Isolation of Moloney Leukemia Virus Temperature Sensitive Mutants, and the Characterization of a Temperature Sensitive Mutant Deficient in the Incorporation of Env Gene Proteins
Gallick, Gary Edward
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Permalink
https://hdl.handle.net/2142/68094
Description
Title
Isolation of Moloney Leukemia Virus Temperature Sensitive Mutants, and the Characterization of a Temperature Sensitive Mutant Deficient in the Incorporation of Env Gene Proteins
Author(s)
Gallick, Gary Edward
Issue Date
1981
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
Techniques for the isolation of temperature sensitive (ts) mutants and revertants of Moloney leukemia virus (M-MuLV) and analysis of ts 7, an M-MuLV ts mutant isolated by Dr. P. K. Y. Wong are presented in this dissertation.
Seven temperature-sensitive (ts) mutants of Moloney murine leukaemia virus (Mo-MuLV) were isolated using a rapid, non-selective replica plating technique designed to select for post-integration mutants. Thymus-bone marrow cells, infected with mutagenized virus, were cloned and incubated at the non-permissive temperature (39(DEGREES)C) for 10 days. The resulting colonies were screened for production of virus by replica plating supernatant from the 'master' tray on to a second tray pre-seeded with fu-1 (a cell line derived from L8 myoblasts) indicator cells. The 'master' tray was shifted to the permissive temperature (34(DEGREES)C) for 48 hr, then re-screened for virus production. Any colony on the 'master' tray which produced syncytia-inducing virus at 34(DEGREES)C but not at 39(DEGREES)C was potentially producing a ts mutant.
Rescreening of these colonies confirmed that seven were producing ts mutants. Analysis of ts 7 revealed that this mutant produces infectious virus at an equivalent rate at both 34(DEGREES)C and 39(DEGREES)C, but that at 39(DEGREES)C the virions are heat labile. Biological studies indicated that after incubation at 39(DEGREES)C, ts 7 was unable to induce syncytia in fu-1 cells, and failed to compete with M-MuSV(M-MuLV) pseudotypes for TB receptors, indicating that the defect of the mutant involved the adsorption process. Biochemical studies demonstrated that ts 7 virions were deficient in the env gene proteins, and that this deficiency was the result of retarded intracellular cleavage of gPr80('env), the precursor to these proteins. No major differences were observed between the tryptic peptide maps of wild type (wt) M-MuLV and ts 7 gp70s and p15(E)s. However, the gag gene protein, p15, or ts 7, which migrated faster than the wt p15 on polyacrylamide gels, displayed an altered tryptic peptide map with respect to the wt protein, suggesting that altered p15 might be responsible for the ts 7 defect. These data offer a model for the role of p15 in the assembly of murine leukemia viruses, suggesting that in infected cells, p15 may influence both the processing of gPr80('env) and the subsequent incorporation of gp70 and p15(E) into budding virions.
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