Infectious Disorders – Drug Targets, 2012, 12, 59-67 59 Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavi- rus Infection

Parisa Younessi, David A. Jans# and Reena Ghildyal*

Faculty of Applied Science, University of Canberra, Canberra; #Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne; Australia

Abstract: 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 syn- thetic, signalling and secretory machinery, and facilitating transcription and translation of viral proteins to achieve in- creased replication and assembly. Among the many cellular pathways affected, recent studies have shown that dis- ruption of nucleocytoplasmic trafficking via inhibition of the functions of the nuclear pore may be a common means of pi- cornavirus-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 mechanism by which evade host cell defences such as 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 in the same genus. This review aims to summarise current understanding of the mechanisms used by picornaviruses to disrupt host cell nucleocytoplasmic trafficking. Keywords: Picornavirus, , poliovirus, , nucleocytoplasmic trafficking, nucleoporins.

1. INTRODUCTION , , , Tremovirus and Avi- hepatovirus (Table 1). The three poliovirus serotypes now Picornaviruses are positive sense single-stranded RNA belong to the species Human C and the species viruses, and include medically significant viruses such as Poliovirus no longer exists. The two human Rhinovirus spe- poliovirus (PV), rhinovirus (HRV), and virus cies are within the genus Enterovirus, with the previous Rhi- (HAV), as well as viruses of veterinary importance such as novirus genus no longer extant. The Sapelovirus, Senecavi- foot and mouth disease virus (FMDV) and encephalomyo- rus, Tremovirus and genera (along with a carditis virus (EMCV) [1]. number of new picornavirus species) were formally recog- PV is the causative agent of poliomyelitis that can affect nised by the ICTV in August 2009 [7]. nerves and thereby lead to partial or full paralysis [2]. HRV Picornaviridae family members replicate in the cyto- is the major cause of common colds and exacerbation of plasm of the infected cell. Infection is initiated by binding of other respiratory diseases such as chronic obstructive pul- virus to its cell surface receptor, followed by release of the monary disease (COPD) and asthma [3, 4]. EMCV is a major viral genome into the cytoplasm from endocytosed vesicles. pathogen of pigs but can also cause disease in several other The genome of picornaviruses is a single positive-stranded animals and has been the cause of outbreaks in zoos world- RNA molecule which is infectious as it is translated on entry wide; human infection, however, is uncommon. EMCV into the cell to produce all the viral proteins required for Mengo (previously named Mengovirus) infects rodents and viral replication. The genomic RNA is translated into a sin- rarely humans, causing mild disease. Theiler's murine en- gle polyprotein, which is cleaved during translation, so that cephalomyelitis virus (TMEV) is responsible for infections the full-length product is not observed. Cleavage is carried of the central nervous system of the mouse and can induce a out by virus-encoded (L , 2A protease and chronic, progressive, demyelinating disease in susceptible 3C/3CD protease) to yield 11 to 12 final cleavage products. mice. have a striking capacity to persist in the Nucleotide sequence analysis of picornavirus genomic RNA central nervous system in spite of a strong and specific im- has revealed a common organisation. The picornavirus ge- mune response [5]. FMDV, the prototypic member of the nome is divided into three regions, the P1 region encodes the genus, is the etiologic agent of FMD, a highly viral capsid proteins, whereas the P2 and P3 regions encode contagious disease that affects wild and domestic cloven- proteins involved in protein processing (2A protease in en- hoofed animals, including swine and cattle [6]. teroviruses, 3C and 3CD proteases in all picornaviruses) and According to the revised classification of 2010, the genome replication (2B, 2C, 3AB, 3BVPg, 3D polymerase) family Picornaviridae belongs to the order Picornavirales [5] (see Fig. 1). In addition, and cardioviruses and consists of 12 genera: Enterovirus, Cardiovirus, Aph- encode a leader (L) protein before the P1 region. thovirus, Hepatovirus, , , , The 2A protease of is responsible for the first polyprotein cleavage. In all other picornaviruses, 2A is *Address correspondence to this author at Rm 3D51, Faculty of Applied not a protease, although in cardioviruses and aphthoviruses Science, University of Canberra, Bruce, ACT 2615, Australia; Tel: +612 2A causes its own release from 2B via an unknown mecha- 6201 5755; Fax: +612 62012328; E-mail: [email protected] nism. The aphthovirus L protease catalyses its own cleavage

2212-3989/12 $58.00+.00 © 2012 Bentham Science Publishers 60 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 1 Younessi et al.

Table 1. Current Classification of the Picornaviridae Family from P1. The majority of the polyprotein cleavages in all [54] picornaviruses are performed by 3C protease, which is es- sential for virus replication and polypeptide maturation, and Genus Species hence a major target for anti-viral therapy [4].

1 Infection by picornaviruses results in “host cell shutoff”, Human enterovirus A,B, C and D wherein host cell transcription and translation are switched Simian enterovirus A off while viral transcription and translation continue unab- ated [5]. Like other positive sense RNA viruses, picornavi- Enterovirus Bovine enterovirus ruses recruit host proteins during replication to assist in Porcine enterovirus A intracellular localization of the viral proteins, replication and assembly [8]. All picornaviruses have an internal ribosome Human rhinovirus A, B, C entry site (IRES) element in their viral genomic RNA, which Encephalomyocarditis virus2 recruits the host cell translation initiation factors to enable Cardiovirus optimal polyprotein translation; at least six host proteins in- Theilovirus3 cluding UNR, a partner of poly (A) binding protein [9], polypyrimidine tract binding protein (PTB) [10], La and Human parechovirus Parechovirus Poly(rC)-binding protein 2 (PCBP2), a member of a group of Ljunganvirus pre-mRNA factors, are involved in virus translation [11]. Recruitment of these factors by the viral IRES results in their Foot-and-mouth disease virus non-availability for cellular functions, probably contributing Aphthovirus Equine rhinitis A virus to host cell shutoff. Notably, some of these factors (e.g. La, Sam68) are normally present in the nucleus, but infection Bovine rhinitis B virus results in their mislocalisation to the cytoplasm (see Section 2.1.1 below) [12]. Furthermore, cleavage of eIF4G in en- Hepatovirus Hepatitis A virus terovirus-infected cells is catalysed by 2A protease, repre- Human Cosavirus senting a major mechanism underlying virus-induced inhibi- tion of host cell translation. Erbovirus Equine rhinitis B virus In recent years, disruption of nucleocytoplasmic transport Aichi virus has emerged as a key mechanism whereby picornaviruses Kobuvirus Bovine kobuvirus induce host cell shutoff [12, 13]. Efficient transport of macromolecules across the nuclear envelope (NE) is essen- Porcine kobuvirus tial for optimal cellular translation as well as the host re- Teschovirus Porcine teschovirus sponse to infection. This review will focus on the various mechanisms used by picornaviruses to disrupt nucleocyto- Porcine Sapelovirus plasmic transport.

Sapelovirus Bovine Sapelovirus 2. NUCLEOCYTOPLASMIC TRANSPORT Avian Sapelovirus Eukaryotic cells sequester their genome in the nucleus, Avihepatovirus Duck hepatitis virus which is surrounded by the double lipid bilayer structure of the NE. The only avenue for transport into and out of the Seneca virus Seneca valley virus nucleus is via the NE-embedded nuclear pore complexes Tremovirus Avian encephalomyelitis-like virus (NPCs) that are made up of over 40 different proteins called nucleoporins (Nups) (see Fig. 2A). Although diffusion of Seal picornavirus (SePV) molecules < 55 kDa can occur, most transport through the Bat kobu-like virus NPC is mediated by members of the importin superfamily, which recognize nuclear localization sequences (NLSs) or Bluegill picornavirus nuclear export sequences (NESs) on cargo molecules for Cosaviruses transport into and out of the nucleus respectively [14-18]. Importins function by binding NLSs and docking transiently Eel picornavirus at various “FG” (Phe-Gly repeat containing) Nups within the Unassigned Human klassevirus / NPC to effect translocation through it, followed by release within the nucleus facilitated by the guanine nucleotide bind- Seal picornavirus ing protein Ran [19]. The best studied nuclear import path- way is mediated by the importin 1 heterodimer, where Tortoise picornavirus / importin  recognizes and directly binds to the NLSs of the Turdiviruses cargo, and importin 1 mediates binding of the import com- plex to Nups. However, most nuclear import cargoes prob- Turkey hepatitis virus ably enter the nucleus through the direct action of importin 1–Human enterovirus C includes all poliovirus serotypes. 1 or homologues thereof, not requiring importin . In all 2–   Mengovirus is now a serotype of EMCV. cases, release within the nucleus occurs through binding of 3–Genus Theilovirus includes TMEV Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavirus Infection Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 1 61

Fig. (1). A. General organization of Picornaviridae genome; adapted from [54]. 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 non-structural 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, Cardio- virus and Aphthovirus species; adapted from [5]. The polyprotein products of the three species are shown with the primary cleavages cata- lysed by specific proteases shown by arrows. L - Leader Protein, VP1 to VP4 - viral capsid proteins, 2A - protease in Enterovirus, 2C - NTPase, 3B – VPg coding region (3 copies in Aphthoviruses), 3C - viral protease, 3D – RNA dependent RNA polymerase. P1, P2, P3 – pro- ducts of primary proteolytic cleavage of the polyprotein.

Ran, in its GTP-bound form, to importin 1 or homologues tion factors are transported specifically into the nucleus to effect dissociation of the import complex. Nuclear export through the action of the importins (see above) to activate is analogous to nuclear import, wherein cargo molecules transcription of IFN-. IFN- mRNA is then exported out of containing NESs bind importin  homologues such as CRM- the nucleus, where it is translated into protein which is ulti- 1 (exportin 1) complexed with Ran in its GTP bound state mately secreted from the cell to induce a secondary cellular and are transported out of the nucleus; release in the cyto- response in an autocrine and paracrine manner, by binding to plasm is facilitated by Ran hydrolysis of GTP to GDP, which the IFN-/ receptor (IFNAR). This in turn leads to activa- leads to dissociation of the export complex [20]. tion of a second cascade of events involving several effectors and transcription factors such as the STAT (signal transducer 2.1. Nucleocytoplasmic Transport in Host Response to and activator of transcription) proteins. These proteins trans- Virus Infection locate into the nucleus, interact with IFN-sensitive response elements (ISRE) and activate transcription of a broad range The innate antiviral response, mediated principally by the of IFN stimulated genes (ISGs). The ISG gene products action of Type-I interferons (IFNs), is one of the earliest mount a concerted immune response which targets and kills responses of the host to viral infection and is activated by the the virus [22]. Thus, optimal nuclear import of specific tran- cellular recognition of viral by-products termed pathogen scription factors/export of specific mRNAs is central to an associated molecular patterns (PAMPs) [21]. Two major effective host response. receptors for intracellular PAMPs (e.g. viral RNA) are cellu- lar retinoic-acid-inducible protein 1 (RIG-1) and 3. INHIBITION OF NUCLEOCYTOPLASMIC melanoma-differentiation-associated gene 5 (MDA-5) [22]. TRANSPORT BY PICORNAVIRUSES RIG-1 and MDA-5 recognise viral PAMPs and initiate a cascade of events resulting in the activation of transcription During infection, picornavirus proteins interact with sev- factors including IFN response factor 3 (IRF-3), nuclear fac- eral host proteins resulting in alterations to the host cell syn- tor (NF) B and activating protein 1 (AP1). These transcrip- thetic, signalling and secretory machinery, and facilitating 62 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 1 Younessi et al. transcription and translation of viral proteins to increase vi- tion of 3C and 2A to overall Nup degradation, their possible rus replication and assembly. Among the many cellular synergistic action and the kinetics of their action in vivo re- pathways affected, recent studies have shown that disruption main to be determined, Nup degradation by 2A and 3C, to- of NPC function may be a common means of picornavirus- gether with cleavage of essential transcription factors by 3C induced pathogenesis, although the mechanisms used and the [27] and of translation factors by 2A [28], is almost certainly viral proteins responsible differ between distinct genera (see pivotal to host cell shutoff. Critically, Nup degradation pre- Table 2). Specific and efficient signal-dependent transport of vents the host cell mounting anti-viral responses that rely on macromolecules into and out of the nucleus is essential for nucleocytoplasmic trafficking of signalling molecules. optimal cell function as well as an effective anti-viral re- 3.1.2. Poliovirus sponse (see Section 2.1 above). Any disruption of nuclear transport would have profound effects on the intracellular PV-infected cells demonstrate profound disruption of signalling required for normal cell function, thus contribut- nucleocytoplasmic trafficking as shown by mislocalisation of ing to the observed pathogenesis. Most of our knowledge normally nuclear proteins to the cytoplasm. The PV-induced regarding the disruption of nuclear transport by picornavi- change in NE permeability caused by Nup degradation is ruses is derived from studies of PV and HRV infection, and accompanied by mislocalisation of various endogenous nu- more recent studies in cardioviruses and aphthoviruses, but clear proteins involved in cellular transcription and transla- there is no information regarding disruption of nucleocyto- tion [29]. Predominantly nuclear mRNA binding proteins, plasmic transport in cells infected by other picornaviruses. such as those of the heterogeneous nuclear ribonucleopro- teins (hnRNP) family and La protein involved in mRNA 3.1. Disruption of Nucleocytoplasmic Trafficking by En- translation, are mislocalised to the cytoplasm in PV infected teroviruses cells [12, 30, 31]. Electron microscopic analysis of PV- infected cells indicated perforation of the NE in comparison HRV and PV infections result in disruption of nucleocy- to mock-infected cells [9]. GFP fused to a strong NLS was toplasmic transport, leading to mislocalisation of endogen- found to localise to the nucleus in HeLa cells, but was distri- ous proteins such that several nuclear proteins are observed buted diffusely throughout the cell following PV infection; in the cytoplasm. Intriguingly, several Nups are degraded this mislocalisation could be reversed by inhibitors of polio- upon infection (see Fig. 2B/C) [12, 13, 23]. It seems likely virus 2A, including the elastase inhibitors methoxysuccinyl- that Nup degradation is a key mechanism to modify/prevent Ala-Ala-Pro-Val-chloromethylketone (MPCMK) and elasti- host cell signal transduction/anti-viral responses, thereby nal, suggesting that 2A is responsible for mislocalisation of leading to evasion of the host immune system and an in- nuclear proteins in PV-infected cells [32]. This is consistent crease in virus replication. with the fact that expression of PV 2A protease in HeLa cells 3.1.1. Rhinovirus results in cleavage of Nup62, Nup98 and Nup153, concomi- tant with disruption of RNA export from the nucleus [33]. HRV infection of cells results in the general disruption of The extent to which altered RNA export may occur in PV- nucleocytoplasmic trafficking and hence affects multiple infected cells has, however, not been established [23]. cellular signalling pathways. This has been shown by exam- ining both endogenous cellular proteins and ectopically ex- HeLa cell extracts treated with recombinant PV 3C pro- pressed reporter proteins such as a green fluorescent protein tease show cleavage of the TATA-binding protein (TBP) as

(GFP) fused to a strong NLS, which is normally strongly determined by Western analysis [34]. Unlike HRV 3C pro- nuclear in uninfected cells, but is diffusely distributed be- tease, PV 3C does not localise to the nucleus of cells when tween the cytoplasm and nucleus in HRV-infected cells [12]. expressed alone in transfected cells, with PV-infection re- General disruption of nuclear import by HRV results in the quired for 3C nuclear localisation. The examination of cells relocalisation of nucleolin, Sam68 and La to the cytoplasm, transiently expressing 3C and 2A fused to the fluorescent and is attributable to the degradation of the specific NPC proteins GFP and mCherry respectively suggests that 3C components Nups 153 and 62 [12, 16, 24, 25] through the nuclear localisation is dependent on 2A proteolytic activity, action of the 2A and 3C proteases. Experiments with HeLa possibly to disrupt NPC function; coexpression of a protease cell lysates imply that HRV 2A protease is able to cleave inactive 2A does not enable nuclear localisation of 3C. PV Nup62 [26], while HRV 3C protease has been shown to be 2A protease localises to the NE in transfected cells, where it able to disrupt nucleocytoplasmic trafficking, apparently via is able to catalyse Nup degradation and effect NPC dysfunc- degradation of specific Nups. Using a semi-intact cell system tion [35]. The degradation of Nups by 2A together with [16], we showed that 3C can disrupt both active and passive cleavage of essential transcription factors by 3C protease nuclear import, dependent on its proteolytic activity. Further, would appear to be central to PV-induced host cell shutoff. endogenous proteins are mislocalised in cells transfected to express GFP-fused 3C. Interestingly, in cells transfected to 3.2. Inhibition of Nucleocytoplasmic Trafficking by Car- express GFP-3C, Nup153, 214 and 358 but not 62 were de- dioviruses graded, raising the possibility that 3C, in addition to 2A, may Cardiovirus infection, in contrast to that by enteroviruses, contribute to the overall degradation of Nups observed in does not result in cleavage of FG Nups, although nuclear HRV-infected cells. Of note, 3C protease has been shown to trafficking is disrupted [36, 37]. Cardioviruses appear to in- localise to the nucleus of HRV-infected cells [27] and hence hibit nucleocytoplasmic trafficking through the activity of is clearly able to access NPC components in infected cells; in their leader (L) protein, a 67- to 76-amino acid (aa) polypep- contrast, the localisation of 2A protease in HRV-infected tide that has no known enzymatic activity. Infection with cells has not been established. Although the precise contribu- TMEV in cell culture results in only low induction of IFN, Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavirus Infection Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 1 63

Table 2. Picornavirus Components and their Cellular Targets

Virus Viral Component Host Protein Target Target Protein Alteration

HRV 3C and 2A protease [24, 55] Nup153 Degradation

HRV 3C protease [27] Nup214 Degradation

HRV 2A protease [24] Nup62 Degradation

PV 2A protease [35] Nup98, Nup153 Degradation

PV 3’ non coding region [29, 30] hnRNP C1/C2 Increased virus replication

PV 3C protease [31, 34] La, TBP Degradation

PV 2A protease [56, 57] eIF4E Degradation/ dephosphorylation

Cardiovirus L protein [58] Nup62 Hyperphosphorylation

EMCV L protein [45, 57] eIF4E Degradation

EMCV 2A protein [45] eIF4E Unknown

FMDV L protease [50] p65/RelA Degradation

FMDV L protease [59] eIF4G Degradation

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 B even though nuclear translocation of the IFN transcriptional ported by experiments using a promoter- activator IRF-3 is induced. Nuclear localisation of IRF-3 is driven luciferase reporter where luciferase activity was sig- probably due to loss of integrity of the NPC induced by the nificantly reduced in the presence of IRES-dependent ex- action of L (see Fig. 2D). This is supported by the finding pression of wild type but not mutated L. EMCV L has a well that, in cells infected with TMEV mutated in L where effects characterised N-terminal zinc finger domain and a C- on the NPC are absent, no nuclear localisation of IRF-3 is terminal acidic domain, deletion of either of which, or a observed [38]. Consistent with this idea, an NLS-containing point mutation disrupting zinc binding, results in loss of GFP is more cytoplasmic in cells infected with TMEV than inhibition of luciferase activity. As L cannot inhibit tran- non-infected cells, presumably through loss of NPC func- scription in vitro, these results suggest that another mecha- tionality resulting in the inability of NLS-containing proteins nism, probably disrupted nuclear entry of key transcription to localise strongly in the nucleus; again, this is not the case factors, is the cause of the reduced expression [42]. EMCV L in cells infected with TMEV containing the mutated L and is localised to the cytoplasmic side of the NE in infected hence possessing an intact, functional NPC. Endogenous cells and is able to bind and pull down RanGTP from whole proteins are similarly observed to be mislocalised; PTB was cell lysates [42]. Since Ran plays a key role in driving nu- rapidly redistributed to the cytoplasm of TMEV-infected clear import/export, altering Ran function would be expected cells but not in cells infected with TMEV with the mutated L to result in profound effects on all nuclear trafficking in the [38]. Importantly, since cytoplasmic PTB forms part of the cell. Infection with EMCV but not EMCV mutated in L re- viral replicative machinery, the effect of wild type L in fa- sults in hyperphosphorylation of Nup62, implying a role for cilitating PTB mislocalisation contributes strongly to viral L in modulating Nup62 phosphorylation; consistent with replication. It must be emphasised here that TMEV is a poor this, recombinant L is able to induce Nup62 phosphorylation inducer of IFN, so that the presence of IRF-3 in the nucleus in digitonin-permeabilised cells, concomitant with disruption is the result of a dysfunctional (“leaky”) NPC that allows of nuclear trafficking [41]. Disruption of nuclear trafficking endogenous proteins to pass through the NPC when this by L can be inhibited by the broad spectrum kinase inhibitor would not normally occur, and not a consequence of initi- Staurosporine, but cannot be overcome by the addition of ation of an anti-viral response. Ricour et al. [39] performed exogenous Ran, implying that the main target of L is not Ran random mutagenesis of L in the context of the whole virus itself [41]. Work by the same group using specific kinase followed by selection for reduced toxicity, demonstrating a inhibitors implies that MAP kinases ERK and p38 are the role for the C-terminus of L in inhibition of cytokine tran- most likely cellular kinases involved [43]. Together, the data scription correlating with effects on NPC integrity as indi- suggest that EMCV L disrupts nuclear trafficking by facilita- cated by mislocalisation of PTB to the cytoplasm [40]. ting phosphorylation of Nup62 and thereby altering NPC funciton, although this requires further examination. Compa- EMCV L is also able to disrupt nuclear trafficking di- rable Nup phosphorylation has been observed in Mengovi- rectly as shown in nuclear import assays using semi-intact rus-infected cells [44], implying a possible common mecha- HeLa cells loaded with a fluorescent NLS-containing re- nism to disrupt nuclear trafficking by cardioviruses. porter protein; the addition of recombinant L resulted in loss of the protein from the nucleus [41]. This was further sup- 64 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 1 Younessi et al.

Fig. (2). Schematic representation of the nuclear pore and nucleocytoplasmic trafficking, and disruption thereof in picornavirus infection. A. Schematic representation of a nuclear pore complex (NPC) showing the cytoplasmic fibrils, cytoplasmic and nuclear rings and nuclear bas- ket, and highlighting some of the key nucleoporin (Nups) components of the NPC. The directionality of transport of transcription factors and mRNA into and out of the nucleus respectively is also shown. B. In Rhinovirus infection, 3C degrades Nup214 and Nup358 located on the cytoplasmic side of the NPC (1), 2A degrades Nup62 and possibly Nup98 embedded in the NPC (2), while both 2A and 3C 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 results in the mislocalisation from nucleus to cytoplasm of nuclear proteins such as La and nucleolin. C. In the case of Poliovirus proteases, 2A cata- lyses 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). Nup degradation results in increased permeability/lack of selectivity of the NPC, and subsequent mislocalisa- tion 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 dys- functional (leaky) NPC that allows non-selective diffusion between nucleus and cytoplasm of cellular proteins such as interferon regulatory factor 3 (IRF-3, 2).

Interestingly, EMCV 2A, which, unlike the enterovirus 3.3. Inhibition of of NFkB Function by FMDV L Protein 2A, lacks proteolytic activity, accumulates in nucleoli shortly after infection. This localisation is facilitated by an Like other picornaviruses, FMDV infection results in host cell shutoff, although it is not clear if disruption of nu- NLS, mutation of which results in 2A cytoplasmic localisa- clear trafficking is part of the host cell shutoff, or indeed, tion. However, the functional significance of EMCV 2A nu- occurs at all [46]. FMDV 3C protease, similar to the PV 3C, cleolar accumulation in host cell shutoff is currently unclear cleaves key nuclear factors resulting in inhibition of mRNA [45]. transcription [47], but how it accesses them is not known. Clearly, cardioviruses, like enteroviruses, disrupt nucleo- Unlike cardioviruses, FMDV L has protease activity and has cytoplasmic transport, but, unlike enteroviruses, may do so a role in virus polyprotein processing, catalysing its release via hyperphosphorylation rather than degradation of specific from P1. L also has a well known role in host cell shutoff, Nups, to alter NPC function without gross effects on struc- cleaving the translation initiation factor eIF4G in a fashion ture. reminiscent of enterovirus 2A. Given these key functions of Modulation of Host Cell Nucleocytoplasmic Trafficking During Picornavirus Infection Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 1 65

L, it is not surprising that FMDV lacking L is severely at- PV = Poliovirus tenuated [48]. L localises to the nucleus of FMDV infected FMDV = Foot and mouth disease virus cells via an as yet unknown mechanism, correlating with a decrease in the nuclear levels of NFB p65/RelA subunit EMCV = Encephalomycocarditis virus [49, 50]. Infection with TMEV that expresses FMDV L pro- TMEV = Theiler’s murine encephalomyelitis tease in the absence of any other FMDV protein results in nuclear localisation of the L protease and degradation of IRES = Internal ribosome entry site p65/RelA; mutation of the L protease active site does not Nups = Nucleoporins affect L nuclear localisation but inhibits cleavage of p65/RelA [51]. The sequences responsible for L nuclear lo- NE = Nuclear envelope calisation were mapped to a SAP (for SAF-A/B, Acinus, and PTB = Polypyrimidine tract binding protein PIAS)-like domain, associated with binding to the nuclear matrix/nuclear retention of molecules involved in transcrip- NPC = Nuclear pore complex tional control [52]; mutation of this SAP domain resulted in NLS = Nuclear localization sequence altered localisation of L protease [53]. FMDV L has no ef- NES = Nuclear export sequence fect on mRNA distribution or Nup98 integrity [33]. Clearly, detailed studies are still required to establish whether aph- COPD = Chronic obstructive pulmonary disease thoviruses, like entero- and cardioviruses disrupt host cell IFN = Interferon nuclear trafficking by targeting Nups, and thereby contribute to host-cell shutoff. REFERENCES 4. CONCLUSION [1] Bedard, K. M.; Semler, B. L. Regulation of picornavirus gene expression. Microb. Infect., 2004, 6(7), 702-713. 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Received: April 27, 2011 Revised: June 23, 2011 Accepted: June 26, 2011