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1 - University of Canberra Research Publication Collection Faculty of Health Peer-Reviewed Journal Article for Dr. Reena Ghildyal (Associate Professor, University of Canberra) This is the peer reviewed version of the following article: Citation: Parisa Younessi, David Jans, Reena Ghildyal. (2012). Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavirus Infection. Infectious Disorders - Drug Targets, 12 (1), pp , doi: / Find this item in the UC Research Repository: Copyright: 2012 Bentham Science This article has been published in final form at: Version: This is the authors final version of a work that was accepted for publication in Infectious Disorders - Drug Targets. Changes resulting from the publishing process, such as editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. University of Canberra, ACT 2601 Australia, Switchboard The University of Canberra is located on Ngunnawal Country. CRICOS number: University of Canberra / University of Canberra College #00212K.

2 Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavirus Infection Parisa Younessi, David A. Jans*, Reena Ghildyal Faculty of Applied Science, University of Canberra, Canberra; *Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne; Australia All correspondence to: Dr Reena Ghildyal, Room 3D51, Faculty of Applied Science, University of Canberra, Bruce, ACT 2615, Australia Tel: ; Keywords: Picornavirus, rhinovirus, poliovirus, cardiovirus, nucleocytoplasmic trafficking, nucleoporins. Abbreviations: HRV: human rhinovirus; PV: poliovirus; FMDV: foot and mouth disease virus; EMCV: Encephalomycocarditis virus; TMEV: Theiler s murine encephalomyelitis; IRES: Internal ribosome entry site; Nups: nucleoporins; NE: nuclear envelope; PTB: polypyrimidine tract binding protein; NPC: nuclear pore complex; NLS: nuclear localization sequence; NES: nuclear export sequences; COPD: chronic obstructive pulmonary disease; IFN; Interferon. 1

3 ABSTRACT Picornavirus infection is characterised by host cell shutoff, wherein host transcription and translation processes are severely impaired. Picornavirus proteins interact with host cell proteins, resulting in alterations in the host cell synthetic, signalling and secretory machinery, and facilitating transcription and translation of viral proteins to achieve increased virus replication and assembly. Among the many cellular pathways affected, recent studies have shown that disruption of nucleocytoplasmic trafficking via inhibition of the functions of the nuclear pore may be a common means of picornavirus-induced pathogenesis. Disruption of nuclear pore functions results in nuclear proteins being relocalised to the cytoplasm and reduced export of RNA, and may be a possible mechanism by which picornaviruses evade host cell defences such as apoptosis and interferon signalling, by blocking signal transduction across the nuclear membrane. However, the mechanisms used and the viral proteins responsible differ between different genera and even between viruses in the same genus. This review aims to summarise current understanding of the mechanisms used by picornaviruses to disrupt host cell nucleocytoplasmic trafficking. 2

4 Introduction Picornaviruses are positive sense single-stranded RNA viruses, and include medically significant viruses such as poliovirus (PV), rhinovirus (HRV), and hepatitis A virus (HAV), as well as viruses of veterinary importance such as Foot and mouth disease virus (FMDV) and Encephalomyocarditis virus (EMCV) [1]. PV is the causative agent of poliomyelitis that can affect nerves and thereby lead to partial or full paralysis [2]. HRV is the major cause of common colds and exacerbation of other respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma [3, 4]. EMCV is a major pathogen of pigs but can also cause disease in several other animals and has been the cause of outbreaks in zoos worldwide; human infection is uncommon. Mengovirus infects rodents and rarely humans, causing mild disease. Theiler's murine encephalomyelitis virus (TMEV) is responsible for infections of the central nervous system of the mouse and can induce a chronic, progressive, demyelinating disease in susceptible mice. These viruses have a striking capacity to persist in the central nervous system in spite of a strong and specific immune response [5]. FMDV, the prototypic member of the aphthovirus genus, is the etiologic agent of FMD, a highly contagious disease that affects wild and domestic cloven-hoofed animals, including swine and cattle [6]. According to the revised classification of 2010, the family Picornaviridae belongs to the order Picornavirales and consists of 12 genera: Enterovirus, Cardiovirus, Aphthovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus, Sapelovirus, Senecavirus, Tremovirus and Avihepatovirus (Table 1). The two original human rhinovirus species are within the genus Enterovirus, with the previous Rhinovirus genus no longer extant. The Sapelovirus, Senecavirus, Tremovirus and Avihepatovirus genera (along with a number of new picornavirus species) were formally recognised by the ICTV in August 2009 [7]. Picornaviridae family members replicate in the cytoplasm of the infected cell. Infection is initiated by binding of virus to its cell surface receptor, followed by release of the viral genome into the cytoplasm from endocytosed vesicles. The genome of picornaviruses is a single positive-stranded RNA molecule which is infectious because it is translated on entry into the cell to produce all the viral proteins required for viral replication. Nucleotide sequence analysis of picornavirus genomic RNA has revealed a common organizational pattern. The genomic RNA is translated into a single polyprotein, which is cleaved during translation, so that the full-length product is not observed. Cleavage is carried out by virusencoded proteinases (mainly 2A protease and 3C/3CD protease) to yield 11 to 12 final cleavage products. The picornavirus genome is divided into three regions, the P1 region encodes the viral capsid proteins, whereas the P2 and P3 regions encode proteins involved in protein processing (2A, 3C and 3CD proteases) and genome replication (2B, 2C, 3AB, 3BVPg, 3D polymerase) [5] (see Figure 1). Aphthoviruses and cardioviruses encode a leader (L) protein before the P1 region. The 2A protease of enteroviruses is responsible for the first polyprotein cleavage. In all other picornaviruses, 2A is not a protease, although in cardioviruses and aphthoviruses 2A causes its own release from 2B via an unknown mechanism. The aphthovirus L protease catalyses its own cleavage from P1. The majority of the polyprotein cleavages in all picornaviruses are performed by 3C protease, which is essential for virus replication and polypeptide maturation, and hence a major target for anti-viral therapy [4]. 3

5 Infection by picornaviruses results in host cell shutoff, wherein host cell transcription and translation are switched off while viral transcription and translation continue unabated [5]. Like other positive sense RNA viruses, picornaviruses recruit host proteins during replication to assist in intracellular localization of the viral proteins, replication and assembly [8]. All picornaviruses have an internal ribosome entry site (IRES) element in viral genomic RNA which recruits the host cell translation initiation factors to enable optimal polyprotein translation; at least six host proteins including UNR, a partner of poly (A) binding protein [9], polypyrimidine tract binding protein (PTB) [10], La and Poly(rC)-binding protein 2 (PCBP2), a member of a group of pre-mrna factors are involved in virus translation [11]. Recruitment of these factors by the viral IRES results in their non-availability for cellular functions, probably contributing to the host cell shutoff. Some of these factors (e.g. La, Sam68) are normally present in the nucleus, but infection results in their mislocalisation to the cytoplasm (see Section below) [12]. Cleavage of eif4g in enterovirus-infected cells is catalysed by 2A protease, representing a major mechanism underlying virus-induced inhibition of host cell translation. In recent years, disruption of nucleocytoplasmic transport has emerged as a key mechanism whereby picornaviruses induce host cell shutoff [12, 13]. Efficient transport of macromolecules across the nuclear envelope (NE) is essential for optimal cellular translation as well as the host response to infection. This review will focus on the various mechanisms used by picornaviruses to disrupt nucleocytoplasmic transport and the subsequent effect on the host. 1. Nucleocytoplasmic transport Eukaryotic cells sequester their genome in the nucleus, which is surrounded by the double lipid bilayer structure of the NE. The only avenue for transport into and out of the nucleus is via the NE-embedded nuclear pore complexes (NPCs) that are made up of over 40 different proteins called nucleoporins (Nups) (see Figure 2A). Although diffusion of molecules < 55 kda can occur, most transport through the NPC is mediated by members of the importin superfamily, which recognize nuclear localization sequences (NLSs) or nuclear export sequences (NESs) on cargo molecules for transport into and out of the nucleus respectively [14-18]. Importins function by binding NLSs and docking transiently at various FG (Phe- Gly repeat containing) Nups within the NPC to effect translocation through it, followed by release within the nucleus facilitated by the guanine nucleotide binding protein Ran [19]. The best studied nuclear import pathway is mediated by the importin α/β1 heterodimer, where importin α recognizes and directly binds to the NLSs of the cargo, and importin β1 mediates binding of the import complex to Nups; most nuclear import cargoes probably enter the nucleus through the direct action of importin β1 or homologues thereof. In all cases, release within the nucleus occurs through binding of Ran in GTP-bound form to importin β1 or homologues to effect dissociation of the import complex. Nuclear export is analogous to nuclear import, wherein cargo molecules with NESs bind importin β homologues such as CRM-1 (exportin 1) and are transported out of the nucleus; release in the cytoplasm is facilitated by Ran hydrolysis of GTP to GDP, which leads to dissociation of the export complex [20]. 4

6 1.1. Nucleocytoplasmic transport in host response to virus infection. The innate antiviral response, mediated principally by the action of Type-I interferons (IFNs), is one of the earliest responses of the host to viral infection and is activated by the cellular recognition of viral by-products termed pathogen associated molecular patterns (PAMPs) [21]. Two major receptors for intracellular PAMPs (e.g. viral RNA) are cellular helicases retinoic acid-inducible protein 1 (RIG-1 and melanoma-differentiation-associated gene 5 (MDA-5) [22]. RIG-1 and MDA-5 recognise viral PAMPs and initiate a cascade of events resulting in the induction of transcription factors including IFN response factor 3 (IRF-3), nuclear factor NFĸB and activating protein 1 (AP1). These transcription factors are then transported specifically into the nucleus through the action of the importins (see above) to activate transcription of IFN-β. IFN-β mrna is then exported specifically out of the nucleus where it is translated into protein which is ultimately secreted from the cell to induce a secondary cellular response in an autocrine and paracrine manner by binding to the IFN-α/β receptor (IFNAR). This in turn leads to activation of a second cascade of events involving several effectors and transcription factors such as the STAT (signal transducer and activator of transcription) proteins. These proteins then translocate into the nucleus, interact with IFNsensitive response elements (ISRE) and activate transcription of a broad range of IFN stimulated genes (ISGs). The ISG gene products mount a concerted immune response which targets and kills the virus [22]. Thus, optimal nuclear import of specific transcription factors/export of specific mrnas is central to an effective host response. 2. Inhibition of nucleocytoplasmic transport by picornaviruses During infection, picornavirus proteins interact with several host proteins resulting in alterations to the host cell synthetic, signalling and secretory machinery, and facilitating transcription and translation of viral proteins to increase virus replication and assembly. Among the many cellular pathways affected, recent studies have shown that disruption of NPC function may be a common means of picornavirus-induced pathogenesis, although the mechanisms used and the viral proteins responsible differ between distinct genera (see Table 2). Most knowledge regarding the disruption of nuclear transport in picornaviruses is derived from studies of PV and HRV infection, and more recent studies in cardioviruses and aphthovirus, but there is no information regarding disruption of nucleocytoplasmic transport in cells infected by other picornaviruses Disruption of nucleocytoplasmic trafficking by enteroviruses HRV and PV infection results in disruption of nucleocytoplasmic transport leading to mislocalisation of endogenous proteins such that several nuclear proteins are observed in the cytoplasm. Intriguingly, several Nups are degraded upon infection (see Figure 2B/C) [12, 13, 23]. It seems clear that Nup degradation is a key mechanism to modify cellular signal transduction leading to evasion of the host immune system and an increase in virus replication Rhinovirus HRV infection of cells results in the general disruption of nucleocytoplasmic trafficking and hence affects multiple cellular signalling pathways. This has been shown by examining both endogenous cellular proteins and ectopically expressed reporter proteins such as green 5

7 fluorescent protein (GFP) fused to a strong NLS which is normally strongly nuclear in uninfected cells, but diffusely distributed between the cytoplasm and nucleus in HRVinfected cells [12]. General disruption of nuclear import results in the relocalisation of nucleolin, Sam68 and La to the cytoplasm, and is attributable to the degradation of the specific NPC components Nups 153 and 62 [12, 16, 24, 25] through the action of the 2A and 3C proteases. Experiments with HeLa cell lysates imply that HRV 2A protease is able to cleave Nup62 [26], while HRV 3C protease has been shown to be able to disrupt nucleocytoplasmic trafficking, apparently via degradation of specific Nups (described below). Using a semi-intact cell system [16], we showed that 3C can disrupt both active and passive nuclear import, dependent on its proteolytic activity. Further, endogenous proteins are mislocalised in cells transfected to express GFP-fused 3C. Interestingly, in cells transfected to express GFP-3C, Nup153, 214 and 358 but not 62 were degraded, raising the possibility that 3C, in addition to 2A, may contribute to the overall degradation of Nups observed in HRV-infected cells. Of note, 3C protease has been shown to localise to the nucleus of HRVinfected cells [27] and hence is clearly able to access NPC components in infected cells; in contrast, the localisation of 2A protease in HRV-infected cells has not been established. At present, the precise contribution of 3C and 2A to overall Nup degradation, their possible synergistic action and the kinetics of their action in vivo, remain to be determined Poliovirus PV-infected cells demonstrate profound disruption of nucleocytoplasmic trafficking as shown by mislocalisation of normally nuclear proteins to the cytoplasm. The PV-induced change in NE permeability induced by Nup degradation is accompanied by mislocalisation of various endogenous nuclear proteins involved in cellular transcription and translation [28]. Predominantly nuclear mrna binding proteins, such as those of the heterogeneous nuclear ribonucleoproteins (hnrnp) family and La protein involved in mrna translation, are mislocalised to the cytoplasm in PV infected cells [12, 29, 30]. Electron microscopic analysis of the PV-infected cells indicated perforation of the NE in comparison to mock-infected cells [9]. GFP fused to a strong NLS was found to localise to the nucleus in HeLa cells, but was distributed diffusely throughout the cell following PV infection; this mislocalisation could be reversed by inhibitors of poliovirus 2A, including the elastase inhibitors methoxysuccinyl-ala- Ala-Pro-Val-chloromethylketone (MPCMK) and elastinal, suggesting that 2A is responsible for mislocalisation of nuclear proteins in PV-infected cells [31]. This is consistent with the fact that expression of PV 2A protease in HeLa cells results in cleavage of Nup62, Nup98 and Nup153, concomitant with disruption of RNA export from the nucleus [32]. Since inhibition of mrna export is not observed in PV-infected cells, this effect may stem largely from the fact that 2A is over-expressed in this system [23]. Hela cell extracts treated with recombinant PV 3C protease show cleavage of TATA-binding protein (TBP) as determined by Western analysis [33]. Unlike HRV 3C protease, PV 3C does not localise to the nucleus of cells when expressed alone in transfected cells, with PVinfection required for 3C nuclear localisation. The examination of cells transiently expressing GFP-3C and mcherry-2a suggests that 3C nuclear localisation is dependent on 2A proteolytic activity, possibly to disrupt NPC function; co-expression of a protease inactive 2A does not enable nuclear localisation of 3C. PV 2A protease localises to the NE in transfected cells, where it is able to catalyse Nup degradation and effect NPC dysfunction [34]. The degradation of Nups by 2A together with cleavage of essential transcription factors by 3C protease would appear to be central to PV-induced host cell shutoff. 6

8 2.2. Inhibition of Nucleocytoplasmic Trafficking by Cardioviruses Cardiovirus infection, in contrast to that by enteroviruses, does not result in cleavage of FG Nups, although nuclear trafficking is disrupted [35, 36]. Cardioviruses appear to inhibit nucleocytoplasmic trafficking through the activity of their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide that has no known enzymatic activity. Infection with TMEV in cell culture results in only low induction of IFN, even though nuclear translocation of the IFN transcriptional activator IRF-3 is induced. Nuclear localisation of IRF-3 is probably due to loss of integrity of the NPC induced by action of L (see Figure 2D). This is supported by the finding that, in cells infected with TMEV mutated in L where effects on the NPC are absent, no nuclear localisation of IRF-3 is observed [37]. Consistent with this idea, an NLScontaining GFP is more cytoplasmic in cells infected with TMEV than non-infected cells, presumably through loss of NPC integrity resulting in the inability of NLS-containing proteins to localise strongly in the nucleus; again, this is not the case in cells infected with TMEV containing the mutated L and hence possessing an intact, functional NPC. Similar effects were observed for endogenous proteins; PTB was rapidly redistributed to the cytoplasm of TMEV-infected cells but not in cells infected with TMEV with the mutated L [37]. Importantly, since cytoplasmic PTB forms part of the viral replicative machinery, the effect of wild type L in facilitating PTB mislocalisation contributes strongly to viral replication. Ricour et al. (2009 #171) performed random mutagenesis of L in the context of the whole virus followed by selection for reduced toxicity, demonstrating a role for the C-terminus of L in inhibition of cytokine transcription correlating with effects on NPC integrity as indicated by localisation/mislocalisation of PTB [38]. EMCV L is also able to disrupt nuclear trafficking directly as shown in nuclear import assays using semi-intact HeLa cells loaded with a fluorescent NLS-containing reporter protein; addition of recombinant L resulted in loss of the protein from the nucleus [39]. This was further supported by experiments using a cytomegalovirus promoter-driven luciferase reporter where luciferase activity was significantly reduced in the presence of IRESdependent expression of wild type but not mutated L. EMCV L has a well characterised N- terminal zinc finger domain and a C-terminal acidic domain; deletion of either, or a point mutation disrupting zinc binding, resulted in loss of inhibition of luciferase activity. As L cannot inhibit transcription in vitro, these results suggest that another mechanism, probably disrupted nuclear entry of key transcription factors, is the cause of the reduced expression [40]. EMCV L is localised to the cytoplasmic side of the NE in infected cells and is able to bind and pull down RanGTP from whole cell lysates [40]. Since Ran plays a key role in driving nuclear import/export, targeting Ran to disrupt the Ran gradient would be expected to result in profound disruption of all nuclear trafficking. Infection with EMCV but not EMCV mutated in L results in hyperphosphorylation of Nup62, implying an indirect/direct role of L; consistent with this, recombinant L is able to induce Nup62 phosphorylation in digitoninpermeabilised cells, concomitant with disruption of nuclear trafficking [39]. Disruption of nuclear trafficking by L can be inhibited by the broad spectrum kinase inhibitor Staurosporine, but cannot be overcome by the addition of exogenous Ran [39]. Work by the same group using specific kinase inhibitors implies that MAP kinases ERK and p38 are the most likely cellular kinases involved [41]. Together, the data suggest that EMCV L disrupts nuclear trafficking via phosphorylation of Nup62 rather than effects on Ran, although this requires further examination. Comparable Nup phosphorylation has been observed in 7

9 Mengovirus-infected cells [42], implying a common mechanism to disrupt nuclear trafficking by cardioviruses. Interestingly, EMCV 2A, which, unlike the enterovirus 2A, lacks proteolytic activity, accumulates in nucleoli shortly after infection. This localisation is facilitated by an NLS, mutation of which results in 2A cytoplasmic localisation. The functional significance of EMCV 2A nucleolar accumulation in host cell shutoff is currently unclear [43]. Clearly, cardioviruses, like enteroviruses, disrupt nucleocytoplasmic transport, but, unlike enteroviruses, may do so via hyperphosphorylation rather than degradation of specific Nups, to alter NPC function without gross effects on structure Inhibition of NFkB function by FMDV L protein Like other picornaviruses, FMDV infection results in host cell shutoff, although it is not clear if disruption of nuclear trafficking is part of the host cell shutoff, or indeed, occurs at all [44]. FMDV 3C protease, similar to the PV 3C, cleaves key nuclear factors resulting in inhibition of mrna transcription [45], but how it accesses them is not known. Unlike cardioviruses, FMDV L has protease activity and has a role in virus polyprotein processing, catalysing its release from P1. L also has a well known role in host cell shutoff, cleaving the translation initiation factor eif4g in a fashion reminiscent of enterovirus 2A. Given these key functions of L, it is not surprising that FMDV lacking L is severely attenuated [46]. L localises to the nucleus of FMDV infected cells via an as yet unknown mechanism, correlating with a decrease in the nuclear levels of nuclear factor (NF)-κB p65/rela subunit [47, 48]. Infection with TMEV that expresses FMDV L protease in the absence of any other FMDV protein results in nuclear localisation of the L protease and degradation of p65/rela; mutation of the L protease active site does not affect L nuclear localisation but inhibits cleavage of p65/rela [49]. The sequences responsible for L nuclear localisation were mapped to a SAP (for SAF- A/B, Acinus, and PIAS)-like domain, associated with binding to the nuclear matrix/nuclear retention of molecules involved in transcriptional control [50]; mutation of this SAP domain resulted in altered localisation of L protease [51]. FMDV L has no effect on mrna distribution or Nup98 integrity [32]. Clearly, detailed studies are still required to establish whether aphthoviruses, like entero- and cardioviruses disrupt host cell nuclear trafficking by targeting Nups, and thereby contribute to host-cell shutdown. 3. Conclusion Viruses frequently disrupt cellular processes such as transcription, translation and DNA synthesis in an effort to augment viral replication. Picornaviruses are especially efficient in inhibition of host cell transcription and translation, such that almost no cellular proteins can be detected by the time of peak replication activity; disruption of nucleocytoplasmic trafficking appears to be a key regulator of these effects that bring about host cell shutoff. Picornavirus infection results in several nuclear proteins being re-localized to the cytoplasm, presumably representing a mechanism by which picornaviruses evade host cell defences such as apoptosis and IFN signalling by interfering with signal transduction to and from the nucleus. Thus far, proteases appear to play major roles in disruption of nucleocytoplasmic trafficking by picornaviruses, with PV and HRV 2A protease, HRV 3C protease, all having been shown to be important in this process. However other proteins may be just as important, as has been shown in the case of the cardiovirus L protein that mediates 8

10 disruption of NPC function rather than structure, and thereby affects IFN signalling. Interestingly, research within these families has focussed primarily on 2A protease and L protein (protease) with little or no focus on the 3C protease. Given the recent findings as to the effect of HRV 3C protease on the NPC/host cell protein nuclear trafficking, and the fact that all picornaviruses have a 3C protease, it may be presumed to have similar roles in infection by other picornaviruses. Research into disruption of nuclear trafficking in other family members within Picornaviridae is still in the early stages. Clearly, understanding of the varied mechanisms used by picornaviruses to disrupt nucleocytoplasmic trafficking is far from complete. Identifying additional targets at the NPC and other viral mediators, however, is central to understanding the complex pathogenic mechanisms of picornaviruses, and the key to developing new strategies to combat them. 9

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14 46. Brown CC, Piccone ME, Mason PW, McKenna TS & Grubman MJ (1996) Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. J Virol 70, de los Santos T, de Avila Botton S, Weiblen R & Grubman MJ (2006) The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mrna and Blocks the Host Innate Immune Response. J Virol 80, de los Santos T, Diaz-San Segundo F & Grubman MJ (2007) Degradation of Nuclear Factor Kappa B during Foot-and-Mouth Disease Virus Infection. J Virol 81, de los Santos T, Segundo FD, Zhu J, Koster M, Dias CC & Grubman MJ (2009) A conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function. J Virol 83, Aravind L & Koonin EV (2000) SAP - a putative DNA-binding motif involved in chromosomal organization. Trends in Biochemical Sciences 25, de los Santos T, Diaz-San Segundo F, Zhu J, Koster M, Dias CCA & Grubman MJ (2009) A Conserved Domain in the Leader Proteinase of Foot-and-Mouth Disease Virus Is Required for Proper Subcellular Localization and Function. J Virol 83, Norder H, De Palma AM, Selisko B, Costenaro L, Papageorgiou N, Arnan C, Coutard B, Lantez V, De Lamballerie X, Baronti C, Solà M, Tan J, Neyts J, Canard B, Coll M, Gorbalenya AE & Hilgenfeld R (2011) Picornavirus non-structural proteins as targets for new anti-virals with broad activity. Antiviral Research In Press, Uncorrected Proof. 53. Bastos R, Lin A, Enarson M & Burke B (1996) Targeting and function in mrna export of nuclear pore complex protein Nup153. J Cell Biol 134, Cammas A, Pileur F, Bonnal S, Lewis SM, Leveque N, Holcik M & Vagner S (2007) Cytoplasmic Relocalization of Heterogeneous Nuclear Ribonucleoprotein A1 Controls Translation Initiation of Specific mrnas. Mol Biol Cell 18, Sukarieh R, Sonenberg N & Pelletier J (2010) Nuclear assortment of eif4e coincides with shut-off of host protein synthesis upon poliovirus infection. J Gen Virol 91, Kleijn M, Vrins CL, Voorma HO & Thomas AA (1996) Phosphorylation state of the cap-binding protein eif4e during viral infection. Virology 217, Porter FW & Palmenberg AC (2009) Leader-Induced Phosphorylation of Nucleoporins Correlates with Nuclear Trafficking Inhibition by Cardioviruses. J Virol 83,

15 FIGURE LEGEND Figure 1. A. General organization of Picornaviridae genome that may differ between species; adapted from [52]. The positive strand RNA genome has a 5 -VPg element and a 3 poly-a tail (An) that are important for efficient translation. The polyprotein encoding region is flanked by 3 and 5 untranslated regions (UTRs). The coding regions for specific structural and nonstructural proteins are indicated. The L protein and 2A protein are not homologous through all the picornaviruses and their coding regions are shown shaded. B. Polyprotein processing in Enterovirus, Cardiovirus and Aphthovirus species; adapted from [5]. The polyprotein products of the three species are shown with the primary cleavages catalysed by specific proteases shown by arrows. L - Leader Protein, VP1 to VP4 - viral capsid proteins, 2A - protease except in cardiovirus, 2C - NTPase, 3B VPg coding region (3 copies in Aphthoviruses), 3C - viral protease, 3D RNA dependent RNA polymerase. P1, P2, P3 products of primary proteolytic cleavage of the polyprotein. Figure 2. Schematic representation of the disruption of the nuclear pore and nucleocytoplasmic trafficking in picornavirus infection. A. Schematic representation of a nuclear pore complex (NPC) showing the cytoplasmic fibrils, cytoplasmic pore, nuclear ring and nuclear basket and highlighting some of the key nucleoporin (Nups) components of the NPC. The directionality of transport of transcipition factors and mrna into and out of the nucleus respectively is also shown. B. In rhinovirus infection, 3C protease degrades the Nup214 and Nup358 located on the cytoplasmic side of the NPC (1), 2A protease degrades Nup62 and possibly Nup98 embedded in the NPC (2), while both 2A and 3C protease are able to degrade Nup153 (3) located on the nucleoplasmic side of the NPC. This is believed to cause increased permeability/lack of selectivity of the NPC, and resultant mislocalisation from nucleus to cytoplasm of nuclear proteins e.g., La and nucleolin. C. In the case of Poliovirus proteases, 2A catalyses the degradation of Nup62, Nup98 and Nup153 (1,2) resulting in nuclear localisation of 3C (3), which degrades the transcription factor TATA binding protein (TBP). This is believed to cause increased permeability/lack of selectivity of the NPC, and result in mislocalisation of cellular proteins such as La, Nucleolin and hnrnp C1/C2 (4) enabling the viral IRES to co-opt these proteins for viral transcription and translation (5). D. In cardiovirus infection, hyperphosphorylation of Nup62 (1), dependent on cardiovirus L protein, results in a dysfunctional NPC that allows diffusion between nucleus and cytoplasm of cellular proteins such as interferon regulatory factor (IRF3, 2). 14

16 Table 1 Current classification of the Picornaviridae family [52]. Genus Enterovirus Cardiovirus Parechovirus Aphthovirus Hepatovirus Cosavirus Erbovirus Kobuvirus Teschovirus Sapelovirus Avihepatovirus Seneca virus Tremovirus Unassigned Species Human enterovirus A,B, C and D Simian enterovirus A Bovine enterovirus Porcine enterovirus A Human rhinovirus A, B, C Encephalomyocarditis virus Theilovirus Human parechovirus Ljunganvirus Foot-and-mouth disease virus Equine rhinitis A virus Bovine rhinitis B virus Hepatitis A virus Human Cosavirus Equine rhinitis B virus Aichi virus Bovine kobuvirus Porcine kobuvirus Porcine teschovirus Porcine Sapelovirus Bovine Sapelovirus Avian Sapelovirus Duck hepatitis virus Seneca valley virus Avian encephalomyelitis-like virus Seal picornavirus (SePV) Bat kobu-like virus Bluegill picornavirus Cosaviruses Eel picornavirus Human klassevirus / Salivirus Seal picornavirus Tortoise picornavirus Turdiviruses Turkey hepatitis virus 15

17 Table 2. Picornavirus components and their targets Virus Viral Component Host Protein Target HRV 3C and 2A protease [24, Nup153 53] HRV 3C protease Nup214 [27] HRV 2A protease Nup62 [24] HRV IRES hnrnp A1 [54] PV 2A protease Nup98, Nup153 [34] PV IRES Nucleolin [12] PV IRES hnrnp C1/C2 [28, 29] PV 3C protease La and TBP [30, 33] PV 2A protease eif4e [55, 56] Cardiovirus L protein Nup62 [57] FMDV L protease p65/rela [48] EMCV L protein eif4e [43, 56] Target Protein Alteration Degradation Degradation Degradation Mislocalisation to the cytoplasm Degradation Mislocalisation to the cytoplasm Mislocalisation to the cytoplasm and degradation Degradation Degradation/dephosphorylation Hyperphosphorylation Degradation Phosphorylation/dephosphorylation HRV human rhinovirus, PV poliovirus, FMDV foot and mouth disease virus, EMCV encephalomyocarditis virus, IRES internal ribosome entry site, Nup nucleoporin, TBP TATA binding protein, eif4e elongation initiation factor 4E, p65/rela 65kDa subunit of nuclear factor κb 16

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