Enhancement of endogenous xenotropic murine retrovirus expression by tuftsin.

The exposure of a high-passage clone of Kirsten sarcoma virus transformed Balb/c (K-Balb) mouse cells to tuftsin (Thr-Lys-Pro-Arg) enhanced the expression of endogenous xenotropic retrovirus. The tetrapeptide increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion (0.001-1000 micrograms/ml). Increased virus expression could be achieved during short-term incubations (3-4 hr), with maximum enhancement occurring over longer time periods (16-18 hr). The enhancement of virus expression by tuftsin was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp 70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. Tuftsin stimulated DNA, RNA, and protein synthesis in K-Balb cells during 16-hr incubations. Increased cellular proliferation was also seen at various time periods. The effects observed using K-Balb cells offer an opportunity to study the modulation of gene expression by tuftsin in a fibroblast culture system.

Cell cycle-specific inhibition by retinoic acid of xenotropic murine retrovirus expression.

Several retinoids were examined for their capacity to block chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus-transformed BALB/c mouse cells. Retinoic acid (RA) was found to inhibit induction of virus by 5-iododeoxyuridine, cycloheximide, and histidinol; inhibition was concentration (10(-4) to 10(-6) M) and time dependent (1 to 7 hr) and not a consequence of cytotoxicity. Following a 6-hr treatment with 10(-4) M RA, [3H]thymidine and [3H]uridine incorporation into total cellular DNA and RNA was reduced 37 and 63%, respectively. Heteronuclear RNA synthesis was reduced 36 and 7% within 4 hr by 10(-4) and 10(-5) M RA, respectively, indicating that inhibition was not the result of a general transcriptional block. Using synchronized cells, it was found that 5 X 10(-5) M RA added in G1 phase and followed by cycloheximide or 5-iododeoxyuridine induction inhibited virus expression 60 and 84%, respectively. Little or no inhibition was observed when RA was added during S phase with the inducers or during G2 phase followed by inducers. Cells synchronized by mitotic arrest showed a RA-mediated restriction point in early-to-mid-G1 phase as indicated by a delay in the onset of DNA synthesis and an inhibition of virus induction during S phase. The results show the presence in Kirsten sarcoma virus-transformed BALB/c cells of a RA-sensitive G1 restriction point for cell progression and suggest that inhibition of retrovirus activation may be related to an extended G1 phase.

Viral susceptibility of skin fibroblasts from patients with Huntington disease.

Cultured skin fibroblasts from patients with Huntington disease (HD) and age-matched controls were tested for susceptibility to vesicular stomatitis virus (VSV) and transformation by Kirsten mouse sarcoma virus (KiMSV). The HD and control cells could not be distinguished on the basis of viral replication, plaque morphology, virus yield, or susceptibility to transformation by KiMSV. These findings suggest that the HD gene product, if expressed within peripheral tissue, does not selectively alter or interfere with viral replication.

Virus RNA species in kirsten murine sarcoma virus-transformed mink cells.

The nuclear and cytoplasmic RNAs from Kirsten murine sarcoma virus (KiMuSV)-transformed non-producer mink cells were studied for the species of virus-specific RNA by fractionation in agarose gels, transfer to diazotized aminophenylthioether paper and hybridization to complementary DNA probe. In both nuclei and cytoplasm, only genome-length KiMuSV-specific RNA was detected. No subgenomic virus RNA species was detected in poly(A+) or poly(A-) RNA fractions. The same observations were made in KiMuSV-transformed mink cells superinfected with feline leukaemia viruses. The significance of these findings is discussed.

Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification.

A wide variety of infected mammalian cell cultures were observed to produce high levels of RNA tumor virus particles in the absence of serum for at least 12 h. Virus production was measured by yields of 50-70 S virus RNAs isolated directly from serum-free culture media by chromatography on oligo(dT)-cellulose. Yields of RNAs from viruses produced in serum-free medium were comparable to yields obtained from purified viruses produced in serum-containing medium. Subunits of viral RNAs were thermally dissociated and separated by a new sedimentation system using sucrose gradients with resolving power (in the relevant size range) equivalent to that obtained with electrophoresis in polyacrylamide gels. RNA subunits isolated directly from serum-free medium appeared to be intact as judged by poly(A) content and resedimentation. The overall approach developed here represents dramatic savings in time and effort over previous ways of producing purified RNA subunits from tumor viruses.