The host cell response to tick-borne encephalitis virus

Biochemical and Biophysical Research Communications xxx (2017) 1e8

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The host cell response to tick-borne encephalitis virus Tea Carletti, Mohammad Khalid Zakaria, Alessandro Marcello* Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 December 2016 Received in revised form 31 January 2017 Accepted 2 February 2017 Available online xxx

Tick-borne encephalitis virus is the most prevalent autochthonous arbovirus in Europe and an important travel-associated virus. Complications of the infection could lead to lethal encephalitis in susceptible individuals. However, despite its clinical relevance and expanding geographical distribution, most of our knowledge on its pathogenesis is inferred from studies on other flaviviruses. Molecular details of the host cell response to infection are scarce leading to a poor understanding of the antiviral pathways and viral countermeasures that are critical to determine the outcome of the infection. In this work the relevant literature is reviewed and the key elements of tick-borne encephalitis virus infection of human cells are identified, which requires further investigation. © 2017 Published by Elsevier Inc.

Keywords: Flavivirus TBEV Interferon Unfolded protein response Integrated stress response

1. Introduction Flaviviridae is a large family of enveloped RNA viruses, which share similarities in virion morphology, genome organization and replication strategies. The genus Flavivirus consists of more than 70 viruses that are transmitted to humans by arthropod vectors. Members of this genus include widespread human pathogens delivered by mosquitoes such as Dengue virus (DENV), Zika virus (ZIKV), Yellow fever virus (YFV), West Nile virus (WNV) and Japanese Encephalitis virus (JEV). Tick-borne encephalitis virus (TBEV) is the most prominent member of the TBEV complex, which includes antigenically related viruses including Omsk haemorrhagic fever virus (Siberia), Kyasanur Forest disease virus (India), Akhrma virus (Saudi Arabia), Louping ill virus (UK), Powassan virus (United States and Russia) and Langat virus (Malaysia). TBEV includes three sub-types, namely Far Eastern, Siberian and Western European. TBEV is transmitted by ticks of the species Ixodes ricinus (Western TBEV) or Ixodes persulcatus (Eastern/Siberian TBEV). Maximum incidence of human infections coincides with seasonal peaks of feeding activity of the ticks, usually in spring. The sylvatic cycle is sustained by small mammals in the forest, which do not generally

* Corresponding author. Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99, 34149 Trieste, Italy. E-mail address: [email protected] (A. Marcello). URL: http://www.icgeb.org/molecular-virology.html

succumb to the infection [1,2]. Humans are occasional dead-end hosts, who become infected by a tick bite or by consumption of raw milk from infected domestic animals. Approximately 5000e12,000 cases of TBE are reported in Europe each year [3]. The incubation period of TBEV is between 7 and 14 days with generally mild symptoms that include fever, fatigue, pain and headache. In some patients the infection causes damage to the central nervous system, which could be fatal particularly in elderly people. As observed in 20e30% of cases, encephalitis caused by European TBEV is biphasic with fever during the first phase and neurological disorders during the second phase. In contrast with severe Eastern subtype virus infection symptoms are usually milder, with case fatality rates as low as 1e2%, mostly without sequelae. TBEV tends to occur focally even within endemic areas. Currently, the highest incidences of clinical cases are being observed in the Baltic States, Russian Federation and Slovenia. However, autochthonous cases are constantly being reported in new areas of Western Europe showing an expansion to non-endemic areas [4]. A protective vaccine derived from inactivated Western TBEV is available and its efficacy is demonstrated by the lower prevalence of TBEV infection in highly endemic Austria, which implemented a program of vaccination with a high coverage of the population [5,6]. No drugs are licensed for TBEV, although some compounds have been tested [7]. The focus of this review is on the host cell response to TBEV infection. The transmission cycle of TBEV between ticks, vertebrate reservoirs and humans is analysed to gain information on the cellular targets in vivo. Also, the interferon response to TBEV

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infection is considered together with the TBEV escape strategies identified so far. Finally, the integrated stress response to TBEV infection and its antiviral role is discussed. The final picture will instruct on the key steps to be implemented in the future research on TBEV infection. 1.1. General features of the virus Mature virions are about 50 nm in diameter and are composed of an electron dense core surrounded by a lipid bilayer containing two envelope glycoproteins, E (envelope) and M (membrane). Capsid (C) protein and a positive sense single stranded RNA of 11kb make up the viral core. The genome encodes a single long open reading frame (ORF) flanked by a 50 and 30 noncoding regions (NCR). The ORF is translated into a polyprotein of about 3400 amino acids, which is cleaved into the viral proteins by host and viral (NS2B/ NS3) proteases. The structural proteins capsid (C), pre-membrane (prM) and envelope (E) precede the nonstructural proteins NS1, NS2A, NS2B, NS3 (helicase and protease), NS4A, NS4B and NS5 (RNA-dependent RNA polymerase RdRp and methyltransferase). The 50 NCR lacks sequence conservation, but secondary structures in this region are conserved among different Flaviviruses, albeit with some differences between mosquito and Tick-borne viruses. These structures are functionally important as cis-acting regulatory elements for genome cyclization, minus-strand synthesis and translation [8,9]. The 30 -NCR of TBEV is extremely variable in length, ranging from about 450 to 800 nucleotides in natural isolates [10]. It is further subdivided into a highly conserved “core” region of about 340 nucleotides at the distal 30 -end and a “variable” region between the core and the end of NS5. The core consists primarily of conserved RNA secondary structures required for cyclization that are essential for viral replication [11,12]. The variable region lacks sequence conservation and can be of different lengths. In some TBEV isolates the variable region contains an internal poly(A) tract of consecutive adenine residues [11,13]. The Neudoerfl strain contains between 30 and 250 adenosine stretches, while the highly virulent Hypr strain counts only few nucleotides [10]. While it has been shown that the components of the variable region are not essential for virus growth in cell cultures, it still remains to be established whether they might have an effect on viral pathogenesis. TBEV genomes sequenced directly from engorged ticks to avoid laboratory adaptation demonstrated that a pool of TBEV quasispecies exist in ticks, which shifts when the virus switches between invertebrate and vertebrate environments [14]. Moreover, an abundant 0.3e0.5 kb non-coding RNA fragment (termed sfRNA for subgenomic flaviviral RNA) has been detected in cells infected by Flaviviruses including tick-borne members [15]. The sfRNA is derived from incomplete degradation of the viral 30 NCR by the cellular 50 -30 exonuclease Xrn1 that stops at specific stem-loop structures (SL2I and SL1 in TBEV) found in the 30 -NCR. It has been demonstrated that the sfRNA regulates multiple cellular pathways to facilitate flaviviral pathogenicity and to inhibit the interferon/ stress response [16,17]. Intriguingly, the sfRNA of Flaviviruses deploys RNA interference (RNAi) suppressor activities in arthropod cells [18,19]. 1.2. TBEV entry and dissemination routes TBEV enzootic transmission cycles are determined by the interaction between viruses, ticks, and their vertebrate hosts [20,21]. Vertical trans-ovarian transmission of TBEV, from an infected adult female tick to its offspring, as well as horizontal transmission to ticks via feeding on an infected vertebrate host has been well documented. However, the most important route of transmission for TBEV in the wild is believed to be non-viremic

transmission by co-feeding ticks [22,23]. Ticks feed in clumps on hosts and simultaneous feeding of infected and uninfected ticks (co-feeding) on the vertebrate host is the pre-requisite for transmission. Skin explants of feeding sites contain migratory dendritic cells (DC) and neutrophils containing viral antigen. Moreover, migratory monocyte/macrophages were shown to produce infectious virus. Therefore, cellular infiltration of tick feeding sites and their migration between sites provides a vehicle for transmission between co-feeding ticks [24]. Intriguingly, the saliva of feeding ticks has been shown to enhance this mode of transmission [22]. During the blood meal, 33e50% of the fluid ingested by the tick is excreted back into the host [25]. Thus, tick feeding involves alternation of blood ingestion and saliva secretion for protracted periods of up to 2 weeks or more. Transmission of TBEV to humans generally occurs following the bite of an infected tick. Ticks remain attached for long periods of time until detected and removed, which is very different from what happens following a mosquito bite. Another documented route of human TBEV infection is associated with the consumption of raw milk, usually from infected goats. The human digestive tract was shown to be an efficient route of infection, which was confirmed in early experiments with mice fed orally with TBEV [26]. Laboratory TBEV infections linked to accidental needle-stick injuries or aerosol infections have also been documented, highlighting the need of implementing accurate safety procedures [27]. Upon inoculation of TBEV into the human skin, initial infection and replication occurs in local DCs, macrophages and neutrophils causing primary viremia [24]. DCs are believed to transport virus to nearby lymph nodes, which is followed by the development of secondary viremia. This picture is mostly inferred from studies on other Flaviviruses since it has not been explored much in the case of TBEV. During the secondary viremic phase, the virus crosses the blood-brain barrier (BBB) and enters the brain [28]. Major hallmarks of TBEV neuropathogenesis are neuroinflammation, followed by neuronal death and disruption of the blood-brain barrier [29]. Neuronal injury may be directly caused by viral infection, but destruction has also been attributed to infiltrating immunocompetent cells (mainly CD8þ T-cells), inflammatory cytokines and activated microglial cells [30]. TBEV infects and replicates in neurons in vitro inducing membrane rearrangements typical of TBEV replication and autophagosome [31e33]. 1.3. The intracellular TBEV life cycle Flavivirus TBEV particles are enveloped in an icosahedral cage of protein E dimers that completely cover the membrane and mediate both receptor binding and membrane fusion [34]. The atomic structure of the TBEV E protein in both its pre- and post-fusion conformation has been resolved as well as the conformational changes that lead to membrane fusion in acidic endosomes [35,36]. However, the uncoating of the nucleocapsid containing the viral protein C and the genomic RNA into the cytosol followed by a Capdependent first round of translation of viral RNA is poorly characterized. The multi-transmembrane domain polyprotein precursor localized on the endoplasmic reticulum (ER) is then co- and posttranslationally cleaved by cellular enzymes (signalase and Furin) and by the viral NS2B-NS3 protease into the three structural and seven non-structural viral proteins. After translation of the genomic input RNA, the NS5 RdRP synthesizes a genome length minus strand RNA, which then serves as a template for the asymmetric synthesis of additional plus strand RNA. The newly synthesized positive strand RNA can be subsequently used for several purposes: for further translation of viral proteins, for synthesis of additional negative strand RNA, or to be incorporated into new viral particles. Hence, the viral RNA genome has three different functions:

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translation, replication and serving as virion-associated genomic RNA. Flavivirus replication occurs in close association with virusinduced membrane structures of the rough ER, predominantly in the perinuclear region. These membrane structures may serve as a scaffold for anchoring the replication complex (RC) and to limit the diffusion of viral/host proteins and viral RNA increasing the concentration of components required for RNA synthesis [37]. In addition, it has also been suggested that these membrane structures may serve to protect the replication intermediate dsRNA from host cellular surveillance [38e40]. Composition and threedimensional organization of TBEV replication vesicles have been recently characterized [39e41]. These appear as spherical invagination of the ER membrane connected to the cytoplasm by a pore and are named replication vesicles (RV), which form vesicle packets (VP). Afterwards, other structures are formed: convoluted membranes (CM) and paracrystalline arrays (PC). Three-dimensional electron tomography studies have also showed that VPs, CM and PC are all part of a single ER-derived membrane network. Moreover, dsRNA, NS5, NS2B-NS3 immunolabelling of cryosections prepared from Flavivirus infected cells revealed that VPs are the sites of RNA replication, whereas CM and PC are possibly the sites of protein translation and proteolytic cleavage [42]. Virion assembly occurs on the ER membrane, budding into the lumen of the ER. Virions are transported via the secretory pathway and released at the cell surface [43]. At the earlier step E and prM proteins are associated as heterodimers through their C-terminal transmembrane anchors. The highly basic C protein interacts with the RNA viral genome in the cytoplasm and forms the nucleocapsid precursor that acquires an envelope by budding into the ER lumen. The maturation of the virus occurs in the trans-Golgi network (TGN) and includes the glycan modification of E and prM and cleavage of prM by Furin. This cleavage renders the mature virion ready for acid catalysed rearrangements of E required for productive entry [43]. 1.4. The interferon response to TBEV infection In order to sense and restrict viral infection, there are two kinds of self-defence mechanisms attained by the mammalian cells: the early innate and the late adaptive immune responses. The former will be the focus of this chapter because it involves a number of intracellular molecular pathways triggered directly by the infection. Innate immunity acts as a first line of defence barrier and brings about activation of the pro-inflammatory cytokines and type I interferons (IFNa/b). The indispensability of IFN in early antiviral immune responses is exemplified by the observation that mice with a defective functional type I IFN succumb to TBEV infection [44]. Early antiviral defence mechanisms mounted by infected cells are based on the identification of characteristic pathogenassociated molecular patterns (PAMP), which trigger the activation of pattern recognition receptors (PRR). PRR then activate antiviral transcription factors that translocate to the nucleus, thereby driving a plethora of virus-induced genes such as primary cytokines, IFNa/b and indirect effector molecules such as interferon-stimulated genes (ISGs) [45]. The toll-like receptors (TLRs) 3 and 7/8 sense viral RNA within endosomes [46,46]. The cytosolic PRRs are the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated 5 (MDA5) [47], collectively called RIG-I-like receptors (RLRs), which sense the viral RNA in the cytoplasm. Flaviviruses like WNV are recognized by both RLR, while interferon induction by TBEV depends only on RIG-I in U2OS cells [48]. Timing of PRR activation is also critical: for WNV it has been demonstrated that RIG-I activation occurs early, while MDA5 is triggered at late time points [49]. PAMP agonists of these receptors are viral RNA replication intermediates such as long stretches of

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dsRNA or 50 triphosphate single-stranded RNA, which are recognized as non-self molecules. Recognition of cytosolic dsRNA by protein kinase R (PKR), a third type of PRR ubiquitously expressed at low levels in all cell types, also results in production of IFN [50]. The expression of PRRs is cell type-dependent. For example, TLR3 is expressed by fibroblasts and conventional dendritic cells (cDC), but not by plasmacytoid DCs (pDCs) that rely on TLR7/8 for virus detection. The RIG-I pathway is important for viral induction of IFN in cDC and in non-dendritic cells, but this pathway is dispensable in pDCs. In the presence of PAMPS, RLRs undergo a conformational change and form oligomers by virtue of their CARD domains, which also interact with the CARD domain of IPS-1 (interferon-b promoter stimulator 1) resulting in the activation of downstream effectors such IRF-3 (interferon regulatory transcription factor 3) and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells). These transcription factors translocate to the nucleus and induce IFN transcription [51]. In turn, secreted interferon helps the nearby cells, or the parent cell, in amplifying such antiviral responses by binding to IFNa/b receptors (IFNAR) in a para- or autocrine manner, respectively. This interaction triggers the activation of the Jak-STAT transduction pathway to induce the expression of hundreds of IFNinducible genes (ISGs) with antiviral activity [52]. Although some ISGs are believed to have broad antiviral activity, some are more specific for certain types of infection. The importance of IFN-associated responses is illustrated by the observation that genetic resistance to flaviviruses in mice could be linked to the antiviral interferon stimulated gene (ISG) Oas1b that encodes a 20 e50 oligoadenylate synthetase (OAS) [53]. OAS is up- regulated in response to IFN stimulation and together with its effector protein, RNase L, functions to cleave RNA. Additionally, more specific to tickborne flaviviruses, the ISG TRIM79a restricts TBEV replication by degrading the NS5 replicase [54], while the ISG Viperin inhibits TBEV RNA synthesis [55]. IPS-1 has been shown to be critical for restricting TBEV infection in the mice brain, since IPS-1 knockout mice developed severe neuropathogenesis. This is by virtue of the ability of IPS-1 to regulate downstream IFN signalling and anti-viral ISGs like Viperin and IRF-1 [56]. Flaviviruses, like other pathogens, have evolved a variety of strategies to evade the host innate immune sensing. Several reports indicate that during TBEV infection the efficiency of nuclear localization of IRF-3 is significantly reduced [40,48]. This sheds light on the fact that the TBEV deploy mechanisms to counteract the innate immune responses. TBEV replicates inside the ER derived vesicles, thus protecting viral RNA replication intermediates from exposure to multiple cytosolic sensors [39,40,48,57]. Also, it has been shown that TBEV NS5 could impair the Jak-STAT pathway specifically during IFN receptor-mediated secondary signalling [58]. Moreover, TBEV is able to down-regulate the level of IFNAR in order to minimise secondary signalling activation. The mechanistic study demonstrates that the NS5A of TBEV inhibits the host prolidase enzyme, which is required for IFNAR maturation and accumulation on the cell surface [59]. DCs represent an early target of TBEV infection and are major producers of IFN. Thus, interactions between DCs, IFN responses, and the virus are likely to substantially influence the outcome of infection. Interestingly, early IFN and DC responses are modulated not only by the virus, but also by immunomodulatory compounds of the tick saliva inoculated with the virus into the skin [60,61]. After virus recognition, DCs migrate to local lymphoid tissues and undergo a process of maturation that involves cytokine production and antigen presentation to activate naive T cells and shape adaptive immunity. Many flaviviruses infect DCs, resulting in impaired DC maturation and T cell priming/proliferation. This manipulation of DC function is thought to be important in virus pathogenesis, although the molecular mechanisms are poorly understood [62,63].

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LGTV infection has been recently shown to affect DC maturation by suppressing the expression of co-stimulatory molecules such as IL12 by a coordinated impairment of IRF-1 and IFN-I signalling [64]. The IFN response is also critical to limit infection at the level of the CNS [44]. In particular, astrocytes have been recently shown to respond fast to TBEV infection by mounting a robust interferon response [65]. Therefore, the interferon response appears to be critical to limit LGTV/TBEV infection in mice both at the periphery, by limiting systemic dissemination of the virus, and in the CNS through a local antiviral response. 1.5. The integrated stress response to TBEV infection The Integrated Stress Response (ISR) is a cellular response to several types of stress, including hypoxia, nutrient deprivation, ER stress but also viral infection [66]. The ISR is designed to limit general cellular translation but it also facilitates the expression of stress-response genes to promote cell survival or apoptosis. The key event of the ISR is the phosphorylation of the a-subunit of the eukaryotic translation initiation factor eIF2a, performed by four different kinases that are activated by different stresses. Those most relevant to viral infection are: (i) PERK (Double-stranded RNAactivated protein kinase (PKR)-like endoplasmic reticulum kinase), a trans-membrane protein of the ER that sense ER stress due to accumulation of unfolded proteins. To note that PERK is also a key regulator of the unfolded protein response (UPR); and (ii) PKR (double stranded RNA activated protein kinase), which is ubiquitously expressed and is activated by sensing dsRNA, playing an important role in antiviral immunity. The phosphorylation of eIF2a upon infection has been demonstrated for several flaviviruses such as DENV [67,68]. Consistently, also TBEV infection triggers the phosphorylation of eIF2a, but the kinase responsible for this process was not investigated [69]. Phosphorylation of eIF2a blocks the loading of tRNAi(met) onto the small ribosomal subunit to initiate protein synthesis. Under these conditions, a stalled pre-initiation complex is formed, which, together with the associated mRNAs, the T-cell restricted intracellular antigen-1 (TIA-1) and TIA-1 related protein (TIAR) form cytoplasmic aggregates called stress-granules (SG) [70,71]. Further studies have demonstrated that SGs are composed also by other translation factors like eIF3, eIF4G and eIF4E as well as the polyA

binding protein (PABP), HuR and G3BP1 among others [72e74]. Together with cytoplasmic processing bodies (PB), SG regulate translation repression and decay of host mRNA [74]. A large numbers of studies have demonstrated that many viruses are able to manipulate the cellular components of the SG to regulate their formation [75]. WNV, DENV and JEV have been shown to interfere with SG assembly by hijacking the cellular localization of SG proteins [76e78] suggesting a common strategy to prevent the inhibition of viral mRNA translation. TBEV recruits TIA-1/R to sites of viral replication via viral RNA and these factors modulate viral translation [69]. However, TBEV is also able to trigger the formation of SG containing other components such as G3BP, eIF3 and eIF4B. Other studies demonstrated that also WNV and DENV are able to induce SG [79]. Taken together these evidences suggest that flaviviruses are able to hijack components of the SG such as TIA-1/R for their replication, but also to induce the formation of SG in infected cells. Studies on the temporal dynamics of SG formation following flavivirus infection could be very informative as it has been already demonstrated for HCV, which induces oscillatory formation of SG in cells stimulated with IFNa [80]. An intriguing additional role for SG in the interferon response has also been proposed. Cells infected with influenza virus showed localization of RIG-I, MDA5, PKR and viral RNA into virus-induced SG [81]. Moreover, depletion of G3BP1 reduced the expression of IFNb following infection. Such structures, dubbed as antiviral SG (avSG), may act as a platform for viral RNA sensing and activation of the interferon response [82]. In a very recent report this model has been further reinforced looking at the spatial and temporal dynamics of RIG-I in cells infected by Newcastle disease virus, a negative-strand RNA virus [83]. RIG-I initially localized to sites of virus replication with minimal induction of IFNb. Subsequently, RIG-I could be found together with viral RNA into avSG induced by the infection. A similar behaviour was observed in TBEV infected cells where RIG-I localized to SG 24 h post infection (Fig. 1), a timepoint when the interferon response to infection is fully activated [48]. These data may suggest a model where viral replication vesicles could be disrupted only at later time points allowing RIG-I sensing, which will be eventually amplified through avSG acting as a platform for IFNb induction. However, further investigations are required to fully analyse this phenomenon in TBEV infected cells.

Fig. 1. TBEV-induced antiviral stress granules. RIG-I localize to TBEV induced stress granules. U2OS cells were either mock infected or infected with TBEV at a MOI of 1. At 24 h post infection (h.p.i.) cells were fixed and immunostained with a G3BP1 antibody to detect stress granules and a RIG-I antibody to study localization of the antiviral protein. Arrows indicate Stress Granules. Scale bar 10 mm.

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1.6. The Unfolded Protein Response Protein homeostasis (proteostasis) is perturbed in several ways by virus infection. Flaviviruses completely remodel the ER-derived membranes and overload the lumen with their own proteins, thus draining the cellular ER chaperones and triggering stress responses. The Unfolded Protein Response is a cellular response to ER stress mediated by three ER trans-membrane receptors: (i) the activating transcription factor 6 (ATF6); (ii) the inositol requiring kinase 1 (IRE1) and (iii) PERK [84]. The ER chaperone protein BiP (Binding immunoglobulin Protein) tightly regulates these pathways in response to ER stress. In physiological conditions, BiP acts as a chaperone for the folding of newly synthesized proteins but also binds the luminal domain of the transmembrane ER stress transducers PERK, ATF6 and IRE1, blocking their activation. However, in presence of an excess unfolded proteins in the lumen of the ER, BiP dissociates from the UPR receptors inducing their activation. The general purpose of the UPR is to removes misfolded proteins either by attenuating general translation or by enhancing the ER folding capacity and ER-associated degradation. However, the UPR may

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also signal to apoptosis ultima ratio. Increasing evidences suggest an intimate relationship between viruses and the UPR. Indeed, if, on one hand, the host activates the UPR in an attempt to restrict virus infection, on the other hand, the virus can manipulates the UPR to facilitate its own replication and promote cell survival. Furthermore, ER stress has been linked to the production of pro-inflammatory cytokines and type I interferon [85]. Flaviviruses activate all the three pathways of the UPR. Infection with JEV or DENV induced the IRE1/Xbp1 pathway protecting cells from virus-induced cytopathic effects [86]. However, DENV induction of IRE1/Xbp1 and ATF6 pathways has also been shown to promote DENV pathogenesis [87]. WNV and JEV trigger the UPR-induced pro-apoptotic transcription factor CHOP (CCAAT/ enhancer-binding protein homologous protein) [68,88]. DENV infection leads to induction of the UPR in a sequential order: early PERK activation followed by IRE1/Xbp1 and ATF6 [67]. Finally, TBEV appears to manipulate the UPR to facilitate its own replication. Indeed, during infection IRE1 and ATF6 pathways are activated and chemical inhibition targeting of the UPR impaired TBEV

Fig. 2. Cellular pathways induced by TBEV. The drawing depicts a simplified life cycle of TBEV infection with the cellular pathways that are activated following infection. TBEV uncoating from endosomes releases the capped ssRNA, which is translated into a single trans-membrane polyprotein at the ER. Replication vesicles (RV) are formed as invagination of the ER membrane, which contain the replicative complex that extrudes newly synthetized viral RNA in a cytosolic compartment. Assembly occurs also on the ER membrane and the virion maturates through the ER, Golgi and Trans-Golgi network until released from the infected cell. Cellular pathways that are triggered by the infection include: the activation of class I IFN and IRF3-responsive ISG; the induction of (antiviral) stress granules (avSG; SG) by the integrated stress response pathway; the activation of the ER stress response and unfolded protein response pathway (UPR).

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replication [89]. The non-structural proteins NS4A and NS4B of WNV induce Xbp-1 transcription and splicing when expressed individually [90]. Consistently, NS2B/3 of DENV induced Xbp-1 splicing [86]. Such non-structural proteins are ER membrane associated proteins, which are required for virus replication. Hence, a pro-viral role of the UPR could be justified, although fine tuning is required to avoid detrimental UPR effects like inhibition of viral translation, mRNA processing or induction of apoptosis. Finally, ATF6 and IRE1/Xbp1 contribute to the inhibition of IFN signalling mediated by STAT1 phosphorylation [91].

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1.7. Final remarks TBEV infection leads to profound modifications of the host mammalian cell (Fig. 2). The virus replicates on ER membranes forming vesicles where the viral polymerase can synthetize new RNA genomes undisturbed by cytoplasmic sensors. Virions are then assembled in the ER and secreted to infect other cells. The principal antiviral response to TBEV is delayed at the cellular level implying viral countermeasures to interferon activation. Intriguingly, several other cellular responses in addition to interferon are triggered by the infection, such as the integrated stress response and the unfolded protein response. How these responses lead to antiviral signalling and how TBEV interferes with this signalling and takes advantage of these pathways is still poorly understood. In the following years it will be important to dissect the temporal and spatial induction of each of these pathways following infection and to study their interconnections and causal relationships. To note, these studies may be conducted in relevant cells of the host, such as dendritic cells, macrophages and CNS cells, to understand the pathology of the infection and to establish novel targets for therapy. These are exciting times for TBEV research, which could also serve as a powerful model for other flaviviruses.

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Acknowledgements We thank present and past members of the Molecular Virology Laboratory for useful discussions. Work on Flaviviridae is funded by the Beneficentia Stiftung and by the Regional grant Flavipoc.

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