Nectin-2-mediated entry of a syncytial strain of herpes simplex virus via pH-independent fusion with the plasma membrane of Chinese hamster ovary cells

Background: Herpes simplex virus ( HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary ( CHO) cells that express glycoprotein D ( gD)- binding receptors are model cells that support a pH- dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without ( FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway. Results: Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH- independent entry at the plasma membrane. In addition, CHO- nectin-2 cells supported pH- dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid ( t(1/2) of 5-10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO. Conclusion: HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.