Fluorescence microscopy images showed a partial co-localization between dynamin and HSV-1 in HOG cells (Figure 4A), only during virus entry 20 min after adhesion, thus discarding the processes described above. cells. are one of the most studied families of viruses. HSV-1 is able to enter several host cell types using different strategies, an ability that might be due to the number of viral glycoproteins involved, the existence of multiple alternative cell receptors, and the varied strategies and mechanisms available [3]. Depending on the cell model, HSV-1 can enter via pH-independent fusion between viral lipid envelopes and host membranes, phagocytosis or endocytosis, or a combination of these routes [4,5]. In all cases, entry is mediated through the interaction of viral glycoproteins D (gD), B (gB), C (gC) and H/L (gH/L) with different Bentiromide cellular receptors [6]. These receptors are grouped according to their affinity to viral glycoproteins: gB and gC recognize and interact with heparan Bentiromide sulfate proteoglycans (HSPGs), whereas gD can recognize herpes virus entry mediator (HVEM), Nectin-1 and 2, or 3-O-sulfated heparan sulfate (3-OH-HS) [7]. Endocytosis begins with a Bentiromide weak interaction between various binding factors of the cell membrane and the envelope of HSV-1, followed by a specific recognition between one or more cell receptors and the viral glycoproteins gD and gH/gL [8]. Multivalent binding leads to a grouping of receptors which can give rise to an association with lipid domains and activation of several signaling pathways [9]. In particular during HSV-1 infection, the first step during adsorption is the recognition of heparan sulfate (HS) moieties by viral gB or gC [10]. Adsorption triggers internalization through endocytosis, which Rabbit polyclonal to ABCA6 may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolin- or flotillin-mediated endocytosis, or a variety of other poorly characterized mechanisms (such as lipid raft-dependent mechanisms) [11]. CME is one of the most well studied and characterized pathways and is a commonly used mechanism for viral entry into cells. Furthermore, in response to different cellular signals, cytoplasmic clathrin molecules are recruited to the membrane where the entry process takes place [12], forming Bentiromide clathrin-coated pits (CCPs) which induce a curvature in the membrane, leading to vesicle formation. Dynamin is a GTPase required for constriction of the vesicle and its excision into the cell cytoplasm. The implication of dynamin in viral endocytosis has been widely accepted [13,14,15], and though there exist endocytic pathways that lack this protein, it is Bentiromide crucial in CME. This dynamin belongs to the fission group, which is cytosolic proteins that are recruited peripherally and reversibly to membranes in the endocytosis pathway [16]. Another common process ending with the formation of a primary vesicle involves lipid rafts and cholesterol as well as phosphatases and kinases [9]. Lipid rafts present in the cell form specialized membrane microdomains enriched in cholesterol and sphingolipids that can alter the fluidity of the membrane, receptor clustering and the assembly of signaling molecules [17]. In the clathrin-independent case of lipid raft-dependent endocytosis [18], dynamin may or may not be necessary, although, in some cases, caveolin can be part of this alternative entry process (caveolin-mediated endocytosis) [19]. In fact, caveolin is a transmembrane protein that clusters in lipid rafts, so unlike clathrin, it does not need to be recruited from the cellular cytoplasm [20]. In this work, we have studied the roles of clathrin, caveolin and dynamin in the endocytic entry of HSV-1 into the established human oligodendroglial (HOG) cell line. Dynamin and clathrin might play significant roles in endocytosis, while caveolin does not appear to be involved in the process. 2. Materials and Methods 2.1. Cell Culture The human HOG cell line [21] was established from a surgically removed human oligodendroglioma and kindly provided by A. T. Campagnoni (University of California, Los Angeles, USA). Cells were cultured in growth medium (GM) containing low-glucose Dulbeccos Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), penicillin (50 U/mL) and streptomycin (50 g/mL) at 37 C in a humidified atmosphere of 5% CO2. To induce differentiation, cells were cultured in serum-free differentiation medium (DM), containing low-glucose DMEM.