Title Epidermal Growth Factor Receptor-PI 3 K Signaling Controls CofilinActivity To Facilitate Herpes Simplex Virus 1 Entry into Neuronal Cells

Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases. IMPORTANCE The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis. Received 18 November 2013 Accepted 19 December 2013 Published 14 January 2014 Citation Zheng K, Xiang Y, Wang X, Wang Q, Zhong M, Wang S, Wang X, Fan J, Kitazato K, Wang Y. 2014. Epidermal growth factor receptor-PI3K signaling controls cofilin activity to facilitate herpes simplex virus 1 entry into neuronal cells. mBio 5(1):e00958-13. doi:10.1128/mBio.00958-13. Invited Editor Carolyn Coyne, University of Pittsburgh School of Medicine Editor Glen Nemerow, The Scripps Research Institute Copyright © 2014 Zheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Yifei Wang, twang-yf@163.com, or Kaio Kitazato, kkholi@nagasaki-u.ac. jp. Viruses have evolved a variety of strategies to interact with host cells to initiate infection. They trigger their own cellular signaling pathways and depend greatly on the nature and physiological state of the host cells and on the interaction of the viruses with their receptors (1, 2). Because the actin cytoskeleton is a dynamic assembly of structures and is involved in many crucial cellular processes, it is not surprising that many viruses interact with actin and actin-regulating signaling pathways within the host cell (3, 4). Co-opting actin dynamics is fundamental to the diverse steps of herpesvirus biology, including entry, intracellular movement, and intercellular spreading (5). Herpes simplex virus 1 (HSV-1), a member of the alphaherpesvirus subfamily, is typically responsible for complications ranging from mucosal lesions to deadly brain infections (e.g., herpes simplex encephalitis [HSE]) (6). Infection by HSV-1 induces neurite damage and neuronal death, and virus-induced neuronal cytoskeletal disruption is the suggested cause of this neurodegeneration. Moreover, HSV-1 has been demonstrated to utilize host actin for the infection of neuRESEARCH ARTICLE January/February 2014 Volume 5 Issue 1 e00958-13 ® mbio.asm. org 1 rons, which are the cells in which the virus establishes latency and reactivates under specific conditions (7). The role of actin during HSV-1 entry appears to be correlated with the entry mechanism and the type of cell infected, and distinct downstream signaling events have been observed (8–10). Cortical actin is the first obstacle encountered by the virus upon infection. Upon viral binding to the cellular receptor, different kinases such as Rho GTPase were triggered to modulate the cytoskeleton. In addition, efficient virus entry was achieved by the active remodeling of dynamic actin through the induction of membrane-associated receptor clustering (11, 12) and promotion of virus surfing, a process in which the virus benefits from the actin-driven mobility of these receptors to reach subcellular sites suitable for membrane fusion (13). For example, studies with human immunodeficiency virus (HIV) have demonstrated that binding of the HIV-1 gp120 protein to CD4 induces a signaling cascade that triggers actin reorganization and actin-dependent clustering of HIV-1 entry receptors to establish signaling platforms for fusion pore formation (14–16). Furthermore, multiple receptor-induced host signal molecules have been subverted by viruses to promote infection. However, in contrast to Kaposi’s sarcoma-associated herpesvirus (17), very limited information is available on the cellular signaling pathways involved in the ability of HSV-1 to induce a favorable environment for entry via changes in the actin cytoskeleton. In particular, the mechanism by which HSV-1 regulates host cytoskeleton rearrangement and enters neuronal cells remains uncertain, particularly when considering that F-actin dynamics may contribute to the pathogenesis of neurological diseases and that viral entry into host cells is the most critical step for the production of virus-induced life-threatening complications (18). In the present study, we examined the process by which HSV-1 enters neuronal cells. The results indicate that cofilin (CFL), a member of the actin depolymerizing factor family, which is important for actin dynamics and cell migration (19, 20), initiates virus infection. The HSV-1 entry-induced early biphasic dynamics of F-actin and cofilin regulation were responsible for mediating both the assembly and disassembly of F-actin. Specifically, epidermal growth factor receptor (EGFR)–phosphatidylinositide-3 kinase (PI3K) signals participated in the HSV-1-mediated early phosphorylation (inactivation) of cofilin, which triggered events facilitating actin-based receptor clustering and virus entry. We also demonstrated that extracellular signal-regulated kinase 1 (ERK) and ERK2 (Erk1/2) acted as a downstream effector controlling cofilin activity and cell ruffle formation. HSV-1 has thus evolved an intricate mechanism for infecting neuronal cells (see Fig. 8).