Reviews in Medical Virology

REVIEW

Rev. Med. Virol. 2014; 24: 274–286. Published online 29 May 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rmv.1796

Viruses exploit the function of epidermal growth factor receptor Kai Zheng1,2, Kaio Kitazato3* and Yifei Wang1** 1

Guangzhou Jinan Biomedicine Research and Development Center, National Engineering, Research Center of Genetic Medicine, Jinan University, Guangzhou, China 2 College of Life Science and Technology, Jinan University, Guangzhou, China 3 Division of Molecular Pharmacology of Infectious Agents, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

S U M M A RY Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that regulates cellular homeostatic processes. Following ligand binding, EGFR activates different downstream signalling cascades that promote cell survival, proliferation, motility, and angiogenesis and induces F-actin-dependent EGFR endocytosis, which relocalises the activated receptors for degradation or recycling. The responses that are induced by ligand binding to EGFR, including cell signalling activation, protein kinase phosphorylation and cytoskeletal network rearrangement, resemble those induced by virus infection. Increasing evidence demonstrates that many viruses usurp EGFR endocytosis or EGFR-mediated signalling for entry, replication, inflammation, and viral antagonism to the host antiviral system. In addition, viruses have acquired sophisticated mechanisms to regulate EGFR functions by interrupting the EGFRrecycling process and modulating EGFR expression. In this review, we provide an overview of the mechanisms by which viruses alter EGFR signalling in favour of their continued survival. Copyright © 2014 John Wiley & Sons, Ltd. Received: 20 January 2014; Revised: 15 April 2014; Accepted: 16 April 2014

INTRODUCTION Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with cytoplasmic kinase activity that transduces important growth factor signals from the extracellular matrix to the cells by binding specific ligands [1]. The EGFR is *Correspondence author: K. Kitazato, Division of Molecular Pharmacology of Infectious Agents, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail: [email protected] **Correspondence author: Y. Wang, Guangzhou Jinan Biomedicine Research and Development Center, National Engineering, Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China. E-mail: [email protected] Abbreviations used AAV, adeno-associated virus; AEV, avian erythroblastosis virus; ASFV, African swine fever virus; CPXV, cowpox virus; EGFR, epidermal growth factor receptor; EV71, enterovirus 71; gB, glycoprotein B; HCMV, human cytomegalovirus; HPIV, human parainfluenza virus; HPV, human papillomavirus; IAV, influenza A virus; IFN, interferon; IHD-J, International Health Department-J; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; N-WASP, neuronal Wiskott–Aldrich syndrome protein; PI3K, phosphatidylinositide 3-kinase; PICV, Clade A New World arenavirus Pichindé; PLCγ1, phospholipase C-gamma 1; RSV, human respiratory syncytial virus; RV, rhinovirus; STAT, signal transducers and activators of transcription; VACV, vaccinia virus; WR, Western Reserve; WT1, Wilms’ tumour 1.

Copyright © 2014 John Wiley & Sons, Ltd.

widely distributed in epithelial, mesenchymal or neuronal tissue and plays an important role in differentiation, the morphogenesis of many organs and tissue homeostasis. Deregulation of this tightly controlled signalling network by EGFR overexpression has been frequently implicated in several types of human cancers, especially breast, ovary, lung, and prostate cancers as well as glioblastoma and head and neck squamous cell carcinoma [2,3]. Multiple therapeutic agents and strategies have been developed to block the strong tumour promoting effects exerted by EGFR [3]. In recent years, increasing evidence has shown that viruses can interact with and modulate EGFR activity to facilitate viral entry, replication or the evasion of host immune surveillance. In this review, we will summarise the current understanding of the interactions between different viruses and EGFR and the corresponding downstream signalling pathways that are required for efficient viral infection. We will discuss the data collected for the various viruses (shown in Table 1) and highlight how these discoveries may provide insight into novel antiviral therapies while attempting to reveal new issues for future consideration.

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Table 1. List of viruses discussed in this review Family Adenoviridae Arenaviridae Asfarviridae Flaviviridae Hepadnaviridae Herpesviridae

Orthomyxoviridae Papovaviridae Paramyxoviridae Parvoviridae Picornaviridae Poxviridae

Retroviridae

Virus

Abbreviation

References

Adenovirus Clade A New World arenavirus Pichindé African swine fever virus Hepatitis C virus Hepatitis B virus Human cytomegalovirus Herpes simplex virus Epstein–Barr virus Influenza A virus Human papillomavirus Human respiratory syncytial virus Human parainfluenza virus Adeno-associated virus Enterovirus 71 Rhinovirus Vaccinia virus Cowpox virus Western Reserve International Health Department-J Shope fibroma virus Human immunodeficiency virus Avian erythroblastosis virus Mouse Cas NS-1 retrovirus

— PICV ASFV HCV HBV HCMV HSV EBV IAV HPV 16 RSV HPIV 3 AAV EV71 RV VACV CPXV WR IHD-J SFV HIV AEV —

[4–6] [7] [8] [9–15] [16,17] [18–27] [28,29] [30–34] [35–39] [40–42] [43–46] [47,48] [49–51] [52] [53–56] [57,58] [58] [59] [59] [60,61] [62–64] [65,66] [42]

MOLECULAR PROPERTIES OF EGFR EGFR belongs to a family of four closely related receptor tyrosine kinases (RTKs): EGFR/ErbB1, HER2/ErbB2/neu, HER3/ErbB3 and HER4/ErbB4 [67]. EGFR contains four conserved domains: an Nterminal EGF binding domain exposed at the cell surface, a short transmembrane domain, a cytoplasmic domain with protein–tyrosine kinase activity, and a C-terminal regulatory domain. Upon ligand binding, these RTKs homodimerise or heterodimerise, triggering transphosphorylation of the dimerised receptor and intracellular tyrosine kinase activation. These tyrosine-phosphorylated sites allow multiple adaptor and effector proteins to bind through their Src homology 2 domains, leading to the activation of downstream signalling cascades that include the mitogen-activated protein kinases (MAPKs), phosphatidylinositide 3-kinases (PI3Ks), phospholipase C-gamma 1(PLCγ1) and Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathways [68,67] (Figure 1). Copyright © 2014 John Wiley & Sons, Ltd.

Consequently, a variety of biochemical changes, including increased intracellular calcium levels, actin cytoskeleton reorganisation, transcriptional activation and protein synthesis, occur within the cell. For example, activation of the PI3K-dependent Akt signalling cascade is a widely studied signal transduction pathway triggered by activated EGFR that plays a pivotal role in the regulation of many cellular processes, including cell growth, motility, proliferation, and survival. Moreover, modulation of the PI3K signalling pathway has been reported in a variety of viral infections [69]. The C-terminal domain of EGFR modulates signalling through the EGFR network by governing the exact second messenger cascades that are elicited, which confer signalling specificity, and the dense positive and negative feedback and feed forward loops, including transcription-independent early loops and late loops mediated by newly synthesised proteins and microRNAs. Moreover, downstream signals of EGFR have been identified as key elements in Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

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K. Zheng et al.

Figure 1. The regulation of epidermal growth factor receptor (EGFR) signalling. Ligand binding to EGFR on the cell surface induces receptor dimerisation and cross phosphorylation, which then recruits several adaptors and effectors, leading to the activation of downstream signalling cascades including the MAPK, PI3K, PLCγ1/diacylglycerol and JAK/STAT pathways. As a result, a variety of biochemical changes take place within the cell, including protein synthesis, cytoskeleton changes, apoptosis inhibition, angiogenesis and cell motility. The misregulation of those pathways is implicated in many cases of cancers

the complex signalling network that is utilised by various classes of cell-surface receptors [70]. Ligand-dependent activation of the EGFR also induces trafficking events that relocalise the receptors from the cell surface to intracellular endocytic compartments that provide signal specification. In the resting state, most EGFRs reside on the cell surface, while a basal level of receptors is internalised by endocytosis. Upon stimulation with ligand, internalisation of ligand–receptor complexes through clathrin-coated pits is enhanced. Following internalisation, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Clathrin-mediated endocytosis is one of the best studied receptor-dependent pathways, characterised by the formation of clathrin-coated pits that bud into the cytoplasm to form clathrin-coated vesicles [71], and it is essential for sustained EGFR signalling and degradation[72]. Clathrin-mediated Copyright © 2014 John Wiley & Sons, Ltd.

endocytosis of EGFR also requires dynamin, a GTPase that has been shown to play an important role in the control of several cell surface receptors [73]. Moreover, increasing evidence has highlighted the facts that preferred EGFR endocytosis is restricted to the dorsal cellular surface and that the actin network is critical to define and maintain asymmetry in EGFR endocytosis [74–76]. Under the stimulation of ligands, such as EGF, asymmetry in the actin cytoskeleton indirectly affects the distribution of clathrin, leading to a spatial restriction of EGFR activation and alterations in actin dynamics that abolish asymmetry in EGF uptake and decrease EGFR-mediated downstream signalling [76]. Actin cytoskeleton regulators, such as endophilin, lamellipodin and the Arp2/3 activator neuronal Wiskott–Aldrich syndrome protein (N-WASP), cooperate to regulate F-actin-dependent EGFR endocytosis [77]. Ligand binding to EGFR rearranges the cytoskeletal network through EGFR endocytosis or Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

Viruses and EGFR EGFR-mediated signalling through a mechanism that is reminiscent of the actin-dependent cellular phenomena that accompany viral infection. In addition, studies have shown that numerous viruses utilise EGFR endocytosis during the infection of host cells to mediate virus internalisation and trafficking to the site of replication [78]. The actin network beneath the cell plasma membrane, which normally prevents virus entry, was modulated by viral proteins during virus infection [79]. Thus, EGFR and EGFR-mediated signalling pathways may facilitate virus entry through regulating actin dynamics to allow viral fusion/penetration or EGFR-mediated endocytosis. EGFR ACTS AS A CORECEPTOR FOR VIRUSES The initial step of the virus cycle is the process of entry into a new cell, during which the viral genome is translocated across the membrane of the target cell [80,81]. This process is characterised by a complex series of events that are initiated through the binding of the viral surface components to specific receptor molecules on the cell surface membrane. Multiple attachment receptors may be used sequentially or in a cell-type-specific manner, and coreceptors may also be involved to increase binding avidity or sequential engagement of distinct receptor moieties, which allows the timing of key events in virus fusion/penetration to be tightly coordinated, leading to productive infection [80]. In recent years, substantial progress has been made toward identifying undocumented cell surface components exploited by viruses, and EGFR has also been confirmed as a coreceptor for the entry of viruses such as human cytomegalovirus (HCMV) and adeno-associated virus serotype 6 (AAV6) [18,19,49]. HCMV is a beta-herpesvirus that causes severe complications in immunocompromised individuals and is suggested as a cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) infection [62]. During HCMV entry into human embryonic lung fibroblasts, EGFR acted as a coreceptor [18]. Aside from the attachment factors heparan sulphate proteoglycans [20], the HCMV envelope glycoprotein B (gB) interacts directly with EGFR though the EGF-like epitope GX4YX3MEX2HX4Q located in the amino-terminal region of gB [18]. Moreover, HCMV entry requires the binding of gH to ανβ3 within lipid rafts and simultaneously induces both EGFR-dependent PI3K Copyright © 2014 John Wiley & Sons, Ltd.

277 and ανβ3-dependent Src signalling. Coordination exists between these two signalling pathways, and disruption of the EGFR-ανβ3 interaction inhibits viral entry [19]. Besides, HCMV infection of other cell types, such as endothelial cells (ECs) [21], trophoblasts [22,23] and monocytes [24], also activated EGFR and different downstream signalling pathways. However, unlike the transient activation observed in fibroblasts and trophoblasts, HCMV stimulates chronic activation of EGFR in ECs, suggesting that a cell-type-specific mechanism is usurped by HCMV for different functional outputs. To add to the complexity of HCMV entry, it has been reported that EGFR does not mediate entry into fibroblasts, and others have shown that platelet-derived growth factor receptor, rather than EGFR, is required for efficient viral entry [82–84]. The reasons for these conflicting results remain unresolved. Another intriguing example of a virus using EGFR as a coreceptor for entry is AAV6 [50]. AAV6 is an attractive gene therapy vector because of its lack of pathogenicity, wide range of infectivity and ability to establish long-term transgene expression [51]. By a bioinformatics-based approach, EGFR expression is identified to have a positive correlation with cells permissive to AAV6, rather than other AAV serotypes. In vivo and in vitro studies suggest that EGFR is a coreceptor for AAV6 [49]. The specific interaction between EGFR and AAV6 and the ability of AAV6 to efficiently transduce EGFR-expressing tumours in vivo present an opportunity to target and deliver cytotoxic transgenes to tumours highly expressing membrane-localised EGFR [85], whereas the roles of other cell surface molecules, such as intracellular adhesion molecule-1, in AAV6 vector internalisation require further study. In the cases of viral receptors, understanding the tropism and signal transduction, an important mechanism that acts as a consequence of a virus binding to its receptor and influences virus cytopathogenicity and the immune response, may provide novel strategies for abrogating these viruses. HARNESSING EGFR SIGNALLING FOR VIRAL ENTRY AND REPLICATION Viral interaction with the receptors triggers signalling transduction cascades coincident with virus entry and overcoming the intrinsic barriers imposed by host cells to set the stage for a Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

278 favourable cellular environment for viral needs. These barriers include the plasma membrane, actin cortex and limiting intracellular membranes, and the plasma membrane represents the major barrier to virus entry. In most cells, the cortex (a thin, cross-linked actin network lying immediately beneath the plasma membrane) provides physical and signalling communication and has the potential to restrict incoming viral particles [79]. Furthermore, reconfiguration and reorganisation of the cellular actin cytoskeleton affect every stage of the viral life cycle [79]. Viruses have evolved various strategies to overcome these barriers, such as pH-independent fusion at the plasma membrane, coupled with receptor-mediated signalling, coordinated disassembly of the actin cortex [86] and receptor-mediated endocytosis [78], the latter of which is the main mechanism exploited by viruses. Because of the role of EGFR in endocytosis and cytoskeleton remodelling, it is not surprising

K. Zheng et al. that viruses harness the EGFR signalling pathway to transit across plasma membrane and entry into host cells (Figure 2). It has been reported that virus-induced EGFR signalling can cause local actin perturbation to allow viruses to penetrate the cortex (Figure 2A). For HCMV, coordination of EGFR-dependent PI3K and ανβ3-dependent Src signalling activates RhoA and cofilin, leading to actin reorganisation and consequent translocation of HCMV capsids to the nucleus [19]. Recently, we also found that entry of herpes simplex virus type 1 (HSV-1) into neuronal cells requires EGFR-mediated actin remodelling [28]. An EGFR-dependent signalling pathway, such as EGFR/PI3K/MEK/Rock/cofilin, is activated to regulate F-actin dynamics and facilitate viral postentry and replication processes. Epstein–Barr virus (EBV), a member of the gamma herpesvirus subfamily, can induce actin filament rearrangement through viral protein latent membrane protein 1

Figure 2. Viruses manipulate the epidermal growth factor receptor (EGFR) signalling network for entry. (A) EGFR-mediated signalling facilitates virus entry through pH-independent fusion by regulating the actin network. glycoprotein B (gB) of human cytomegalovirus (HCMV) binds to EGFR and activates PI3K-dependent RhoA and cofilin, leading to F-actin reorganisation and, as a consequence, translocation of HCMV capsids to the nucleus. By activation of EGFR, HCMV also induces angiogenesis in trophoblast cells and modulates blood monocyte motility. Binding of herpes simplex virus 1 (HSV-1) activates a similar EGFR-dependent signalling cascade to regulate F-actin dynamic and benefits its post entry. (B) Hepatitis C virus (HCV) and influenza A virus (IAV) undergo receptor-mediated endocytosis in coordination with EGFR activation. Upon HCV infection, EGFR colocalises with CD81 and induces endocytosis, while GTPase HRas bridges the interaction and mediates PI3K signalling transduction. IAV activates EGFR in a sialic acid-dependent manner

Copyright © 2014 John Wiley & Sons, Ltd.

Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

Viruses and EGFR (LMP1)-mediated PI3K activation [30]. Because EGFR is the direct upstream kinase of PI3K and LMP1 also activates EGFR [31], it is easy to envision that EGFR may mediate the EBV-induced actin rearrangement, and further experiments are required to confirm this possibility. The endocytic pathway is the main mechanism utilised by the majority of viruses to enter host cells for productive infection because endocytosis can assist viruses in bypassing the plasma membrane, allowing direct transport of viruses to subcellular sites of viral replication and helping viruses overcome host immune surveillance by leaving minimal evidence on the cell surface. EGFR expression on the cell surface is dynamic, undergoing continuous endocytosis, and many viruses have shown that their endocytic entry relies on EGFR activation accompanied by a complicated signals triggered by viruses for their own benefits (Figure 2B). For example, by using RNAi screening, EGFR is characterised as previously an unrecognised cofactor for hepatitis C virus (HCV) endocytic entry [9,10]. HCV entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane [11]. More detailed studies have shown that binding to CD81 results in cross linking of CD81 and EGFR activation, which, in turn, induces cointernalisation of HCV-CD81-EGFR [12]. Subsequently, the colocalisation between EGFR and CD81 induces endocytosis to enhance the rate of HCVentry. In addition, the GTPase HRas associates with CD81–CLDN1 and activates downstream of EGFR signalling, which acts as a key host signal transducer for EGFR-mediated HCV entry [13]. HRas regulates CD81 diffusion and provides a physical link between the EGFR/ Shc1/Grb2/HRas signalling pathway and the HCV entry cofactor complex. Influenza A virus (IAV) can enter cells either by clathrin-mediated endocytosis or by a clathrin/ caveolin-independent mechanism. Because of the incapability of sialic acids—a direct receptor for IAV—to transmit signals across the plasma membrane, additional signalling receptors, such as EGFR, are required for IAV uptake [35–37]. IAV attachment results in the clustering of lipid rafts and the activation of EGFR in a sialic acid-dependent manner, which subsequently recruits PI3K, which is necessary for endocytosis triggering [36]. In addition, EGFR is also involved in the endocytic entry Copyright © 2014 John Wiley & Sons, Ltd.

279 and infection of other viruses such as human papillomavirus type 16 (HPV16) [40,41], variants of vaccinia virus (VACV; the Western Reserve and International Health Department-J strains) [59], and respiratory syncytial virus (RSV) [43]. African swine fever virus is a large and highly pathogenic DNA virus, and its entry requires a sodium/proton (Na+/H+) exchanger, activation of EGFR and PI3K, and phosphorylation of Pak1 kinases together with activation of Rho-GTPase Rac1 and relies on actindependent blebbing/ruffling formation [8]. All these events are fully linked with activation of macropinocytosis, which is another important type of endocytic route used by several viruses to enter host cells. It is defined as an actin-dependent endocytic process associated with a vigorous plasma membrane activity in the form of ruffles or blebs induced by activation of kinases and Rho GTPases [87]. Further studies to elucidate if the same EGFR signalling pathways are triggered by viruses with different receptor specificity would be of great interest. In addition, to promote viral entry, EGFR-mediated different signalling cascades have also been implicated to facilitate viral replication or viral pathogenesis, such as EBV [31,32] and Pichindé virus [7]. Viruses can initiate intracellular signalling cascades, which occur at particle concentrations that may mirror physiological conditions even in the absence of virus entry [88]. For example, within a minute after HIV-1 exposure, more than 200 phosphorylation sites are modified in T-cells, with the potential to alter several cellular processes immediately after infection and thus to support viral replication [63]. Previous studies have reported that rapid activation of EGFR occurs in various cell lines following HCMV infection [18,19,21–24] and that the activation of EGFR signalling is required for efficient infection [18,23]. The EGFR expression and ensuing downstream signalling that follow viral engagement provide cell-type specificity for viral internalisation. For example, HCMV entry into human peripheral blood monocytes rapidly activates the EGFR signalling pathway to modulate the motility of monocyte via the specific upregulation of N-WASP, a highly active actin nucleator controlling actin growth, in a PI3K-independent manner [24]. EGFR also acts as a viral tropism receptor for targeting HCMV entry into trophoblast cells [22], where HCMV-induced angiogenesis is dependent on the activities of EGFR and Src family tyrosine Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

280 kinases, along with the activation of the downstream PI3K and MAPK pathways. This difference highlights how unique pathways originating from the same cellular receptor in biologically distinct cell types are used by HCMV. Furthermore, other viruses, such as enterovirus 71 (EV71), induce a timedependent oxidative stress production during viral replication through EGFR/Rac1 signalling; thus, investigating the relationship between oxidative stress generation and virus virulence is of great value in developing therapeutic interventions for the effective pharmacological treatment of EV71 infection [52]. These data suggest that EGFR internalisation may be a common mechanism utilised by viruses to enter cells and that different intracellular signalling cascades are triggered to induce a favourable environment for infection and viral replication via the activation of EGFR. All these studies endorse the development of preventive and therapeutic antiviral strategies, such as targeting EGFR, an approach that would be less likely to induce drug resistance, which is a significant problem when conventional antiviral agents are administered for a prolonged period. ACTIVATING EGFR SIGNALLING FOR INFLAMMATION AND VIRAL ANTAGONISM OF THE HOST ANTIVIRAL SYSTEM EGFR signalling has also been usurped by viruses to antagonise the host immune response. Interferons (IFNs) are cytokines produced by the immune system that stand as the first line of defence against viral infection. Therefore, many viruses have evolved mechanisms to resist IFN activity, including blocking downstream signalling events that occur after the IFN cytokine binds to its receptor, preventing further IFN production, and inhibiting the functions of proteins that are induced by IFN [89]. For instance, IAV and rhinovirus (RV) infection activate EGFR and reduce IFN-λ production through suppressing IFN regulatory factor via an unknown mechanism while inhibition of EGFR augments IFN-λ, resulting in reduced viral titres [38,53]. On the other hand, downstream effectors of EGFR signalling—for example, STAT1 and STAT3, two members of the STAT protein family stimulated by ligand binding to EGFR—have opposing effects on immune response, and viruses may interrupt the balance or activate one to counteract the others. STAT1 has a proinflammatory and antiproliferative function, and HCV infection induces EGFR-dependent STAT3 activation to Copyright © 2014 John Wiley & Sons, Ltd.

K. Zheng et al. antagonise the effect of STAT1, resulting in reduced expression of suppressors of cytokine signalling 3 and IFN response gene and reduced antiviral activity [14]. Moreover, the virulence protein C, encoded by human parainfluenza virus type 3, inhibits STAT1 activity by interacting directly through its C-terminal domain, blocks type 1 IFN signalling and bridges the EGFR to extracellular signal-regulated kinase (ERK) pathway to induce an inflammatory response [47,48]. These results reveal a previously undiscovered role for EGFR and may open new avenues of improving the efficacy of IFN-based antiviral therapies. Mucins are the host innate components and the main glycoproteins present within mucus. They are thought to be able to trap and eliminate microorganisms from the lung. The upregulation of mucins by respiratory virus infection contributes to the pathogenesis of virus-induced diseases and disease exacerbations. In vitro and in vivo studies demonstrate that IAV upregulates epithelial cellderived mucin production, both at the transcriptional and translational levels, which depends on a protease-EGFR-ERK-specificity protein 1 signalling cascade [39]. RV16 and RV1A (a minor group of RV) also induce mucin production through a different EGFR-dependent pathway [54,55]. Upon RV infection, a pattern recognition receptor TLR3, which specifically recognises viral double-stranded RNA, is activated, leading to the production of TGF-α and subsequent EGFR activation. Complete elucidation of the EGFR-dependent pathways will advance our understanding of viral-induced airway remodelling and disease exacerbation. It is increasingly appreciated that symptoms and signs of many viral diseases are caused less by viral cytopathic effects than by the host response to infection and that the peak of viral infection often precedes the release of inflammatory mediators. RSV is frequently identified in exacerbations of inflammation, infection of which triggers benign or pathogenically intense inflammation; the induction of chemokine and cytokine release by RSV primary infection is mediated by EGFR [44–46]. RSV infection activates EGFR, possibly by activating a membrane-localised metalloprotease resulting in cleavage of EGFR proligands, which, in turn, initiates ERK signalling that is required for IL-8 production [45]. Moreover, EGFR-dependent ERK activation contributes to the sustained survival of RSV-infected cells through modulation of Bcl2 Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

Viruses and EGFR proteins. Similarly, RV16 serotype infection rapidly promotes induction of EGFR ligands and utilises EGFR-Erk1/2-STAT signalling to increase IL-8 levels [56]. The EGFR signalling mediated escalation of inflammation most likely amplifies viral growth and spread, and targeting EGFR may offer an effective therapy that reduces the inflammatory burden associated with viral-induced disease, which operates independently of the antiviral defences of the hosts. VIRAL REGULATORS MANIPULATE EGFR EXPRESSION OR RECYCLING Another avenue of research that has highlighted the importance of EGFR signalling is the study of manipulating EGFR expression by viral proteins [90]. Several viruses constitutively regulate EGFR signalling by expressing viral proteins that transcriptional regulates EGFR gene expression or acts as an

281 active component of EGFR signalling or by interfering with the EGFR degradation pathway (Figure 3). A few viruses regulate EGFR expression at a transcriptional level (Figure 3A). Hepatitis B virus (HBV) [16] and EBV [33,34] both upregulate EGFR expression during the invasion process by transactivation from the EGFR promoter mediated by HBV X-gene product and by the induction of p50/p50/Bcl-3 complexes mediated by EBV-encoded viral protein LMP1 C-terminal activating region 1, respectively. However, some viruses, such as HCMV [25,26] and adenovirus [4,5], downregulate EGFR expression to isolate the infected cell from host-specific signals, forcing the cell to respond solely to viral signals, which specifically optimise the cellular environment for productive infection. HCMV inhibits EGFR expression through upregulation of Wilms’ tumour 1 (WT1) protein, a known transcriptional

Figure 3. Viral regulators modulate EGFR expression or the EGFR recycling process. (A) The X-gene product of hepatitis B virus (HBV) upregulates transcription from the EGFR promoter. Likewise, the viral protein lysosomal-associated membrane protein 1 of Epstein–Barr virus (EBV) induces the formation of a p50/Bcl-3 complex, which subsequently upregulates EGFR expression. In contrast, human cytomegalovirus (HCMV) activates a transcriptional suppressor WT1, as well as the adenovirus E1 protein, to deregulate EGFR expression. Poxviruses encode EGF-like ligands, which significantly increase pathogenicity when expressed at sites of infection. (B) Different virus-encoded proteins monitor the EGFR recycling process. AEV encodes a truncated form of EGFR, which forms covalent dimers at the cell surface in a ligand-independent manner and activates various EGFR target proteins. The herpes simplex virus 1 (HSV-1) and adenovirus enhance EGFR degradation. ICP0 of HSV-1 forms a complex with CIN85 and Cbl, leading to reduced EGFR cell surface expression and enhanced EGFR degradation, whereas adenovirus E3 protein enhances EGFR degradation by causing constitutively internalised EGFRs to accumulate in a prelysosomal compartment. The recycling rate of EGFR is increased by human immunodeficiency virus 1 (HIV-1) Gag protein through retaining more EGFR in late endosomes or human papillomavirus (HPV) E5 protein through the inhibition of an endosomal proton ATPase. In addition, the mouse Cas NS-1 retrovirus encodes a dominant active form of c-Cbl to increase the rate of EGFR recycling back to the cell surface

Copyright © 2014 John Wiley & Sons, Ltd.

Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

282 repressor of EGFR expression [26]. Similarly, E1A, a protein encoded by adenovirus, downregulates the EGFR via repression of its promoter [5]. Besides, HBV X protein can upregulate miR-7 expression to target 3′UTR of EGFR mRNA, which, in turn, results in the reduction of EGFR protein expression [17]. In contrast, other viruses modulate the expression of EGFR by interrupting the recycling process (Figure 3B). HIV-1 decreases the rate of EGFR downregulation by retaining more EGFR in late endosomes through association of the conserved PTAP (Pro-Thr-Ala-Pro) binding motif within the C-terminus of viral Gag protein with TSG101 (tumour susceptibility gene 101 protein) [64]. The increased intracellular retention of EGFR results in prolonged EGFR-mediated signalling, which may contribute to the enhanced HIV-1 replication and/ or infectivity. Most members of the poxviruses family, the largest group of DNA viruses, encode EGF-like ligands that bind to the EGFR family and promote virulence without any influence on viral replication [60,61]. In contrast, avian erythroblastosis virus (AEV) encodes a truncated form of EGFR, which carries many intracellular mutations and is constitutively active [65,66], whereas HPV encodes a protein E5, which inhibits EGFR degradation through inhibition of an endosomal proton-ATPase [42]. This returns internalised receptors to the plasma membrane where they may again bind ligand and stimulate proliferative pathways. In addition, the mouse Cas NS-1 retrovirus, which induces pre-B cell lymphomas and myeloid leukaemia, encodes a dominant active form of c-Cbl, an ubiquitin ligase that targets EGFR proteins to lysosomal degradation, to increase the rate of receptor recycling back to the cell surface [91]. It has also become evident that viruses may encode some viral proteins that promote EGFR degradation and decrease its expression on cell membrane. Adenovirus E3 protein enhances EGFR degradation by causing constitutively internalised EGFRs to accumulate in a prelysosomal compartment [6]. The HCV NS5A protein colocalises with the EGFR and alters its distribution, resulting in a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment [15]. In addition, an IE protein of HSV-1, infected cell protein 0 (ICP0), has been shown to repress EGFR cell surface expression by forming a complex with CIN85 and Cbl [29]. In summary, those chronic stimulations of the EGFR signalling or expression may be involved in the development of cancers or viral pathogenesis. Copyright © 2014 John Wiley & Sons, Ltd.

K. Zheng et al. EGFR-TARGETED AGENTS: NOVEL ANTIVIRAL THERAPEUTICS The current antiviral therapy approach is to use treatments that selectively inhibit the replication cycle of viruses. The mode of action of antiviral agents can be divided into three classes: (i) those that bind to or become incorporated into the viral nucleic acid thereby inhibiting its function; (ii) those that interfere with viral particle maturation, assembly or release; and (iii) those that interfere with the viral enzyme and inhibit their function. However, because of the variability in viral genomes, drug-resistant strains occur frequently in clinical therapy, which makes exploring novel antiviral agents with different mechanisms of action urgent [92]. The central role of the EGFR network in the life cycle of viral infections, its availability to extracellular manipulation, and detailed understanding of the underlying biochemistry have made the EGFR pathway an attractive target for antiviral therapy. The three most common agents for targeting EGFR are monoclonal antibodies (mAbs), vaccines and small molecule inhibitors. Up to now, only small molecule inhibitors display potential antiviral activity. Firstly, EGFR inhibitor CI-1033 (Canertinib) has demonstrated the ability to block viral spread of variola virus in vitro as well as strong antiviral activity in a VACV mouse model [57]. Likewise, an FDA-approved EGFR inhibitor gefitinib (IressaTM) and two other inhibitors, PD153035 and vandetanib, which are still either in experimental or in clinical investigation for anticancer therapy, successfully inhibit virus spreading in cowpox virus and VACV-infected epithelial cells in vitro and MCMV infection in vivo [58,27]. Another recent intriguing example is a licensed EGFR inhibitor, erlotinib, which exhibits significant in vitro and in vivo antiviral activity towards HCV infection [9]. The discovery of the correspondent EGFR inhibitors as candidate antivirals defines a potential new strategy for clinical prevention and treatment of HCV infection. In addition, EGFR inhibitors represent promising drug as therapies for human arenavirus infection [93]. Further studies should be performed to validate these EGFR-targeted mAbs as valuable leads for the treatment of viral infection. Likewise, specific studies should be undertaken to examine antiviral drug resistance in viral infection. Additionally, combinational therapies involving specific antiviral agents, immunotherapy and anticancer agents that interfere with cellular processes essential Rev. Med. Virol. 2014; 24: 274–286. DOI: 10.1002/rmv

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for viral replication, which may overcome the drug resistance, such as EGFR-targeted agents, should be carefully considered. In addition, consideration of the potential for small molecule inhibitorinduced EGFR mutations to occur in normal cells should prompt the scientist to undertake studies along this path.

responses, either by mimicking EGF function through binding to EGFR directly or indirectly or by encoding EGFR homologs. Although most viruses use broadly similar tactics to breach host cell barriers, many have developed unique approaches that ensure their delivery to optimal cellular sites for replication and survival. Exploitation of the EGFR signalling pathway by viruses is quite likely to have had considerable evolutionary impact, and discrepancies that activate different EGFRmediated signal pathways are starting to emerge. Therefore, it will be of particular interest in the future not only to determine if other viruses or viral proteins target the EGFR as part of their infectious effects but also to continue to explore the relevance of EGFR signalling by these viruses or viral oncoproteins in cellular pathogenesis. In addition, EGFR inhibition may present an enormous opportunity for developing novel treatments for a wide array of viral infections.

PERSPECTIVES Viral attachment on plasma membrane and subsequent entry into host cells is a crucial step in the pathogenesis of viral infections as well as a topic of great relevance to the development of successful strategies for gene therapy based on transductional targeting of oncolytic viruses. EGFR is overexpressed on a high percentage of human carcinomas and is employed for targeted gene delivery, either with viral or non-viral gene delivery systems [94,95]. To date, studies have shown that activation of EGFR by ligand binding and entry of microbes, not only viruses but also bacteria [96–100], shares a number of similarities in the resulting cell signalling cascade and that EGFR plays an important role in the viral invasion process. Rather than attempting to fully review the current knowledge on microbial interactions with EGFR, this article discusses EGFR-dependent binding and subsequent intracellular signalling events usurped by viruses. Viruses exploit the EGFR-mediated endocytosis or EGFR-mediated signalling for entry, replication and the antagonism of host antiviral

CONFLICT OF INTEREST The authors have no competing interest. ACKNOWLEDGEMENTS This work was supported by the Twelfth Five-Year National Science and Technology Support Program (2012BAI29B06), the National Natural Science Foundation of China (81274170), and the Foundation for High-level Talents in Higher Education of Guangdong, China ([2010]NO.79).

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