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Control of IFN-inducible MxA gene expression in human cells
1Molecular Biology Programme, National Public Health Institute, Helsinki, Finland.
Abstract:
MxA is an IFN-induced human protein which is located in the cytoplasm of induced cells. MxA makes the cells resistant to infection by influenza and vesicular stomatitis viruses. In the present work we used baculovirus expression system to produce MxA protein. The protein was purified to homogeneity and highly specific polyclonal anti-MxA antibodies were prepared. In human mononuclear cells, and A549 lung carcinoma cells expression of MxA protein is induced by very low (< 1 IU/ml) doses of leukocyte IFN-alpha (nIFN-alpha), whereas IFN-gamma does not seem to induce it or potentiate the induction by IFN-alpha. In mononuclear cells stimulated with high doses of leukocyte IFN-alpha concentrations, the amount of MxA mRNA was induced 10-fold at 4 h after IFN induction and up to 10-fold higher MxA protein levels were observed at 24-48 h postinduction. The gene can be reinduced by IFN-alpha 24 h after the initial induction suggesting for a lack of negative feedback after this time point. The protein is very stable, the half-life being approximately 2.3 days. Flow cytometric analysis revealed that monocytes have higher basal and induced MxA protein levels than lymphocytes but the dose-dependency of MxA expression is very similar in both cell types. Granulocytes are producing very low amounts of MxA protein.
Insights
MxA protein, induced by low-dose interferon-alpha (IFN-α), confers resistance to viral infections like influenza. This stable protein shows dose-dependent expression in human cells, with monocytes expressing higher levels than lymphocytes.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- MxA is an interferon (IFN)-induced protein found in the cytoplasm.
- MxA confers cellular resistance against viruses such as influenza and vesicular stomatitis virus.
Purpose of the Study:
- To produce and characterize MxA protein using a baculovirus expression system.
- To investigate the induction of MxA protein expression by different types of interferon (IFN) in human cells.
Main Methods:
- Baculovirus expression system for MxA protein production and purification.
- Generation of specific polyclonal anti-MxA antibodies.
- Flow cytometry for MxA protein level analysis in human mononuclear cells and A549 lung carcinoma cells.
Main Results:
- MxA protein expression induced by low-dose leukocyte IFN-alpha (< 1 IU/ml) in human mononuclear and A549 cells; IFN-gamma showed no induction.
- IFN-alpha induced a 10-fold increase in MxA mRNA at 4 hours and up to 10-fold higher MxA protein levels at 24-48 hours.
- MxA protein is stable with a half-life of approximately 2.3 days; monocytes showed higher MxA levels than lymphocytes.
Conclusions:
- Leukocyte IFN-alpha is a potent inducer of MxA protein expression in human cells.
- MxA protein plays a significant role in cellular antiviral defense.
- The stability and differential expression of MxA protein in various immune cells highlight its importance in the innate immune response.