αvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

Campadelli-Fiume G (2013) avb6- and avb8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion. PLoS Pathog 9(12): e1003806. doi:10.1371/journal.ppat.1003806 avb6- and avb8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion Tatiana Gianni 0 Stefano Salvioli 0 Liudmila S. Chesnokova 0 Lindsey M. Hutt-Fletcher 0 Gabriella Campadelli-Fiume 0 Rozanne M. Sandri-Goldin, University of California, Irvine, United States of America 0 1 Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum-University of Bologna , Bologna , Italy , 2 Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center , Shreveport, Louisiana , United States of America Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as EpsteinBarr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that avb6- and avb8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which avb6-integrin was expressed in integrin-negative cells. avb6- and avb8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of avb8, but not avb6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of avb6- or avb8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion. - Funding: This work was supported by the Italian Association for Cancer Research (AIRC), Milan, Italy; by grants from the Department of Experimental Pathology, University of Bologna (Pallotti funds); by the University of Bologna RFO (Ricerca Fondamentale Orientata); the Italian Ministry for University and Research (PRIN project) and by Public Health Service grant DE016669 (to LMHF). 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 declared that no competing interests exist. The glycoproteins of enveloped virions fulfill three major functions to enable virus entry into target cells; the attachment of virions to cells, a step that partly determines the type of cells that the virus targets, hence the viral tropism; the triggering of fusion, i.e. the activation of the fusion machinery, and the execution of fusion. For a number of viruses, a fourth event occurs between these steps, virion internalization by endocytosis, or macropinocytosis. The domains responsible for all these activities are often localized in one or two glycoproteins; this is the case for example for ortho-, paramyxo- and retroviruses. Virion glycoproteins can be considered ready-to-use machines that need to undergo a transition in conformation from the metastable fusion-inactive to the fusionactive form, in order to induce the merging of the two membranes that of the virion and that of cell - so that lipids are mixed and fusion is executed [1]. A fundamental aspect of the process is that the steps are sequentially ordered and coordinated, to ensure that the glycoprotein transition takes place only after the virus has attached to the cells. Indeed, a premature activation would irreversibly exhaust the fusogenic potential of the virion glycoproteins, and lead to failure to infect. A key question is therefore how the timing of glycoprotein transition and activation is controlled. Essentially, there are two strategies. Either the glycoprotein transition is dependent on the glycoprotein encounter with the cognate cellular receptor, or on the low pH of the endosomal compartment. These levels of control guarantee that the virion fusion machinery is only active after the virus has attached to cells, or, for those viruses which undergo internalization, after they have been endocytosed and the endosomal pH has been lowered. According to this view, two major functions of cellular receptors are determination of viral tropism and triggering of fusion. Herpes simplex virus (HSV), and herpesviruses in general, exhibit a high level of complexity since they encode a multipartite entry/ In order to infect their hosts and cause disease, viruses must enter their host cells. The human pathogen herpes simplex virus (HSV) - and herpesviruses in general - are equipped with a complex, multipartite entry apparatus, made of four glycoproteins gD, gH/gL, gB. These glycoproteins must be activated in a timely, coordinated manner. According to the current model, the flux of activation goes from receptor-bound gD, to gH/gL and gB. The premature activation, and hence exhaustion of the glycoproteins must also be prevented. We report on a checkpoint at the gH/gL level. Specifically, avb6- and avb8integrins serve as receptors for HSV entry into keratinocytes and other epithelial and neuronal cells. Both bind gH/gL with high affinity. The interaction profoundly affects the pathway of HSV entry, promoting HSV endocytosis into acidic endosomes. For avb8-integrin, the portal of entry is at lipid microdomains and requires dynamin 2. We propose that, by placing the activation of gH/gL under control of an integrin trigger point, HSV can synchronize virion endocytosis with the cascade of activation that culminates in the execution of fusion between the virion envelope and cellular membranes. fusion machinery [2,3]. Some of the herpesvirus glycoproteins are species-specific; they play a role in the initial steps of virus entry and interact with receptors that belong to variety of molecular (...truncated)