The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells

et al. (2014) The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells. PLoS ONE 9(2): e89141. doi:10.1371/journal.pone.0089141 The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells Raquel Bello-Morales 0 Antonio Jesu s Crespillo 0 Beatriz Garca 0 Luis A ngel Dorado 0 Beatriz Martn 0 Enrique Tabare s 0 Claude Krummenacher 0 Fernando de Castro 0 Jose Antonio Lo pez-Guerrero 0 Deepak Shukla, University of Illinois at Chicago, United States of America 0 1 Universidad Auto noma de Madrid, Departamento de Biolog a Molecular , Edificio de Biolog a, Darwin 2, Cantoblanco, Madrid, Spain, 2 Centro de Biolog a Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, Madrid , Spain , 3 Universidad Auto noma de Madrid, Facultad de Medicina , Madrid , Spain , 4 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia Pennsylvania, United States of America, 5 Grupo de Neurobiolog a del Desarrollo-GNDe, Hospital Nacional de Paraple jicos , Toledo , Spain Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs. - Funding: The work was partially supported by the Ministerio de Economa y Competitividad-MINECO (SAF2012-40023 and RD12-0032-12 -partially funded by FEDER- European Union/Una manera de hacer Europa). FdeC is hired by Gobierno de Castilla-La Mancha-SESCAM. CK is supported by Public Health Service grant AI-097171 from the National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journals policy and have the following conflicts: Fernando de Castro and Claude Krummenacher are Academic Editors in PLOS One. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Several infectious agents, ranging from mycobacteria to retroviruses, have been proposed to be associated with demyelinating diseases such as Multiple Sclerosis (MS), in which oligodendrocytes (OLs), the myelin-forming cells in the central nervous system (CNS), may be the initial target for the pathogenic onset [1,2,3]. Of all studied organisms, members of the viral family Herpesviridae are among the most promising candidates [3,4,5,6,7,8]. In addition to other herpesviruses (for example Epstein-Barr virus or human herpesvirus 6), herpes simplex virus type 1 (HSV-1), has been linked to the possible aetiology or development of several neurodegenerative diseases and virusinduced demyelination [9,10,11,12]. Previous reports have shown that a human oligodendrocyte-derived cell line is highly susceptible to HSV-1 [13], and that the virus may play a role in triggering MS relapses during clinical acute attacks of MS, at least in the most frequent clinical presentation of the disease, the relapsing-remitting form. [14]. Besides neurodegenerative diseases, HSV-1 may also be involved in cognitive alterations in bipolar or schizophrenia dysfunctions [15]. Herpesviruses usually infect their hosts for life, after the initial infection of epithelial cells, the virions spread to neurons and establish latent infections in sensory ganglia [16]. In some cases, the virus spreads into the CNS to cause encephalitis or meningitis [17]. HSV-1 entry into a diverse range of cell types has been described [18]. The entry of HSV into various cell types follows a complex process [19,20]. The initial attachment of HSV-1 to the cell surface is mediated by glycoproteins B (gB) and C (gC). This interaction with heparan sulfate proteoglycans (HSPGs) enables the binding of viral gD to one of its receptors on the host cell surface. This binding triggers conformational changes in gD that allow the activation of gH/gL, which in turn activate the fusion effector gB [21,22]. Cellular proteins binding to HSV gB have also been identified but their roles in the entry process or in cell tropism remains unsolved [23,24,25]. Molecules derived from three structurally different groups have so far been described as gD receptors in the host, Herpes Virus Entry Mediator (HVEM), a member of the tumor necrosis factor receptor family, nectin-1 and 22 from the immunoglobulin superfamily and distinctive sites in heparan sulfate (HS) generated by a specific 3-O-sulfotransferase (3-OST) [26,27,28,29]. Nectin-1 and HVEM appear to be the principal gD-binding entry receptors although they bind distinct regions of the gD ligand [20]. They are coexpressed in many cells and used by the majority of tested clinical strains of HSV-1, as well as HSV-2 [30]. HVEM expression has been found in liver, kidney, lymphoid tissues, lung and in several cell lines. Nectin-1 is the main, although not exclusive, HSV receptor on epithelial and neuronal cells, whereas nectin-2 use seems to be limited to only few viral mutant strains [27,30,31,32,33]. It is worth noting that nectin-1 is an adhesion molecule present at adherent junctions in polarized cells, such as epithelial and neurons cells, and in cell-cell contact in some cultured cells [34]. 3-O-ST HS can be used as an entry receptor for HSV-1 but not HSV-2 in multiple cell lines like neuronal or endothelial cells [27,35]. Although in all cases, binding of gD to a specific receptor is required during HSV entry, membrane fusion can take place directly at the cell surface or, in some cases, following virus endocytosis. Why the virus chooses one or another pathway is largely unknown. However, studies with cell cultures of different origin SY5Y, HeLa or Vero cell lines suggest that nectin-1-mediated internalization may direct HSV to the endocytic pathway, possibly with the cooperation of integrins [36,37,38]. Finally, binding of H (...truncated)