Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions

We have studied the virus produced by a clone, termed 8A, that was isolated from a culture of murine sarcoma virus-transformed mouse cells after superinfection with Moloney murine leukemia virus (MuLV-M). Clone 8A produced high levels of type C virus particles, but only a low titer of infectious murine sarcoma virus and almost no infectious MuLV. When fresh cultures of mouse cells were infected with undiluted clone 8A culture fluids, they released no detectable progeny virus for several weeks after infection. Fully infectious MuLV was then produced in these cultures. This virus was indistinguishable from MuLV-M by nucleic acid hybridization tests and in its insensitivity to Fv-1 restriction. It also induced thymic lymphomas in BALB/c mice. To explain these results, we propose that clone 8A is infected with a replication-defective variant of MuLV-M. Particles produced by clone 8A, containing this defective genome, can establish an infection in fresh cells but cannot produce progeny virus at detectable levels. Several weeks after infection, the defect in the viral genome is corrected by back-mutation or by recombination with endogenous viral genomes, resulting in the formation of fully infectious progeny MuLV. The progeny MuLV's that arose in two different experiments were found to be genetically different from each other. This is consistent with the hypothesis that, in each experiment, the progeny virus is formed as a result of an independent genetic event. DNA was isolated from clone 8A cells and assayed for infectivity by the calcium phosphate transfection technique. No detectable MuLV was produced by cells treated with this DNA. This finding, along with positive results obtained in control experiments, indicates that clone 8A cells do not contain a normal MuLV provirus.

Section: Resultsmentioning

confidence: 99%

A replication-defective variant of Moloney murine leukemia virus. I. Biological characterization

Rein¹,

Gerwin²,

Bassin³

et al. 1978

“…6. DISCUSSION Some temperature-sensitive mutants of R-MuLV are partially characterized (15,16,18,19,23,27). Among them are the mutants ts17, ts25, ts26, ts28, and ts29, which were used in this study.…”

Section: Methodsmentioning

confidence: 99%

“…ts25 and ts26 are postintegration defective mutants whose defect is known to involve the cleavage of the gag gene-related precursor polypeptides (2,19). Mutant ts29 possesses a thermolabile reverse transcriptase (23) and ts28 has an unidentified ts defect at a late step in its replication (15,27).…”

Section: Methodsmentioning

confidence: 99%

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus

Ven¹,

Zaane²,

Onnekink³

et al. 1978

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31°C) with temperaturesensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulsechase experiments at the permissive and nonpermissive (39°C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants tsl7 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39°C, showed a normal turnover of the gag and env precursor polypeptides.

“…The pellets were then suspended in 0.1 ml of 0.01 M Tris-0.05 M KCl-0.001 M dithiothreitol (pH 7.4) at 200C and assayed. Portions (30 p1) were assayed in 100-pl incubation mixtures containing 50 mM Tris (pH 8.0), 60 mM KCl, 1 mM MnCl2, 2.5 mM dithiothreitol, 0.05% Nonidet P-40, and 0.04 optical density unit (260 nm) of polyribocytidylic acid* oligodeoxyguanylic acid [12][13][14][15][16][17][18] (rC:dG). To start the reaction, 10 id of 0.2 mM [3H]dGTP (10 Ci/mmol) was added.…”

Section: Methodsmentioning

confidence: 99%

Preliminary characterization of a temperature-sensitive mutant of Moloney murine leukemia virus that produces particles at the restrictive temperature

Wong¹,

Gallick²

1978

The isolation and preliminary characterization of a new temperature-sensitive mutant of Moloney murine leukemia virus, designated ts7, are reported. The infectivity of ts7, determined by a focus-forming unit assay, was reduced at least 100-fold when the virus was assayed at the nonpermissive temperature (39°C) as compared with assay at the permissive temperature (340C). However, several lines of evidence indicated that the diminution of ts7 titer at 390C is not due to its inability to form virus particles at that temperature. The supernatant from ts7-infected cells grown at 390C showed significant infectivity when assayed at 34°C; only small reductions in reverse transcriptase activity and fusion ability were observed when compared with supernatant from 340C ts7-infected cultures. That particles are produced at the nonpermissive temperature was confirmed by transmission electron microscopy of the supernatant from ts7-infected cells at 390C and by transmission electron microscope observations of mature particles trapped in the intercellular spaces of pelleted thin cell sections. A possible explanation for the productivity at 390C of particles that are infectious at 34°C but not at 390C is that the virus is heat labile at the nonpermissive temperature. Consistent with this hypothesis is the extreme heat lability of virus harvested at 340C. Such virus, when incubated at 390C, has a half-life one-sixth that of identical virus incubated at 340C, or that of wild-type virus at either temperature.