Hidesaburo Hanafusa

The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant.

Abstract A temperature-sensitive mutant, Ts-68, was isolated from a stock of the Schmidt-Ruppin strain of Rous sarcoma virus which had been grown in the presence of 5-fluorouracil. At the nonpermissive temperature, the mutant was unable to induce or maintain morphological transformation of chick embryo cells, but virus reproduction remained unaffected. In addition to the morphological changes, cells infected with Ts-68 could be either “transformed” or “normal” with respect to cell division under agar medium and the rate of sugar uptake depending on the incubation temperature. Kinetic studies, using the rate of sugar uptake and cell morphology as indices, showed that the changes toward either direction proceeded linearly without a time lag and 50% conversion was obtained at 5–6 hr after a reciprocal shift of temperature. The presence of cycloheximide or puromycin interfered with the appearance of cell transformation when cultures were shifted from a nonpermissive to a permissive temperature, but thymidine, cytosine arabinoside, or actinomycin D had no effect on this process. These results strongly suggest that a virus-coded, heat-labile protein, which is not a structural component of the virus, plays an essential role in cell transformation by the mutant.

Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus; alteration in the characteristics of sugar transport

Abstract A quantitative as well as sequential analysis of the uptake of certain sugars during the process of cell transformation with Rous sarcoma virus has been made. Significant alteration in the kinetics of sugar transport occurs in the infected cells concomitant with changes in cell morphology. These alterations appear to be expressed by the transforming gene(s) of RSV and are not found in cells infected with an avian leukosis virus.

Proteins of helper-dependent RSV.

Abstract The proteins of purified avian leukosis and sarcoma viruses grown in C/O(chf+) and C/O′(chf−) chicken cells were analyzed by polyacrylamide SDS-gel electrophoresis. RAV-2, RAV-60, andSR-RSV all contained seven proteins, one of which was a major glycoprotein with a low electrophoretic mobility. Bryan RSV differed from RAV-2, RAV-60, and SR-RSV primarily in possessing multiple glycoproteins of low electrophoretic mobility. The heterogeneity of the carbohydrate-labeled material varied according to the growth of Bryan RSV in C/O(chf+) or C/O′(chf−) chicken cells. In the absence of chf, a major, slowly migrating glycoprotein was missing from Bryan RSV.

Genetic recombination with avian tumor virus.

A recombinant that has the transforming activity of a Rous sarcoma virus (helper-independent) and the subgroup specificity of an avian leukosis virus has been isolated from cells infected with these two types of virus. The temperature sensitivity of a sarcoma virus mutant was unaffected following alteration of the host range by recombination. The high frequency of recombination observed seems to support the assumption that the virus genome is composed of several subunits. On the other hand, the helper-independent nature of the Bryan strain of Rous sarcoma virus was not altered following replication with leukosis virus. The recombination may be restricted in this strain, or it may be insufficient to restore the defective function of the virus. Nontransforming viruses were found in all preparations of genetically purified transforming recombinants, suggesting that the loss of the gene(s) related to cell transformation is not a rare event.

Further studies on RSV production from transformed cells

Abstract In order to determine whether or not every Rous-infected cell produces infectious progeny in the absence of helper virus, a number of cloned lines of L-R cells (leukosis virus negative Rous cells, formerly called NP cells) were tested for spontaneous virus production. It was found that there were two classes of L-R lines, one producing RSV infectious for Japanese quail embryo or C A type chick embryo cells and another producing virus particles that were not detectably infectious for any cell line tested.

Penetration and intracellular release of the genomes of avian RNA tumor viruses

Abstract Attachment and penetration of avian sarcoma and leukosis viruses into chick embryo fibroblasts was examined by a combination of electron microscopy, autoradiography, and physical-chemical procedures. Adsorption occurred for 60 min at low temperature and, upon warming to 37°, disposition of virions in cells was studied in samples taken at intervals over a 120-min period. Monolayer cultures were either preserved in their undisturbed state or cells were scraped and centrifuged into pellets. In both preparations, adsorption of most viruses occurred at surfaces coated by a dense reticulum of material, termed attachment sites. Individual virions or groups of them were internalized by viropexis and moved within vacuoles to the vicinity of nuclear envelopes. Ten minutes after warming, at the time that inoculum particles were first observed in the perinuclear zone, the radioautograms indicated the occurrence of 3 H-uridine-labeled viral RNA within the nucleus. Maximum concentration of this tracer was reached by 60–120 min after warming, although only about 5–10% of all cell-associated inoculum radioactivity became intranuclear. The phenol extracted nuclear labeled RNA possessed a sedimentation value of 60 S, the same as that of parental virions. Experiments in which 3 H-thymidine was employed to examine by autoradiography DNA synthesis of uninfected and virus-infected cells failed to demonstrate “reverse transcriptase” activity in the cytoplasm. These observations, together with the evidence for rapid transfer of inoculum genomes into the nucleus, are best explained by assuming that virion-directed DNA transcription and reduplication occurs in the nucleus.

A type of chick embryo cell that fails to support formation of infectious RSV.

Abstract Although all C 0 type chick embryo cells can be transformed by Rous sarcoma virus (RSV), cells derived from about 20% of these embryos (termed C 0′ ) fail to produce infectious RSV(0). Production of infectious RSV(0) is uniformly suppressed in all cells originating from C 0′ embryos. These transformed C 0′ cells, however, do produce noninfectious particles containing complete RSV genomes, and they issue infectious RSV when superinfected with avian leukosis virus. The noninfectious particles are capable of establishing infection in either chick or quail cells under conditions of cell fusion generated by UV-irradiated Sendai virus, suggesting that the particles have lost the capacity of cell penetration after growth in C 0′ cells. Other avian leukosis-sarcoma viruses are able to grow normally in C 0′ cells, and thus far this characteristic of C 0′ cells is confined to RSV(0).

Presence of antigen common to avion tumor viral envelope antigen in normal chick embryo cells

Abstract Antiserum against the envelope antigen of avian tumor virus of subgroup E was obtained from chickens bearing tumors induced by RSV(f). The capacity to absorb neutralizing activity from the antiserum was used to demonstrate the presence or absence of the envelope antigen in various types of cells. The specificity of the antigen was also confirmed by immuno-electron microscopy. Cell extracts or viable cells from uninfected chick embryos were generally positive for the envelope antigen when the embryos were also positive in both the formation of gs-antigen and helper activity. Chick embryo cells negative for these viral functions were also deficient in the envelope antigen. However, two exceptional embryos were found in which the amount of envelope antigen and the level of helper activity do not directly correlate with each other. The antigen common to that on the viral envelope appeared to be located on or or in the fuzzy coat at the cell surface.

Plaque assay for some strains of avian leukosis virus

Abstract Some strains of avian leukosis virus were found to produce plaques at 41 ° in cultures of chick embryo cells which had been fully infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV) which fails to induce morphological transformation at the elevated temperature. The leukosis viruses caused no discernible lesion in normal chick embryo cultures. Macroscopically visible plaques appeared within 4 days after infection in the mutant-infected cultures, and by 7 to 10 days the plaques became nonstainable by neutral red. The titer of plaque-forming units of these virus preparations was comparable to the titer of virus-infectivity estimated by the interference assay. Avian leukosis viruses of subgroups B and D thus far tested produced plaques under these conditions, while viruses of subgroups A, C, and E did not. Some preparations of avian sarcoma virus, subgroups B and D, also induced plaques, but whether or not the sarcoma virus itself can induce plaques has not been clearly determined. Conditions required for plaque formation, and possible mechanisms are discussed.

Electrophoretic analysis of the RNA of avian tumor viruses

Abstract The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37°, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 × 106 daltons and at least one minor RNA species of slightly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, RAV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of Schmidt-Ruppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions.