Sol

Sol., soluble; Ab, antibody. ACKNOWLEDGMENTS We thank Gabriele Drescher for technical assistance with the preparation of samples for electron microscopy. for some retroviruses (1), or with endosomal membranes after endocytosis, as is the case for influenza computer virus (2). The same computer virus can enter different cell types either by direct fusion at the cell surface or by the endocytic route, where the latter can be pH dependent or pH impartial. At the same time, one cell type may allow initiation of contamination by different entry pathways for related or unrelated viruses (3C5). Alphaherpesviruses have been shown to enter cells by a number of different pathways that, with a few notable exceptions that include varicella zoster computer virus, are dependent on the same subset of viral glycoproteins, namely, glycoprotein D (gD), gB, gH, and gL, as well as cellular receptors and coreceptors (3, 6C9). Previous studies with herpes simplex virus type 1 (HSV-1) have shown that the computer virus can enter many cell types, including primary neurons and Vero cells, via fusion with the plasma membrane at neutral pH (10C12). Furthermore, HSV-1 can enter other cell types, such as HeLa and CHO cells, through a pH-dependent endocytic pathway, while it enters C10 (mouse melanoma cells expressing nectin 1) through a pH-independent endocytic pathway (13C15). In addition, phagocytosis-like uptake through macropinocytosis has been suggested for nectin 1-expressing CHO cells (16). Recently, it has been shown that V3 integrin determines the entry pathway of HSV-1 into cells. In the presence of V3 integrin, HSV-1 enters nectin 1-expressing CHO cells through a pathway dependent on lipid rafts, dynamin II, and acidic pH that is impartial of caveolin 1 (Cav-1) (17). The effect of V3 integrins on entry seems to be dependent on their ability to relocalize the nectin 1 receptor to lipid rafts independently of computer virus binding (18). Equine herpesvirus type 1 (EHV-1) and (S)-(-)-5-Fluorowillardiine EHV-4 are members of the subfamily and are assigned to the genus (19). Although the two viruses are highly comparable in terms of genetic and antigenic structure, differences in cell tropism, host range, and clinical disease are well known (20C22). As is the case with HSV-1, EHV-1 can enter some cells, such as rabbit kidney (RK13) and equine dermal (ED) cells, through direct fusion with the plasma membrane at neutral pH, a process that is mediated by gC, gD, gB, and the gH/gL complex (23C25). In addition, EHV-1 can enter CHO-K1 cells, peripheral blood mononuclear cells, and equine brain microvascular endothelial cells through pH-dependent or -impartial endocytic pathways (26C28). However, the viral and cellular factors that govern the entry process and route viruses to various compartments are still unknown. Integrins are cell surface proteins that can trigger endocytosis and mediate cell-cell and cell-matrix adhesion (29). Several viruses, including some herpesviruses, utilize integrins for entry into cells, and examples include Epstein-Barr virus (EBV) (30), human cytomegalovirus (HCMV) (31), and Kaposi’s sarcoma-associated herpesvirus (KSHV) (32). Recently, we showed that different integrins, including V3, V5, 41, and 47, have no measurable effect on EHV-1 or EHV-4 infection (20, 33). Integrin interaction with extracellular matrix proteins lead to a series of signaling events that involve the activation of focal adhesion kinase, c-Src kinase, phosphatidylinositol 3-kinase, and cytoskeletal proteins such as paxillin (26, 29, 34, 35). Here, we address the entry of two alphaherpesviruses into cells where gH and integrins apparently play a decisive role in the choice of the entry route. We make use of fluorescently labeled (mutant) viruses, inhibitors of different cellular functions, and confocal microscopy combined with electron microscopy to identify virus-containing compartments. Our results indicate that EHV-1 and EHV-4 employ different entry pathways during infection of epithelial (ED) cells although utilizing the same receptor, major histocompatibility complex class I (MHC-I), in either case. EHV-1 enters equine epithelial cells via fusion at the plasma membrane and EHV-4 fuses with (S)-(-)-5-Fluorowillardiine the membrane of an endocytic vesicle. Replacement of EHV-1 gH with that of EHV-4 redirects EHV-1 into an endocytic pathway that is dependent on dynamin II,.Acad. mutation in the SDI integrin-binding motif of EHV-1 gH also directed EHV-1 to the endocytic pathway. Cumulatively, we show that viral gH and cellular 41 integrins are important determinants in the choice of alphaherpesvirus cellular entry pathways. INTRODUCTION Viruses are obligatory intracellular organisms that attach to and then enter cells in order to establish infection. For enveloped viruses, productive entry into cells is mediated by fusion either with the plasma membrane, as is the case for (S)-(-)-5-Fluorowillardiine some retroviruses (1), or with endosomal membranes after endocytosis, as is the case for influenza virus (2). The same virus can enter different cell types either by direct fusion at the cell surface or by the endocytic route, where the latter can be pH dependent or pH (S)-(-)-5-Fluorowillardiine independent. At the same time, one cell type may allow initiation of infection by different entry pathways for related or unrelated viruses (3C5). Alphaherpesviruses have been shown to enter cells by a number of different pathways that, with a few notable exceptions that include varicella zoster virus, are dependent on the same subset of viral glycoproteins, namely, glycoprotein D (gD), gB, gH, and gL, as well as cellular receptors and coreceptors (3, 6C9). Previous studies with herpes simplex virus type 1 (HSV-1) have shown that the virus can enter many cell types, including primary neurons and Vero cells, via fusion with the plasma membrane at neutral pH (10C12). Furthermore, HSV-1 can enter other cell types, such as HeLa and CHO cells, through a pH-dependent endocytic pathway, while it enters C10 (mouse melanoma cells expressing nectin 1) through a pH-independent endocytic pathway (13C15). In addition, phagocytosis-like uptake through macropinocytosis has been suggested for nectin 1-expressing CHO cells (16). Recently, it has been shown that V3 integrin determines the entry pathway of HSV-1 into cells. In the presence of V3 integrin, HSV-1 enters nectin 1-expressing CHO cells through a pathway dependent on lipid rafts, dynamin II, and acidic pH that is independent of caveolin 1 (Cav-1) (17). The effect of V3 integrins on entry seems to be dependent on their ability to relocalize the nectin 1 receptor to lipid rafts independently of virus binding (18). Equine herpesvirus type 1 (EHV-1) and EHV-4 are members of the subfamily and are assigned to the genus (19). Although the two viruses are highly similar in terms of genetic and antigenic structure, differences in cell tropism, host range, and clinical disease are well known (20C22). As is the case with HSV-1, EHV-1 can enter some cells, such as rabbit kidney (RK13) and equine dermal (ED) cells, through direct fusion with the plasma membrane at neutral pH, a process that is mediated by gC, gD, gB, and the gH/gL complex (S)-(-)-5-Fluorowillardiine (23C25). In addition, EHV-1 can enter CHO-K1 cells, peripheral blood mononuclear cells, and equine brain microvascular endothelial cells through pH-dependent or -independent endocytic pathways (26C28). However, the viral and cellular factors that govern the entry process and route viruses to various compartments are still unknown. Integrins are cell surface proteins that can trigger endocytosis and mediate cell-cell and cell-matrix adhesion (29). Several viruses, including some herpesviruses, utilize integrins for entry into cells, and examples include Epstein-Barr virus (EBV) (30), human cytomegalovirus (HCMV) (31), and Kaposi’s sarcoma-associated herpesvirus (KSHV) (32). Recently, we showed that different integrins, including V3, V5, 41, and 47, have no measurable effect on EHV-1 or EHV-4 infection (20, 33). Integrin interaction with extracellular matrix proteins lead to a series of signaling events that involve the activation of focal adhesion kinase, c-Src kinase, phosphatidylinositol 3-kinase, and cytoskeletal proteins such as paxillin (26, 29, 34, 35). Here, we address the entry of two alphaherpesviruses into cells where gH and integrins apparently play a decisive role in the choice of the entry route. We make use of fluorescently labeled (mutant) viruses, inhibitors of different cellular functions, and confocal microscopy combined with electron microscopy to identify virus-containing compartments. Our results indicate that EHV-1 and EHV-4 employ different entry pathways during infection of epithelial (ED) cells although utilizing Comp the same receptor, major histocompatibility complex class I (MHC-I), in either case. EHV-1 enters equine epithelial cells via fusion at the plasma membrane and EHV-4 fuses with the membrane of an endocytic vesicle. Replacement of EHV-1 gH with that of EHV-4 redirects EHV-1 into an endocytic pathway that is dependent on dynamin II, cholesterol, and tyrosine kinase activity. Blocking of 41 integrins on the surface of equine epithelial cells also redirects EHV-1 to the same endocytic pathway. In all cases, we find caveolae to be the main viral entry port. MATERIALS.