Oncornavirus Expression in Human × Mouse Hybrid Cells Segregating Mouse Chromosomes

Abstract

Human X mouse hybrid clones obtained by fusing transformed human (VA2) cells with embryonic mouse brain cells were tested for their ability to spontaneously express type C virus particles. It had been previously shown that these hybrid cells preferentially retained human chromosomes while mouse chromosomes were lost. The culture fluid from one cell line was found to contain type C particle markers in abundance, and typical budding C particles were observed in the cells by electron microscopy. In contrast, no particle markers were detected in the culture fluid from parental cells and several other hybrid cell lines. Subelones of the virus-positive cell line continued to lose mouse chromosomes and were found to vary more than 100-fold in their culture fluid DNA polymerase activity. The hybrid cell viruses, termed HMV1, banded in a sucrose gradient between 1.14 and 1.16 g/ml, possessed viral group-specific antigens, and exhibited Btropic host range for replication in mouse embryo cells, but did not replicate in human cells when directly applied. The virus did not transform mouse cells but was able to rescue the defective murine sarcoma virus from sarcomapositive, helper-virus-negative cells. Activity of the DNA polymerase associated with HMV1 was similar to the activity of Rauscher murine leukemia virus (MuLV) DNA polymerase in its preference for poly(rA) over poly(dA) as a template, use of endogenous template, detergent requirement, and inhibition by antiserum directed against MuLV. DNA polymerase. The results suggest that human X mouse hybrid cells segregating mouse chromosomes can spontaneously express endogenous type C viruses and that such hybrid cell lines may be used for the isolation of latent mammalian oncornaviruses and analysis of viral gene regulation.

Differential response of type C and intracisternal type A particle markers in cells treated with iododeoxyuridine and dexamethasone

“…DNA polymerase assays. The assay for C-particle DNA polymerase activity was performed as described previously (16); unless indicated otherwise, each reaction contained: 50 mM Tris-hydrochloride, pH 7.7, at 37°C; 20% glycerol; 400 ,ug of bovine plasma albumin per ml; 0.05% Nonidet P-40; 2 mM dithiothreitol; 1 mM p-hydroxymercuribenzoate; 20 mM glycylglycine; 0.4 mM EDTA; 5 mM magnesium acetate; 40 mM KCl; 40 ,ug of (dT)12 ,8per ml; 200 jig of poly(rA) per ml; 0.5 mM [methyl-3H]dTTP (300 cpm/pmol); and 5 ,.1 of culture fluid pellet fraction. The assay for A-particle DNA polymerase activity was performed as described previously (21); reactions contained either 3 pi of culture fluid pellet fraction or 1 to 3 Ag of cell extract protein.…”

“…Because the catalytic properties of the type A particle and type C particle-associated DNA polymerases are different, reaction conditions may be selected for the specific assay of each enzyme (16,21,22). A comparison of the two DNA polymerase assays is shown in Table 1.…”

Appearance of C‐type virus‐like particles after co‐cultivation of a human tumor‐cell line with rat (XC) cells

“…accomplished by co-cultivation or fusion of non-producer cells carrying the viral genome with permissive cells (Svoboda et a[. 1963); or co-cultivation, by fusion of cell lines containing endogenous but unexpressed viral infor-mation (X-tropic or S-tropic viruses) with heterologous cells permissive for viral replication (Levy, 1973;Todaro et a/., 1973) or alternatively as a result of segregation of the chromosomes bearing viral repressor genes of one parent cell in a somatic cell hybrid (Minna, 1974) allowing expression of endogenous viral information.…”

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.

“…Thirty-one restricted hybrids were tested for cell surface associated viral , who observed diminution of particle-associated RNA-dependent DNA polymerase in culture fluid below parental levels in hybrids between virus-producing rodent cells and fresh human leukocytes. The action of the human restriction of murine virus is still unclear, however, since both exogenously applied murine ecotropic viruses and endogenous induced murine viruses replicated at high levels in different human X mouse hybrids containing a full human genome (36,37).…”