Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: identification, location, and phenotypic characterization in cat X mouse somatic cell hybrids.

Abstract

Somatic cell hybrids were constructed between BALB/c-RAG mouse cells and feline lymphoma cells by the hyoxanthine-aminopterin-thymidine selection scheme. RAG cells spontaneously produce an endogenous B-tropic type C virus. Cat-mouse hybrids preferentially segregate feline chromosomes and retain murine chromosomessdemonstrable by karyotypic and isozyme analyses. Despite the presence of the complete mouse genome, including the viral genome, virus production was diminished to 1-5% of the levels observed in RAG parents based upon particle-associated RNA-dependent DNA polymerase (reverse transcriptase) activity in the culture fluid. Thirty-seven hybrids made on four different occasions had suppressed virus levels, and no hybrids expressed parental virus levels. Reverse selection experiments on 6-thioguanine demonstrated that a restriction gene, tentatively named Bvr-1, was linked to the feline structural genes for hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.2.8) and glucose~-phosphate dehydrogenase (D-glucose-6phosphate:NADP+ 1-oxidoreductase; EC 1.1.1.49) in cats, probably on the X-chromosome. The genetic mode of action of Bvr-1 is trans dominant in restriction of murine leukemia virus. The restriction locus results in a block late in virus maturation but prior to release, since expression of antigens for viral structural proteins and mature budding particles is apparent on surfaces of restricted hybrid cells but not in high-speed pellets from culture fluid of restricted cells.

Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: pleiotropic restriction of endogenous BALB virus in cat X mouse somatic cell hybrids.

“…Monolayer cultures and hybrids were grown on either Dulbecco-Vogt MEM (DV) or Ham's F12M medium supplemented with 10% fetal bovine serum. Hybrids were selected and maintained on hypoxanthine aminopterin thymidine (HAT) medium, and back selected on Ham's F12M medium supplemented with 0.1 mM 6-thioguanine as described (13).…”

“…Somatic Cell Hybridization. The construction of RAG × FL-74 hybrid clones by Sendai virus cell fusion has been described (13). Hybrid colonies were analyzed genetically by karyological and isozyme procedures, and generally retained the full murine genome, but had segregated the majority of feline chromosomes.…”

“…Neither of the parental species will support replication of the alternative parent's virus (Table II and * Criteria of type C virus production include: particulate RDDP activity in cell supernates, budding particles on examination in transmission electron microscopy, and viral antigens (p30, gp70) on infected cell surfaces (14,36). * FL-74 is a lymphoid suspension cell which was a selective parameter used in hybrid production (13,27,36). § RAG and VA-2 are 6-thioguanine resistant and die on HAT medium.…”

“…Karyological analysis and tests of 25 gene enzyme systems which are distinguishable between cat and mouse cells indicated that the majority of feline chromosomes were lost in the hybrids while the murine genome remained intact (13). FeLV and FeLV group-specific p27 antigen detected by a competition radioimmunoassay have been found in only 2 of 18 hybrids tested thus far (C. Sherr and S. O'Brien, unpublished observations).…”

“…thine guanine phosphoribosyl transferase (HPRT) and glucose-6-phosphate dehydrogenase (G-6-PD) (13). The physiology of the HAT selective system lends itself to a back selection experiment whereby individual hybrid colonies can be selectively maintained in the presence or absence of Bvr-1 (Table III).…”