Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

RESEARCH ARTICLE Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells Raquel Bello-Morales1,4, Antonio Jesús Crespillo1, Beatriz Praena1, Enrique Tabarés2, Yolanda Revilla3, Elena García3, Alberto Fraile-Ramos4, Wia Baron5, Claude Krummenacher6, José Antonio López-Guerrero1* 1 Universidad Autónoma de Madrid, Departamento de Biología Molecular, Edificio de Biología, Darwin 2, Cantoblanco, 28049, Madrid, Spain, 2 Universidad Autónoma de Madrid, Facultad de Medicina, Arzobispo Morcillo 4, 28029, Madrid, Spain, 3 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 5, Cantoblanco, 28049, Madrid, Spain, 4 Universidad Complutense de Madrid, Facultad de Medicina, Plaza de Ramón y Cajal, s/n Ciudad Universitaria, 28040, Madrid, Spain, 5 University of Groningen, Faculty of Medical Sciences, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands, 6 Department of Biological Sciences and Department of Biomedical and Translational Sciences, Rowan University, Glassboro, NJ, 08028, United States of America * OPEN ACCESS Citation: Bello-Morales R, Crespillo AJ, Praena B, Tabarés E, Revilla Y, García E, et al. (2016) Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells. PLoS ONE 11(1): e0147885. doi:10.1371/ journal.pone.0147885 Editor: Deepak Shukla, University of Illinois at Chicago, UNITED STATES Received: November 4, 2015 Accepted: January 8, 2016 Published: January 25, 2016 Copyright: © 2016 Bello-Morales et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus–unable to replicate in ICP4-defficient cells- showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells. Data Availability Statement: All relevant data are contained within the paper. Funding: Financial support for the study was provided by Plan Nacional de I+D+I SAF2012-40023 Subprograma de Proyectos de Investigación Fundamental, Ministerio de Economía y Competitividad and BIO2013-46605-R Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad. A F-R and R B-M were supported by grant BFU2012-35067 (Ministerio de Economía y Competitividad). The funders had no role in study design, data collection Introduction Herpes simplex virus type 1 (HSV-1) is a highly prevalent human pathogen belonging to the neurotropic alphaherpesviruses. HSV-1 infects epithelial cells and establishes latency in neurons in sensory ganglia [1, 2], but is also capable of spreading to the central nervous system (CNS) and causing meningitis or encephalitis [3]. Heparan sulfate glycosaminoglycans act as attachment receptors for the viral glycoprotein gC [4]. Although gC is not essential for viral entry, its absence decreases infectivity, due to a reduced efficiency of viral binding to cells [5]. In the absence of gC, gB can mediate attachment PLOS ONE | DOI:10.1371/journal.pone.0147885 January 25, 2016 1 / 13 Role of PLP in HSV-1 Oligodendrocytic Infection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal's policy and have the following conflicts: C. Krummenacher is an Academic Editor for PLOS One. to heparan sulfate [3]. Four viral glycoproteins, gB, gD, gH, and gL are necessary for viral entry into cells [5, 6]. HSV gD binding to its receptors triggers the viral membrane fusion process which requires the heterodimer gH/gL and the fusion protein gB. Fusion of the viral envelope may occur with the plasma membrane in a pH-independent manner or with endosomal membrane after endocytosis [7, 8] to deliver the nucleocapsid and tegument into the cell cytoplasm. The major entry receptors for gD include HVEM [9], nectin-1 [10] and 3-O-sulfated heparan sulfate [11]. HVEM (herpesvirus entry mediator) is a member of the TNF receptor family expressed in several tissues and cell types, including epithelial cells, fibroblasts, monocytes and lymphocytes [9, 12, 13]. Nectins are molecules that mediate cell-cell adhesion in adherens junctions [14]. They are also expressed in a variety cell types, including epithelial cells, fibroblasts and neurons [15, 16]. A third viral receptor, 3-O-sulfated heparan sulfate, which appears when certain D-glucosaminyl-3-O-sulfotransferases modify heparan sulfate, has been shown to be active in CHO cells [11]. Other HSV-1 gB receptors, which have been found out recently, include paired immunoglobulin-like type 2 receptor (PILR) alpha [17] and myelin-associated glycoprotein (MAG) [18]. It has been recently reported that the interaction of HSV gH/gL heterodimer with its receptor αvβ6- or αvβ8-integrin results in the dissociation of gL from the heterodimer and its release in the medium, a process that requires the presence of gD, nectin1, and gB [19]. The broad range of animal species infectable by HSV-1 suggests that surface receptors for this virus are highly conserved or that different receptors might be used by HSV to enter different cell types [9, 20]. Indeed, data show that utilization of alternative receptors by HSV-1 is quite significant, since it can use different receptors according to the target cell [12]. Moreover, HSV-1 can also enter different cell types not only using different receptor, but also by different pathways: in many cultured cell lines, such as Vero and HEp-2, HSV-1 enters cells by a pHneutral fusion with the cell surface, but entry into HeLa and CHO-K1 cells does depend on endocytosis and subsequent exposure to a low pH [8]. Oligodendrocytes (OLs) are the glial cells that produce myelin,–the electrically insulating layer that surrounds axons [21, 22]–in the CNS [23]. Proteolipid protein (PLP), together with DM20, (...truncated)