The presence and location of DNA sequences related to the U3 and U5 portions of the infectious exogenous feline leukemia virus (FeLV) long terminal repeat (LTR) in various cat DNAs have been determined by hybridization experiments. In uninfected cat DNAs, the U5 LTR segment from the Gardner-Arnstein strain B virus is present at approximately 150 copies per cell. This level is approximately 10-fold greater than that of endogenous internal FeLV sequences. The U5 sequences differ in copy number and, to some extent, in location from one animal to another. For any one animal, the sequence organization of the U5 segments is the same among different tissues, showing that the pattern is inherited through the germ line. Most importantly, the viral U3 LTR probe hybridizes only very weakly with uninfected cat DNAs. Both the U3 and the U5 regions of the LTR from the Gardner-Arnstein strain of virus cross-hybridize with DNA derived from four other infectious FeLVs representing A, B, and C subtypes. Thus, the C3 region may be used as a probe for studying the number and location of exogenously acquired FeLV proviruses in infected cat tissues. In some cases exogenously acquired proviruses are present in unique sites in the genome of virus-positive cat lymphosarcomas, indicating a monoclonal origin for the tumor. In other tumors, the proviral sequences are randomly distributed over many sites. Lymphosarcomas of virus-negative cats have no exogenous U3 sequences despite epidemiological evidence of an association of virus-negative leukemia with exposure to FeLV.

We examined 14 different feline leukemia virus proviruses from the productively infected human cell line RD(FeLV)-2 after cloning in the modified lambda vector Charon 4A. Each isolate was characterized by restriction digestion and Southern blot analysis. The DNA of each isolate was tested for competence to express virus after uptake by sensitive animal cells (transfection). All but one isolate contained an apparently complete provirus, but only four were infectious. Seven isolates (four noninfectious, three infectious) were studied by heteroduplexing followed by electron microscopy or by S1 nuclease treatment and gel electrophoresis. No regions of nonhomology between proviruses were detected by either criterion, and in no case did we observe homology between flanking sequences. Random shearing or removal of flanking sequences by S1 nuclease had no effect on the status of infectivity of the clones. Thus, we were unable to find molecular differences between infectious and noninfectious proviruses. Our data are consistent with either of the following hypotheses: (i) that there is a short host sequence which is essential as a promoter for virus expression; or (ii) that lack of infectivity is due to small mutations within the proviral genome.

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.