Analysis of the conformational changes of viral glycoprotein that is involved in inducing syncytium formation.

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As reported previously, the fusion protein (F) of simian virus 5 (SV5) strain WR induces cell fusion only when coexpressed with the hemagglutinin-neuraminidase protein (HN), while the F of SV5 strain W3A can induce cell fusion independently of HN. When Leu-22 in WR F is replaced with the counterpart (Pro-22) in W3A F, the resulting L22P mutant is able to induce the HN-independent cell fusion. In the present study, I have shown that there is difference in conformation between L22P and WR F, in which the epitope in WR F for monoclonal antibody (MAb) 21-1 is cryptic while it is exposed in L22P. Furthermore, a 36-kDa polypeptide was discovered in the L22P-expressing HeLa cells but not in the WR F-expressing HeLa cells, which proved to be derived from the L22P that has been transported to the cell surface. The cell surface-localized L22P but not WR F was internalized into the cell in a cholesterol dependent manner as analyzed by biotin internalization assay. Furthermore, the 36-kDa polypeptide was not generated when the cells were treated with bafilomycin A1. These results suggest that the 36-kDa polypeptide is derived from the L22P which has undergone endocytosis and has been degraded in the lysosome. By heating at 47℃ for 2 min, WR F acquired the HN-independent cell fusion activity and underwent endocytosis, suggesting that an internalization signal is displayed by the WR F that is in the "postfusion" conformation. The heat treatment also resulted in an exposure of the MAb 21-1 epitope in WR F, suggesting that the head/neck domain of F, which harbors the MAb 21-1 epitope, undergoes a conformational change during the induction of cell fusion.