Ultrathin sections of fibroblasts were cut, stained with uranyl acetate and lead citrate, and analyzed in a JEOL 1200 EX II

Ultrathin sections of fibroblasts were cut, stained with uranyl acetate and lead citrate, and analyzed in a JEOL 1200 EX II. Flow cytometric analysis. prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be EMT inhibitor-2 highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells, suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism suggests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts. IMPORTANCE Herpes simplex virus (HSV) is a human pathogen which infects its host via mucosal surfaces or abraded skin. To understand how HSV-1 overcomes the protective barrier of mucosa or skin and reaches its receptors in tissue, it is essential to EMT inhibitor-2 know which receptors contribute to the entry into individual skin cells. Previously, we have explored the contribution of nectin-1 and herpesvirus entry mediator (HVEM) as receptors for HSV-1 entry into murine epidermis, where keratinocytes form the major cell type. Since the underlying dermis consists primarily of fibroblasts, we have now extended our study of HSV-1 entry to dermal fibroblasts isolated from nectin-1- or HVEM-deficient mice or from mice deficient in both receptors. Our results demonstrate a role for both nectin-1 and HVEM as receptors and suggest a further receptor which appears much less efficient. INTRODUCTION To initiate infection, herpes simplex virus 1 (HSV-1) enters its human host via mucosal surfaces or abraded skin. HSV-1 entry into individual cells involves the interaction of several viral glycoproteins with various cell surface receptors (1, 2). Rabbit Polyclonal to TSPO The first step during entry is the attachment of virions to glycosaminoglycans, which facilitates the interaction with cellular receptors, leading to the fusion of the viral envelope with a EMT inhibitor-2 cellular membrane. Fusion can either occur with the plasma membrane or with vesicle membranes after virions are internalized via endocytosis (3, 4). Only after binding of the envelope glycoprotein D (gD) to a receptor is fusion with cellular membranes induced (5). The primary gD receptors mediating entry into mouse and human cells are nectin-1 and herpesvirus entry mediator (HVEM) (6,C8). The 3-O-sulfated heparan sulfate (3-OS-HS) represents a further gD receptor, which may also contribute to HSV-1 entry into various cell types (9, 10). How each of these receptors contributes to the entry process of HSV-1 into natural target sites such as skin or mucosa is not well understood. Since the absence of both nectin-1 and HVEM prevents HSV pathogenesis in the mouse model, nectin-1 and HVEM are reported to be the dominant functional gD receptors in the murine host (11,C13). Using nectin-1- or HVEM-deficient mice, we recently investigated HSV-1 entry into murine epidermis. Our infection studies identified nectin-1 as the major receptor in the epidermis, whereas HVEM has a more limited role (14). Since the epidermis represents only the outermost layer of skin and mucosa, we address here the contribution of nectin-1 and HVEM as receptors in the underlying dermis. Fibroblasts are the major resident cell type of the dermis, which is connected to the epidermis through the basement membrane, a specialized layer of extracellular matrix that anchors the keratinocytes (15). Nectin-1 is a Ca2+-independent immunoglobulin-like EMT inhibitor-2 cell-cell adhesion molecule involved in the formation of adherens junctions in epithelial cells and fibroblasts (16). In fibroblasts, nectin-1 is detectable at cell-cell adhesion sites and perhaps also diffusely distributed on the free surface of the plasma membrane of migrating cells (17). EMT inhibitor-2 As a member of the tumor necrosis factor receptor superfamily, HVEM can activate either proinflammatory or inhibitory signaling pathways (18). This receptor is expressed mainly by T lymphocytes but is also present on B cells, natural killer cells, dendritic cells, and fibroblasts (19,C21). HVEM is only expressed at low levels on human dermal fibroblasts (22). Using nectin-1- or HVEM-deficient murine dermal fibroblasts, we investigated the role of nectin-1 and HVEM as receptors for HSV-1 and characterized.