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Simian virus 40 signalling promotes viral entry to caveolae andmAb against MHC class I induces adhesion of fibroblasts to T cells

Yuzhi Chen, University of Massachusetts Amherst


Simian virus 40 (SV40) entry through caveolae represents a novel form of endocytosis. SV40 translocation to caveolae was mainly examined by cell fractionation and electron microscopy (EM) in CV-1 African green monkey kidney fibroblast cells. SV40 translocated to the caveolin-enriched low density Triton-insoluble complexes as identified by Western blotting. SV40 partitioning into this fraction was inhibited by nystatin. EM analysis indicated that SV40 translocation to caveolae was prevented by the tyrosine kinase inhibitor genistein (thin sections), but SV40 was not blocked from entering the Triton-insoluble low density fraction (negative staining). Northern analysis suggested that an SV40 signal that upregulates c-myc is transmitted through caveolae. The role of the major histocompatibility complex (MHC) class I proteins was not clear in SV40 translocation to caveolae. This is partly because of the high background level of class I molecules in the low density fraction isolated by sodium carbonate sucrose gradients (LDF-C). Class I proteins were enriched in the LDF-C relative to the whole cell extract or to the plasma membrane isolated by silica coating. The clustering of class I molecules was independent of any induction. Indirect immunofluorescence labeling of class I proteins and caveolin indicated that class I molecules colocalize with caveolae. MHC class I signaling and its physiological relevance were analyzed using a monoclonal antibody (mAb) W6/32. By immunoprecipitation and thin layer chromatography, ras was found to be activated by W6/32. W6/32 did not seem to increase the tyrosine-phosphorylation level in phospholipase C-$\gamma1.$ Engagement of class I molecules did not seem to induce apoptosis, but resulted in a distinct physiological response, the adhesion strengthening in CV-1 cells to Jurkat cells. The adhesion strengthening seemed to be sensitive to the protein kinase C inhibitor calphostin C, but enhanced by the tyrosine kinase inhibitor genistein. Furthermore, the mAb-induced adhesion strengthening was transient, antibody-concentration dependent, but independent of the Fc portion of the mAb. A serum factor was found to inhibit the basal level of adhesion in CV-1 cells to Jurkat cells. These findings represent one of the first investigations of MHC class I signaling in non-lymphoid cell lines.

Subject Area

Cellular biology|Microbiology|Immunology

Recommended Citation

Chen, Yuzhi, "Simian virus 40 signalling promotes viral entry to caveolae andmAb against MHC class I induces adhesion of fibroblasts to T cells" (1997). Doctoral Dissertations Available from Proquest. AAI9809317.