Related Experiment Videos
The effect of glucocorticoid on the subcellular localization, oligomerization, and processing of mouse mammary tumor
1Department of Pathology, University of Southern California Health Sciences Center, Los Angeles 90033.
Abstract:
Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Glucocorticoids induce mouse mammary tumor virus envelope proteins gp52 and gp33 from precursor Pr74 in W7MG1 cells, but this processing is inefficient and requires new gene expression. The hormone promotes conversion by enabling new gene expression, not by inhibiting existing genes.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Mouse lymphoma W7MG1 cells infected with mouse mammary tumor virus produce precursor Pr74.
- Mature viral envelope proteins gp52 and gp33 are derived from Pr74 via posttranslational processing.
- Hormonal induction by glucocorticoids is necessary for efficient processing.
Purpose of the Study:
- To investigate the mechanism and efficiency of Pr74 processing to gp52 and gp33 in W7MG1 cells.
- To determine the role of glucocorticoids in Pr74 posttranslational modification.
- To elucidate the subcellular localization and molecular state of Pr74 and its processed forms.
Main Methods:
- Cell culture with and without glucocorticoid agonists.
- Pulse-chase labeling and analysis of protein processing.
- RNA synthesis inhibition studies.
- Subcellular fractionation and coimmunoprecipitation.
- Analysis of Pr74 aggregation state.
Main Results:
- Glucocorticoid induction of gp52 and gp33 in W7MG1 cells is inefficient, requiring at least 2 hours of exposure.
- Hormonal induction of gp52 and gp33 requires new gene expression, as indicated by inhibition with RNA synthesis inhibitors.
- Pr74 localizes to the ER, while gp52 and gp33 are found in the Golgi and plasma membrane.
- Pr74 in W7MG1 cells forms heterogeneous aggregates and binds BiP/GRP78, unlike Pr74 in more efficient GR3A cells.
Conclusions:
- Glucocorticoid treatment promotes the expression of new genes essential for Pr74 processing into gp52 and gp33.
- The inefficient processing in W7MG1 cells is associated with Pr74 aggregation and ER retention.
- Subcellular localization and protein aggregation play roles in the posttranslational processing of viral glycoproteins.