Journal of Cell Science, Vol 109, Issue 6 1585-1596, Copyright © 1996 by Company of Biologists

JOURNAL ARTICLES

Transport pathway, maturation, and targetting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers

P Rahkila, A Alakangas, K Vaananen and K Metsikko
Biocenter, University of Oulu, Finland.

We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39 degrees C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 10(9)/ml. When we infected the myofibers with the tsO45 mutant at 39 degrees C, the viral glycoprotein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32 degrees C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39 degrees C to 20 degrees C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32 degrees C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endoglycosidase H-sensitive, suggesting its retention in the endoplasmic/sarcoplasmic reticulum endomembranes.
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