Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3.3 kilobase pairs). We isolated the proviruses of the 8.3and 3.3kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avian myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.

The structure of unintegrated spleen necrosis virus DNA was characterized by using various chemical and enzymatic treatments in conjunction with denaturing gels and nucleic acid hybridization probes. Throughout the course of the viral infection, the predominant species of viral DNA was that of a linear double-stranded molecule containing ribonucleotides covalently joined to the DNA. The majority of both - and + strands were continuous. The ribonucleotide linkages appeared to be relatively short, and the base composition and distribution of the ribonucleotide linkages were heterogeneous. On the average, the - strand had fewer of the ribonucleotide linkages than did the strand. Viral DNA containing ribonucleotide linkages was infectious in DNA transfection assays. The structure of spleen necrosis virus DNA was different from that of Schmidt-Ruppin Rous sarcoma virus-D, and mixed infections demonstrated that the observed differences are a result of cis-acting functions.