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Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not
N G Copeland1, N A Jenkins, B Nexø
1Bionetics Research, Inc., National Cancer Institute, Frederick, Maryland 21701.
Abstract:
DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction.
Insights
Murine leukemia virus Emv-3 expression in DBA/2 mice is regulated by a gag gene defect. This defect, a single nucleotide substitution, inhibits protein myristylation, crucial for virus particle formation and expression.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- DBA/2 mice possess an endogenous ecotropic murine leukemia provirus, Emv-3.
- Emv-3 expression is typically low in weanling and adult mice, with limited induction by standard chemical agents.
Purpose of the Study:
- To elucidate the molecular basis for the restricted expression of the Emv-3 provirus in DBA/2 mice.
- To identify the specific defect hindering Emv-3 replication and expression.
Main Methods:
- DNA sequencing and peptide mapping of Emv-3.
- Metabolic labeling with [3H]myristate to assess protein modification.
- Analysis of gag gene defect and its impact on Pr65gag myristylation.
Main Results:
- A single nucleotide substitution in the p15gag gene was identified as the cause of the Emv-3 defect.
- This mutation prevents N-terminal myristylation of the Pr65gag precursor protein.
- Myristylation is essential for Pr65gag association with the plasma membrane and virus particle formation.
Conclusions:
- The identified gag gene defect provides a molecular explanation for the low basal and induced expression of Emv-3.
- Understanding this defect offers insights into the regulation of murine leukemia virus expression and pathogenesis.