of the viral DNA in the 2 clones was virtually identical, except that 1 clone apparently contained 2 copies of the terminal redundancy that occurs in linear viral DNA isolated from infected cells; the other clone probably contained only 1 copy of the redundant sequence. To recover infectious virus from the cloned DNA, a transfection procedure was developed that compensated for the defectiveness of AEV in replication. This was accomplished by ligating cloned AEV DNA to the cloned DNA of a retrovirus (Rous-associated virus type 1) whose genome could complement the deficiencies of AEV. Ligation of the 2 viral DNA was facilitated by using a neutral fragment of DNA as linker between otherwise noncompatible termini. Cloned AEV DNA gave rise to infectious AEV capable of transforming fibroblasts and bone marrow cells in culture and of inducing both sarcomas and erythroleukemia in chickens. The cloned DNA apparently represent the authentic genome of AEV undisturbed by the cloning procedure. Molecular cloning offers a powerful approach to the identification and characterization of retrovirus genomes.