It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type–specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.
It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defi ned. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and traffi cking of the viri ons in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type-specifi c. Clathrin-coated vesicles had no detectable role in virion traffi cking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.C. Clement and V. Tiwari contributed equally to this work.Correspondence to Deepak Shukla: dshukla@uic.edu Abbreviations used in this paper: CHO, Chinese hamster ovary; CF, corneal fi broblasts; CM, confocal microscopy; Cyto D, cytochalasin D; EEA1, early endosome antigen 1; HS, heparan sulfate; HSK, herpetic stromal keratitis; HSPG, heparan sulfate proteoglycans; HSV-1, herpes simplex virus-1; Lat B, latrunculin B; nectin-1-EGFP, nectin-1 with enhanced green fl uorescent protein; TM, trabecular meshwork; TEM, transmission electron microscopy; SEM, scanning electron microscopy.The online version of this article contains supplemental material.
The human eye is an important target for infection with herpes simplex virus 1 (HSV-1). Damage to cells forming the trabeculum of the eye by HSV-1 infection could contribute to the development of glaucoma, a major blinding disease. Primary cultures of human trabecular meshwork cells were used as an in vitro model to demonstrate the ability of HSV-1 to enter into and establish a productive infection of the trabeculum. Blocking of entry by anti-herpesvirus entry mediator (HVEM) antibody implicated HVEM as the major receptor for HSV-1 infection.The trabecular meshwork (TM), a specialized eye tissue, is the major site for regulation of the aqueous humor outflow. Inflammation of cells in the TM (trabeculitis) due to herpes simplex virus 1 (HSV-1) infection during corneal endotheliitis and uveitis leads to elevated intraocular pressure, which is a major risk factor for glaucoma, a major blinding disease (1,19,21,25,32). Recently, immunoreactivity for HSV-1 in the trabeculum of the human eye was demonstrated during corneal endotheliitis (1, 19). HSV-1-induced inflammation of human TM cells impedes aqueous outflow and increases intraocular pressure, as has also been reported with Posner Schlossman syndrome (11, 38). The virus has been detected in aqueous humor (40), tears (14), and ciliary ganglia (2) that innervate the cells of the trabeculum. It is, however, ironic that the ability of the virus to invade and productively infect TM cells remains poorly understood and that several aspects of the virus, including the identity of the mediators of the virus entry into these cells, remain unknown. Since HSV-1 infection of TM cells is a risk factor for glaucoma and uveitis, understanding HSV-1 entry and major entry mediators in TM cells becomes important for designing novel strategies to prevent blindness. Therefore, using primary cultures of human TM cells as a model, the present study was undertaken to determine the susceptibility and the mediator(s) of productive HSV-1 entry into TM cells.HSV entry into cells is a complex process that is initiated by specific interaction of viral envelope glycoproteins and host cell surface receptors (31). The virus uses glycoproteins B and C (gB and gC, respectively) to mediate the initial attachment to cell surface heparan sulfate proteoglycans (12,27). Binding of herpesviruses to heparan sulfate proteoglycans likely precedes a conformational change that brings viral glycoprotein D (gD) to the binding domain of host cell surface gD receptors (7,8,15). Thereafter, a concerted action involving gD, its receptor, three additional HSV glycoproteins (gB, gH, and gL), and possibly an additional gH coreceptor triggers fusion of the viral envelope with the plasma membrane of host cells (22,23,26). To date, three classes of HSV entry receptors have been identified (30, 31). They include herpesvirus entry mediator (HVEM) (20), a member of the tumor necrosis factor receptor family (17); nectin-1 and nectin-2 (3, 5, 10), two members of the immunoglobulin superfamily; and specific sites in heparan su...
Findings report that nectin-1 is widely expressed in murine ocular tissues. Only fibroblasts in the corneal stroma and sclera of uninfected tissues were devoid of nectin-1 expression. HSV-1-infected inflamed corneas contained some stromal fibroblasts with detectable nectin-1 expression, which potentially could be targeted by the virus. Widespread nectin-1 expression in the eye suggests that this receptor may play a role in the pathogenesis of ocular HSV infections.
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