Paul T. Peebles

An in vitro focus-induction assay for xenotropic murine leukemia virus, feline leukemia virus C, and the feline-primate viruses RD-114/CCC/M-7

A rapid focus-induction assay has been developed for certain replicating (r+) but nontransforming (t−) mammalian type C viruses including xenotropic murine leukemia virus (X-MuLV), feline leukemia virus C (FeLV-C), and feline-primate viruses RD-114/CCC/M-7. Mink cells infected only with r−t+ defective Moloney murine sarcoma virus (MSV) were established into a phenotypically flat cell line (S+L− MiCl1). When S+L− MiCl1 monolayers were superinfected with dilutions of the r+t− type C viruses, foci formed with one-hit kinetics. Focus induction was enhanced by treatment of the cells with either DEAE-dextran or polybrene. Quantitation of r+t− virus by use of S+L− MiCl1 cells was comparable to that of other biological assay systems.

Reversion of Murine Sarcoma Virus Transformed Mouse Cells: Variants without a Rescuable Sarcoma Virus

Murine sarcoma virus transformed mouse 3T3 cells, which are negative for murine leukemia virus and which yield sarcoma virus after superinfection with murine leukenmia virus, spotaneously give rise to flat variants front which murine sarcoma virus can no longer be rescued. The revertants support leukemia viruis growth and show an enhanced sensitivity to murine sarcoma superinfection and, like normal cells, do not release RNA-dependent DNA polymerase activity. Because revertants could be obtained with high frequency from progeny of single transformed cells, each cell that containts the sarconma virus genome seems to have the capacity to suppress or eliminate an RNA tumor virus native to its species of origin.

Revertants of mouse cells transformed by murine sarcoma virus: I. Characterization of flat and transformed sublines without a rescuable murine sarcoma virus

Abstract Murine sarcoma virus (MSV)-transformed mouse clonal cell lines produced variants with some properties of nontransformed cells. Such variant cells were epithelioid, contact inhibited, and grew to low density, and their low cloning efficiency in soft agar was similar to that of normal parental 3T3 cells. However, they contained murine leukemia (MuLV) group-specific antigen(s) without demonstrable virus production and reverse transcriptase activity. MSV could no longer be rescued from these flat variant cells by superinfection with MuLV, by cocultivation with normal 3T3 cells or by transspecies rescue into cat cells. An enhancement of sensitivity to MSV and MuLV infection was observed in all flat variant cultures. Flat variant clones spontaneously gave rise to retransformed cells during extended cultivation. Morphology, saturation density, and cloning efficiency in soft agar of cloned spontaneous retransformed cell lines were similar to the original MSV-transformed cells. However, they failed to demonstrate MuLV gs antigen(s), virus production, reverse transcriptase activity and a rescuable MSV genome. The spontaneously retransformed cells were susceptible to MSV and MuLV infection. After treatment with 5-iododeoxyuridine (IUrd), reverse transcriptase activity and virus particles were only rarely induced in flat variant or spontaneously retransformed clones. These particles were not infectious for the original host cells and were not induced in normal 3T3 cells or a majority of the variant clones. Chromosome studies of these variants suggested that the partial or complete loss of expression of transformation in variants might have been associated with an imbalance in the number of chromosomes mediating expression or suppression in these cells.

727 ISOLATION OF FOUR UNUSUAL PEDIATRIC SOLID TUMOR CELL LINES

Four well-characterized, unique cell lines are now available for cancer, immunological, and cell differentiation research. They are derived from malignant melanoma, leiomyoblastoma, osteosarcoma, and Wilms tumor in P-3 containment facilities in the absence of viruses and other human-animal cell lines. True Wilms tumor and leiomyoblastoma cell lines, and a malignant melanoma line faithfully producing melanin, and a highly transformed non-fibroblastic osteosarcoma cell line have not been previously existent. All have been in culture for greater than two years and are transformed growing easily in soft agar suspension. They are human by immunofluorescence, karyotype, and G-6-PD (type B) isoenzymes. All cell lines have been studied extensively by electron microscopy. Extensive virological, immunological, and biochemical studies demonstrate little evidence for viruses similar to known RNA and DNA tumor viruses. Both early and late passages are now available from The American Type Culture Collection, Rockville, Md.

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.

Murine sarcoma virus defectiveness. Viral polymerase expression in murine and nonmurine host cells transformed by S + L - type murine sarcoma virus.

Sarcoma virus-positive, leukemia virus-negative (S + L -) mouse cells transformed by defective but rescuable murine sarcoma virus (MSV) release noninfectious type C virions with a quantitative deficiency of viral-type RNA-dependent DNA polymerase (RDDP). Human S + L - cells containing the same genome fail to express any detectable viral-type reverse transcriptase. To study this difference in MSV expression, second-generation MSV from the S + L - human cells is recloned in dog, mink, and back into mouse cells. Heterologous host dog and mink S + L - cell clones, like human S + L - cell clones, fail to release viral-type reverse transcriptase into culture supernatant fractions. All second-generation homologous mouse S + L - cell clones again release viral reverse transcriptase in supernatant fractions, indicating that the MSV genome has not been altered in this function. Using (dT)(12-18)-cellulose and phosphocellulose chromatography of cellular polymerase preparations, no intracellular buildup of unreleased murine reverse transcriptase is detected in the S + L - clones. The data presented here suggest that the MSV genome is defective in the information for the viral core protein RDDP. The implications of these findings for the genetic study of MSV are discussed.

Murine sarcoma virus defectiveness: Serological detection of only helper virus reverse transcriptase in sarcoma virus rescued from nonmurine S+L− cells

Abstract Murine sarcoma virus (MSV) defectiveness was studied by examining MSV rescued by RD-114 virus superinfection of both dog and human heterologous host cells nonproductively transformed by the S+L− strain of Moloney MSV (S+L− cells). Biological assays showed that the rescued MSV pseudotype was present in excess over the RD-114 helper virus. The biologically determined virus ratio corresponded to that determined by virus nucleic acid hybridizations. Antisera distinguishing the DNA polymerase of murine type C virus from that of RD-114 virus demonstrated that the only detectable RNA-dependent DNA polymerase present in the rescued MSV virions was that of RD-114 helper virus. No murine virus polymerase was detectable within the virions. Previous data have demonstrated that no murine-viral polymerase is associated with S+L− heterologous human, dog, and mink host cells. The work presented here demonstrates that cells producing infectious MSV and helper virions still do not express murine polymerase within released MSV virions. These findings suggest that heterologous host-cell control mechanisms are not preventing MSV polymerase expression in these cells. Instead, the rescued MSV virions acquire the polymerase protein from the rescuing helper virus. These data provide evidence that the MSV genome lacks the information necessary for polymerase, and requires for the production of infectious progeny the functioning of a helper virus replication gene set supplying at least viral envelope antigen(s) and virion core protein polymerase.

RETINOIDS AND IMMUNOMODULATION SUCCEED AGAINST HUMAN PAPILLOMA VIRUS INDUCED NEOPLASIA....AT LEAST FOR NOW. 949

RETINOIDS AND IMMUNOMODULATION SUCCEED AGAINST HUMAN PAPILLOMA VIRUS INDUCED NEOPLASIA....AT LEAST FOR NOW. 949