Interplay Between Poxviruses and the Cellular Ubiquitin/Ubiquitin-Like Pathways

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FEBS Letters 583 (2009) 607–614

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Minireview Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways

Leiliang Zhang, Nancy Y. Villa, Grant McFadden *

Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, 1600 SW Archer Rd., ARB Room R4-295, P.O. Box 100266, Gainesville, FL 32610, United States

article info abstract

Article history: Post-translational polypeptide tagging by conjugation with ubiquitin and ubiquitin-like (Ub/Ubl) Received 3 December 2008 molecules is a potent way to alter protein functions and/or sort speciﬁc protein targets to the Revised 15 January 2009 proteasome for degradation. Many poxviruses interfere with the host Ub/Ubl system by Accepted 18 January 2009 encoding viral proteins that can usurp this pathway. Some of these include viral proteins of the Available online 25 January 2009 membrane-associated RING-CH (MARCH) domain, p28/Really Interesting New Gene (RING) ﬁnger, Edited by Noboru Mizushima ankyrin-repeat/F-box and Broad-complex, Tramtrack and Bric-a-Brac (BTB)/Kelch subgroups of the E3 Ub ligase superfamily. Here we describe and discuss the various strategies used by poxviruses to target and subvert the host cell Ub/Ubl systems. Keywords: Ubiquitin Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Poxvirus RING MARCH PRANC/F-box BTB-BACK-Kelch

1. Introduction es have evolved complex molecular counteracting strategies to promote infection and disable or re-configure the host antiviral re- Members of the Poxviridae family of viruses, which infect di- sponses [3]. Among the myriad of cellular pathways that can be verse members of the animal kingdom, are double-stranded DNA manipulated by poxviruses, the ubiquitin/ubiquitin-like (Ub/Ubl) viruses, each encode around 200 proteins. Although biologically system has drawn considerable attention because many new spe- very diverse, poxviruses were originally discovered to be associ- cific poxvirus proteins implicated in modulating the host Ub/Ubl ated with diseases that produced characteristic pox in the skin system have been recently identified. [1]. Taxonomically, the Poxviridae family can be classified into Ub is a 76-amino acid containing protein that is covalently at- two subfamilies: the poxviruses of vertebrates (Chordopoxvirinae) tached to target proteins and it is the first member of a larger and the poxviruses of insects (Entomopoxvirinae). Chordopoxvirinae Ubl protein family [4]. One complexity of the Ub/Ubl-conjugation subfamily currently contains eight genera: Orthopoxvirus, Para- pathway is the existence of many Ubl molecules, such as small poxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, ubiquitin-like modifiers (SUMO), interferon stimulated gene 15 Molluscipoxvirus and Yatapoxvirus. Entomopoxvirinae subfamily (ISG15), neural-precursor-cell-expressed developmentally down- comprises three genera: Entomopoxvirus A, Entomopoxvirus B regulated 8 (NEDD8) and the list of new Ubl family members con- and Entomopoxvirus C [2]. tinues to expand [5]. The conjugation of Ub/Ubl to target protein Unlike other DNA viruses, the replication of poxviruses occurs substrates regulates diverse biological processes, including: pro- exclusively in the cytoplasm of the infected host cell. Due to the tein turnover, protein targeting, receptor internalization and regu- existence of many diverse host innate antiviral defenses, poxvirus- lation of cell signaling. A generic Ub/Ubl-conjugation pathway involves deubiquitinating enzymes (DUBs) or Ub/Ubl-specific proteases (USPs), Ub/Ubl-activating enzyme (E1), Ub/Ubl-conjugating Abbreviations: RING, Really Interesting New Gene; Ub, ubiquitin; Ubl, ubiquitin- like; SUMO, small ubiquitin-like modifiers; ISG15, interferon stimulated gene 15; enzyme (E2), and Ub/Ubl-protein ligase (E3) enzymes [6]. The DUB, deubiquitinating enzyme; USP, Ubl-specific proteases; SCF, Skp1-Cul1-F-box; extraordinary diversity of E2, E3 and DUB/USP enzymes reflects PRANC, Pox proteins Repeats of ANkyrin-C terminal; BTB, Broad-complex, the fact that Ub/Ubl-conjugation and de-conjugation is a highly Tramtrack and Bric-a-Brac; APC/C, anaphase-promoting complex/cyclosome; IAP, conserved cellular regulatory process that needs to be tightly inhibitors of apoptosis; BIR, Baculoviral IAP repeat; MARCH, membrane-associated controlled. RING-CH * Corresponding author. Fax: +1 352 273 6849. The E3 Ub/Ubl-protein ligases can be divided into five major E-mail address: grantmcf@ufl.edu (G. McFadden). groups. The first group consists of HECT domain-containing

0014-5793/$34.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.01.023 608 L. Zhang et al. / FEBS Letters 583 (2009) 607–614 proteins [7]. The second group includes c-Cbl, Mdm2, inhibitors of 2.1. MARCH poxviral E3 ubiquitin ligases apoptosises (IAPs), and other Really Interesting New Gene (RING) finger-containing proteins, which usually contain both the E2- The function of virus-encoded E3 Ub ligases was originally binding domain and the substrate recognition motif in a single linked with the downregulation of cell surface major histocompat- polypeptide chain [8]. In contrast, the third group of E3 ligases con- ibility complex class-1 (MHC-1) molecules, with the concomitant sists of large protein complexes that contain a minimal ligase core inhibition of antigen presentation at the virus-infected cell surface consisting of a cullin and RING-H2 heterodimer, such as the SCF, [23,25]. For example, the open reading frames (ORFs) K3 and K5 APC and the VBC complexes [9,10]. The fourth group is comprised from Kaposi’s sarcoma associated virus (KSHV-K3 and KSHV-K5, of U-box-containing proteins [11]. The fifth group is called mem- respectively), and the ORF K3 from murine gamma-herpesvirus brane-associated RING-CH (MARCH) [12], which contains a RING- 68 (MHV68-K3), two members of the gamma-herpesvirus family, CH domain at the amino terminus followed by two transmembrane have been reported to downregulate the expression of MHC-1 mol- domains. ecules by inducing the rapid internalization and accelerated degra- The larger subject of how other viruses and pathogens interact dation of MHC-1 molecules in either the lysosomal (e.g. KSHV-K3 with the host Ub/Ubl system has been reviewed previously [5,13– or KSHV-K5), or in the proteasome (MHV68-K3) compartments 16]. In this mini-review, we consider the components of the Ub/Ubl [19,21,22,24,26]. Likewise, myxoma virus infection also downregu- system for which poxviruses have evolved functional modulators. lates host MHC-1, as well as certain other cell surface immuno- Specifically, we focus on the virus-encoded E3 ubiquitin ligases modulatory molecules like CD4 and CD95/Fas [23,25]. and examine how poxviruses target and exploit the Ub/Ubl path- One characteristic that defines these viral E3 Ub ligases is the way to evade or subvert the host responses. Finally, we discuss presence of a RING-CH finger domain. While the RING-finger how various classes of poxviral proteins interfere with the host domain is usually at the carboxy-terminus of the E3 Ub ligase pro- Ub/Ubl degradation pathway. tein, the RING-CH domain is found at the amino terminus and is generally followed by two putative transmembrane domains 2. Poxvirus-encoded E3 ubiquitin ligases [27–29]. Different names have been used to refer to the family of viral membrane-associated proteins with potential E3 Ub ligase To be able to successfully colonize a susceptible host, all viruses activity, example MIRs [12], scrapins [25,30], or K3 family of im- have evolved sophisticated strategies to evade the host anti-viral mune evasion proteins [23]. Now the term MARCH protein is responses, or to eliminate the blockade generated by innate host widely accepted to describe this family. A schematic model for cell defenses that attempt to override virus replication [3,17,18]. the MARCH poxviral E3 Ub ligase is shown in Fig. 1A. For years, one intriguing conundrum relates to how viruses can Myxoma virus (MV), a member of the Poxviridae family that cope with the various host innate and acquired immune responses causes the lethal disease called myxomatosis in European rabbits and survive to successfully replicate and disseminate. The recent (Oryctolagus cuniculus), has a canonical double-stranded DNA discovery of a novel family of virus-encoded proteins with Ub/ genome whose replication occurs exclusively at the cytoplasm Ubl ligase activity (called viral E3 Ub ligase enzymes) has started [31]. Among the 160 proteins encoded by the MV genome, those to shed light into the strategies used by viruses to evade or subvert that are expressed by genes localized within or close to the ter- the host anti-viral responses [12,13]. To date, viral E3 Ub ligase minal inverted repeats are frequently implicated in the regula- gene family members have been identified in the genomes of sev- tion of the host response to infection [32]. In 2002, the M153R, eral different large DNA viruses, like the gamma-herpesviruses and a RING-CH containing protein was shown to be a virulence factor poxviruses [19–24]. The poxvirus-encoded E3 Ub ligases described for myxomatosis in rabbits [25] (Table 1). This MV protein was to date are presented in Table 1. implicated in the downregulation of the host cell surface MHC- 1, the pro-apoptotic factor CD95 (Fas) and the activated leuko- cyte cell adhesion molecule (ALCAM) [33]. M153R is expressed Table 1 as an early protein that localizes to the endoplamic reticulum Representative E3 ubiquitin ligase family members encoded by poxviruses. (ER) and trans Golgi network in transiently transfected cells

E3 Examples of virus protein Substrate Reference [23,25] which translocates to the trans Golgi network during ubiquitin virus infection [23]. The previously observed endocytosis and ligase lysosomal degradation of the host MHC-1 in MV-infected cells MARCH M153 (myxoma) CD4 [25] [34] is now thought to be mediated by the M153R protein. In MHC-I [25] 1995, it was reported that cell surface protein receptor CD4 CD95/ [33] was downregulated and degraded following infection of rabbit FAS CD4+ T-cells with MV [35]. Later, Mansouri et al. [23] demon- ALCAM [40] strated that M153R was responsible for the inhibition of CD4 p28/RING EVM012 (ectromelia) ? [41] in a fashion similar to that for MHC-1. M143 (myxoma) ? [43] VV p28 (vaccinia) ? [65] The question that then arises is how M153R removes the CD4 receptor from the MV-infected T-cell surface. Although the an- PRANC/F- M-T5 (myxoma) p27/Kip- [68,69] box 1 swer for this question is still incomplete, it is clear that internal- M148, M149, M150 (myxoma) ? [71] ization by endocytosis followed by degradation of CD4 in the B18R (vaccinia) ? [70] lysosomes is mediated or accelerated by M153R. Some light ORFV008, ORFV123, ORFV126, ? [72] has been shed about the requirements for the CD4 degradation ORFV128, ORFV129 (orf virus) EVM002, EVM005, EVM154 ? [76] [23]. First, the cytosolic tail of CD4 has to possess at least one (ectromelia) residue of lysine, which is recognized and ubiquitinated when CP77 (cowpox) NFjB Personal M153R is present. Second, the RING-CH domain in the M153R communication protein is necessary to ubiquitinate CD4, suggesting that the BTB/Kelch EVM150, EVM167 (ectromelia) ? [77] RING-CH domain acts as a bona ﬁde viral E3 Ub ligase. These (BBK) D11L, C18L, G3L, A57R (cowpox) ? [32] requirements are also extended to other M153R substrates, like M006, M008, M009, M014, M140 ? the cell surface MHC-1 molecules, whose degradation is also (myxoma) mediated by the M153R protein. L. Zhang et al. / FEBS Letters 583 (2009) 607–614 609

Fig. 1. A schematic of different classes of poxvirus E3 ubiquitin ligases: (A) Membrane-associated RING-CH (MARCH) E3 ubiquitin ligase. Transmembrane regions of MARCH and its targets mediate an interaction, then the N-terminal RING-CH domain of the MARCH recruits E2, and transfer the ubiquitin to the cytosolic tail of the target. (B) p28 RING E3 ligase. C-terminal Really Interesting New Gene (RING) domain of p28 ubiquitin ligase recruits E2 and facilitates the ubiquitination of target. (C) Skp1-Cul1-F-box (SCF) E3 ligase. Scaffold module Cul1 interacts with skp1 and the catalytic module RING domain protein Roc1. Skp1 binds to the C-terminal F-box domain of viral substrate recognition module, which links the substrate through N-terminal Ankyrin (ANK) repeats. (D) BTB-BACK-Kelch (BBK)/Cul3 E3 ubiquitin ligase. Cul3 is a scaffold module that interacts with the RING domain of protein Roc1 (the catalytic module) and with the N-terminal BTB domain of the viral substrate recognition module. This binding occurs through C-terminal Kelch domain.

2.2. Poxviral p28/RING protein strated that the expression of this protein is required for optimal EV replication in murine peritoneal macrophages [40,41,43,45]. Ectromelia virus (EV) is an orthopoxvirus and the causative These observations suggest that the role of p28 may also be related agent of mousepox [36]. One of the EV proteins implicated in alter- to virus host range, in addition to evasion of the innate anti-viral ing the host response during viral infection is p28. This viral pro- response. tein contains a RING zinc finger at the carboxyl terminal domain Other studies have revealed that during infection of T cells and (p28-RING containing protein), and is considered a significant vir- macrophages with EV, p28 is polyubiquitinated and localized in ulence and anti-apoptotic factor in mice infected with EV [37–40] cytoplasmic viral factories [43]. Furthermore, these studies also (Table 1). demonstrated that, although ubiquitination of p28 appears to be Orthologs of the p28-RING containing protein have also been RING-dependent, the localization of p28 in viral factories is identified in other orthopoxviruses, like variola major virus RING-independent. This last observation suggests that p28 is (VARV), cowpox (CPXV), monkeypox (MPXV), and the strain IHD- ubquitinated itself in these compartments [43,45]. W of vaccinia virus (VV) [41]. A related family member has also In vitro studies showed that RING-finger proteins of different been found in the genomes of Shope fibroma virus (SFV) and MV, members of the Orthopoxvirus genus, like p28 from EV (EVP28), two members of the Leporipoxvirus genus [42]. Ten ORFs with its homolog from variola virus (SP28), and M143R from MV, are homology to the p28-RING protein also exist within the Fowlpox all viral E3 Ub ligases [45]. Moreover, the RING domain of both virus genome [23,43]. The conservation of this protein throughout EVP28 and SP28 proteins was found to be necessary to maintain different poxvirus genera, but not all, supports its role in virus the E3 Ub ligase activity [43,45]. As a viral E3 Ub ligase, p28 ap- pathogenesis and/or host range [44]. pears to catalyze the ubiquitination of multiple protein substrates, Different observations have correlated the pathogenesis of pox- as illustrated in Fig. 1B. The p28 dependent ubiquitination of its viruses that express p28 members with the presence of the RING substrate(s) occurs in concert with E2 Ub conjugating enzymes like zinc finger motif. In fact, this motif has been linked to the promo- Ubc4, UbcH5c, and UBc13/Uev1A [46]. tion of virus virulence in vivo and the inhibition of cellular apopto- Although significant advances have been made in elucidating sis during virus replication in vitro. It is known that apoptosis is an the connection between the role of the E3 Ub ligase p28 with viral innate cellular defense mechanism to block virus replication [39]. replication in vitro, and the evasion of the host anti-viral re- Although p28 is not essential for poxvirus replication in cell cul- sponses in vivo, there are other aspects that remain to be better ture, this protein is necessary for EV pathogenesis in mice [40], defined. For example, the nature and number of its key target where the host morbidity was reduced and even eliminated upon substrates (i.e. host and/or viral), the mechanisms that govern infection with the mutant p28 (p28) compared to the infection its catalytic activity, and basis of the anti-apoptotic responses with EV wild-type [40]. In addition to this, mutation of p28 demon- are all unknown. 610 L. Zhang et al. / FEBS Letters 583 (2009) 607–614

2.3. Poxviral IAPs (Skp1, Cul1 and F-box) complex, which facilities the ubiquitination of proteins targeted for degradation by the proteasome. The F-box Originally discovered in insect viruses [47], proteins with Bacul- motif interacts with the Skp1 adaptor protein directly, and cellular oviral IAP repeat (BIR) motifs have been thought to function pri- F-box proteins commonly carry other protein-protein interaction marily as inhibitors of apoptosis (IAP). Subsets of IAPs contain a motifs such as leucine-rich repeats and WD repeats [63]. RING domain and maintain Ub ligase (E3) activity [48]. In this case, In PRANC/F-box proteins, the F-box is at the C-terminus, which the BIR domain brings targeted molecules into proximity with Ub is not in the case for cellular F-box proteins. Also, cellular F-box protein ligase for ubiquitination. proteins are generally not associated with ankyrin-repeat domains Viral IAPs exist in two insect poxviruses: Amsacta moorei ento- although they are sometimes associated with SOCS box domains. mopoxvirus (AmEPV) and Melanoplus sanguinipes entomopoxvi- Given that F-box domains in host proteins are linked to E3 Ub li- rus (MsEPV). AMV021 from AmEPV (AMV-IAP) contains two BIR gase activity, it is fair to hypothesize that poxvirus PRANC/F-box motifs and a C-terminal RING-finger domain whereas MSEV248 proteins may interact with cellular SCF complexes (Fig. 1C). The from MsEPV contains one BIR domain and RING-finger domains first experimental evidence that supports this hypothesis came [48]. It was confirmed that AMV-IAP functions as a bona fide from work with M-T5, one of four MV PRANC/F-box proteins. M- anti-apoptotic protein [49]. In vitro studies have revealed that T5 was originally identified as a virus virulence factor, based on AMV-IAP inhibits the activities of caspases 3 and 9 [47]. Other the observation that the deletion of both copies of the viral M-T5 studies performed in Drosophila have shown that IAP-binding mo- gene attenuated myxomatosis dramatically in susceptible rabbits tif proteins (IBMs) protect the cells from apoptosis induced by the infected with the M-T5 knockout virus [64]. Moreover M-T5 was proteins Hid and Grim [49]. In this case, in vitro and in vivo studies shown to be a critical determinant of cell tropism in vitro for both have revealed that the AMV-IAP domains BIR1 and BIR2 interact rabbit T-lymphocytes and a subset of human cancer cells [65].M- with Grim and Hid, respectively [50]. T5 encodes a protein with seven ANK repeat domains at the N-ter- Future studies will likely focus on whether the RING domain of minus, and a C-terminal F-box motif. M-T5 can be co-immunopre- IAP members is involved in the ubiquitination of pro-apoptotic cipitated with host cell cullin-1 (cul-1) and co-localizes with cul-1 substrates. in the nucleus [65]. Increased transition out of G0/G1 arrest induced by serum deprivation was observed following MV infection 2.4. APC family E3 ubiquitin ligases of poxviruses of permissive cells. However, this effect was not observed following infection by M-T5 knockout MV. These results suggest that Orderly progression through cell cycle relies on the sequential M-T5 functions as a virus-encoded cell cycle regulator capable of degradation of cell cycle regulatory proteins mediated by the mito- overcoming induced growth arrest at the G0/G1 checkpoint. Regu- tic ubiquitination system. In this system, one key E3 Ub ligase is lation of the cell cycle at the G0/G1 checkpoint by host cul-1 in- the anaphase-promoting complex (APC) or cyclosome, which cata- volves control of the expression and activity of p27/Kip1 [66].In lyzes the attachment of Ub to the lysine side chains of securin and this process, transition through G0/G1 requires ubiquitination of mitotic cyclins [51]. The minimal ligase module of APC is com- p27 by cul-1/SCF and subsequent degradation through the 26S pro- prised of APC2 (a distant member of the cullin family) and teasome pathway [67]. Thus, M-T5 promotes the phosphorylation, APC11 (a RING-H2 finger protein) [52]. ubiquitination, and degradation of p27 in MV-infected cells. Fol- Genome analysis has revealed the presence of homologs of cat- lowing the expression of M-T5, p27 is phosphorylated at Thr187 alytic core subunit of cellular APC/C subunit 11 (Apc11) in poxvi- and then the degradation of p27 is subsequently observed. Addi- ruses, such as Crocodile poxvirus protein CRV047 [53], tionally, Akt is another cellular binding partner for M-T5 [68]. molluscum contagiosum virus (MOCV) MC026L [54], parapox virus However, whether p27 (or any other SCF complex member) be- 014 [55] and squirrelpox virus 026L [56]. All of these poxvirus pro- comes a substrate of activated Akt remains to be determined. teins contain a modified RING-finger motif similar to the RING-H2 M150 is another ANK repeat/PRANC viral protein with nine N- motif of Apc11. A functional role for these viral proteins in the for- terminal ANK repeats and a C-terminal F-box and which is essen- mation and regulation of the E3 Ub ligase complex, however, re- tial for MV pathogenesis in rabbits. When transiently expressed mains to be investigated. in transfected cells, M150 protein was found to localize in the nucleus with a punctate pattern which was eliminated when the 2.5. Ankyrin-repeat/F-box poxviral proteins eighth ANK repeat was deleted [69]. In TNFa-stimulated cells, NFjB p65 co-localized with M150 in the nucleus, which suggests Ankyrin (ANK) repeats and F-box domains were discovered sep- that M150 interferes with the NFjB pathway. In this regard, the arately in poxvirus genomes. The ANK repeat is a 33-residue motif cellular inhibitor of NFjB, called IjB, also contains ANK repeats, named after the cytoskeletal protein, ankyrin, which contains 24 but how M150 modulates NFjB signaling remains to be elucidated copies of the repeat and was first demonstrated to have a role in The M148 protein of MV contains 10 ANK repeats and is local- signaling with yeast Cdc10 [57,58]. ANK repeats mediate a diverse ized in both the cytoplasm and the nucleus [69]. Nine ANK repeats range of protein-protein interactions and have been found in a have been identified in M149 protein and a punctuate pattern with wide variety of archaeal, bacterial and eukaryotic proteins [59]. a uniform distribution in the cytoplasm was observed for this MV The presence of Pox protein Repeats of ANkyrin-C terminal protein. Neither M148 nor M149 proteins are required for virus (PRANC) domain was reported soon after the motif was named replication in tissue culture, but they are both virulence factors [60]. Shchelkunov et al. analyzed the coding sequences of variola in MV-infected rabbits [70]. The binding partners and potential tar- major virus strain India-1967 genome and vaccinia virus strain get substrates for M148, M149 and M150 need to be reported. Copenhagen genome and found a group of viral proteins which In orf virus, a member of parapox virus genus that causes local- contain ANK repeats [61]. Because two vaccinia host range pro- ized skin infections in goats, sheep and humans, five PRANC/F-box teins, M1 and K1, are PRANC-containing proteins it was proposed proteins have been identified to interact with host cell Skp1 in an that the expression levels of different sets of the PRANC proteins F-box dependent manner [70]. Compared with most cellular F-box allow the fine regulation and determination of tissue tropism. motifs, poxviral F-box domains tend to be shorter, sometimes The F-box motif is a sequence domain of about 50 amino acids spanning only the first two of three a-helices in cellular F-box pro- named after cyclin F, which contains one F-box [62]. F-box protein teins. All five orf virus proteins interact with endogenous Skp1, was originally discovered in one of three components of the SCF cul1 and Roc1. Moreover, ORFV008 association with SCF does not L. Zhang et al. / FEBS Letters 583 (2009) 607–614 611 inhibit the enzymatic activity of E3 Ub ligase functions of the cel- infection of viral BBK gene deletion mutants showed reduction of lular SCF complex [71]. Taken together, it is likely that poxviruses the virulence, indicating an important role for BBK proteins in viral utilize these ANK/F-box proteins to recruit novel target proteins to pathogenesis. Deletion of Cowpox BTB/Kelch genes (D11L, C18L, SCF1 Ub ligases and exploit the host Ub-proteasome machinery to G3L and A57R) also reduces the cytopathic effect as well as the for- degrade specific cellular (and possibly viral) proteins to favor virus mation of cellular protrusions in vitro (Shchelkunov, personal com- replication in some fashion. munication). In the Sheeppox virus (SPPV) BBK protein SPPV-019 Vaccinia B18R is highly conserved among orthopoxviruses and has been reported to be a significant virulence factor [78]. When also contains ANK repeats and an F-box. From yeast-two-hybrid infected with the SPPV-019 knockout virus, cells exhibited a reduc- screenings, Sperling et al. reported that Skp1a is a binding partner tion in calcium-independent cell adhesion, suggesting that SPPV- for B18R [72]. They confirmed this interaction by co-IP and such 019 may modulate cellular adhesion. When inoculated into sheep interaction was shown to be F-box dependent. by the intranasal or intradermal routes, the SPPV-019 knockout In EV, the causative agent of lethal mousepox, the ANK-repeat/ virus became attenuated [78]. F-box proteins EVM002, EVM005 and EVM154 were shown to be a Vaccinia virus encodes three BBK family proteins: C2L, F3L and part of the cellular SCF complex [72]. Barry et al. also showed that A55R [79–81]. Knockout virus constructs for individual BBK pro- EVM005 protein co-localizes with cellular cul-1 [72]. EVM005 was teins were attenuated in the murine intradermal model, but their co-precipitated with cul-1, Skp1 and Roc1. In addition to this, roles in any host cell Ub/Ubl pathway is still elusive. EVM005 was associated with conjugated Ub. The interaction between EVM005 and Skp1/cul-1 is F-box dependent. Finally, 3. Ubiquitin-like (Ubl) pathways EVM002 and EVM154 interact with Skp1 and conjugated Ub [72]. All of these results are consistent with the systematic modulation Since Ub conjugation was discovered in mid-1970s [4,82],a of the host cell SCF complexes to target novel substrates for ubqui- number of small proteins related to Ub have been identified [6]. tination and degradation. The Ub-like proteins (Ubl) share a similar 3D structure, featured Cowpox protein CP77 is a PRANC/F-box protein that contains by the Ub or beta-grasp fold, despite the low similarity in amino nine ANK repeats and a relatively short C-terminal 13aa F-box mo- acid sequences [83]. tif. Chang et al. have recently found that CP77 inhibits the NFjB activation induced by TNF or IL-1 (Chang, personal communica- a 3.1. SUMOylation tion). Interestingly, the 13aa F-box in CP77 is shorter than most other poxvirus F-box motifs, which suggests it could define a min- As a post-translation modification, the Small Ubiquitin-like imum motif requirement for binding to the cellular SCF complex. Modifiers (SUMO-1, SUMO-2 and SUMO-3) are covalently attached Although CP77 protein acts as a host range protein for vaccinia to target protein substrates to influence their sub-cellular parti- virus in some cell lines, this short F-box motif, which is important tioning and protein stability [84,85]. for binding SCF complex, is not required for the host range function A vaccinia protein called A40 was found to be associated with of CP77. the cytosolic site of the ER, but SUMO-1 conjugation induced the As discussed, poxviruses encode a family of PRANC/F-box pro- relocation of A40 into the DNA-replication site and lysine 95 was teins which can bind Skp1 through the viral F-box domain the key SUMOylation residue for A40 [86]. The role of the SUMO- (Fig. 1C). Cullin-based Ub E3 ligases contain three functional com- 1 modified form of A40 could be in late viral transcription that is ponents: a catalytic module composed of a small RING domain pro- known to occur at vaccinia virus replication sites or it could be tein that recruits the Ub-conjugating enzyme E2, cullin as a an integral component in the process of replication itself [87]. scaffold module, and a substrate recognition module that binds Vaccinia protein E3 is a double-stranded RNA binding protein to the substrate and brings it within proximity to the catalytic that has been shown to interact with SUMO-1 by yeast two hybrid module [73]. assays [88]. However, E3 is not thought to become SUMO-1 conjugated itself because during virus infection the observed molecular 2.6. BTB/Kelch (BBK) poxviral proteins mass of the E3 protein is not overtly changed, as measured by gel electrophoresis. It is likely that SUMO-1 modifies other proteins in Recently, the molecular composition and function of the cullin- a way that favors viral replication, but the role of E3 protein re- 3-dependent E3 ligase complex has been described [74]. In this mains to be better defined. complex, Broad-complex, Tramtrack and Bric-a-Brac (BTB) domain-containing proteins mediate binding of the cullin to the substrate. Poxviruses are the only family of viruses known to encode 3.2. ISGylation BTB-BACK-Kelch (BBK) proteins [75], suggesting that poxvirus might also modulate the host Ub pathway through interaction with ISG15 encodes a 17 kDa small Ubl protein that is transcription- cullin 3 (Fig. 1D). ally regulated by interferon (IFN) a and IFN b and forms covalent The BTB domain of EV BBK protein EVM150 and EVM167 are conjugates (ISGylation) with targeted proteins [89]. By utilizing necessary and sufficient for interacting with N-terminal region of activating, conjugating and ligating enzymes, ISG15 facilitates the cullin 3 [75]. Moreover, EVM150 and EVM167 interact with conju- addition of ISG15 to specific lysine residues of substrate proteins gated Ub and Roc1, the RING-finger protein that functions as the and may alter the subcellular localization, structural stability, or Ub ligase. Taken together, EVM150 and EVM167 support the for- the activities of them [6]. For instance, ISG15 targets many innate mation of active cullin-3-based Ub ligases recruiting still-unidenti- antiviral proteins, including: PKR, STAT1 [90], JAK1 and RIG-I [91], fied substrates for ubiquitination (Table 1). Another two BBK and may regulate their activity during viral infection. proteins, EVM18 and EVM27, also interact with cullin 3 (Wilton ISG15 mRNA of Hela cell was upregulated after infection with and Barry, personal communication). Whether all BTB/Kelch pro- MVA and NYVAC but not with the WR strain of vaccinia virus teins in poxvirus are involved in the Ub/Ubl pathway, or target [92,93]. Moreover, mRNA of ISG15 was increased when infected other host pathways, needs to be investigated. with VVDE3 compared with WR [94]. ISG15 interacts with the Functional evidence indicates important roles for BBK proteins C-terminal domain of E3, by which E3 suppresses the antiviral during the poxvirus life cycle. Deletion of BBK genes in cowpox re- function of ISG15 [95].VVDE3L caused significant disease and duces the number of mature progeny [77]. In mice, intranasal mortality in ISG15-deficient mice, which is not observed in 612 L. Zhang et al. / FEBS Letters 583 (2009) 607–614

ISG15+/+ mice. Compared with ISG15+/+-infected mice, ISG15- The interaction between p53 and Mdm2 is upregulated by phos- deficient mice infected with VVDE3L or with an E3L mutant virus phorylation of p53 at its N-terminus. The vaccinia B1R kinase is lacking the C-terminal domain triggered an enhanced inflamma- an early protein required for viral DNA replication that phosphor- tory response in the lungs [95]. ylates p53 and facilitates the recruitment of p53 to Mdm2 for ubiquitination [108]. 4. Miscellaneous poxvirus strategies to modify Ub/Ubl pathways 5. What is the role of the host cell proteasome? 4.1. Deconjugating enzymes While it is apparent that poxviruses encode a wide variety of Removal of Ub or Ubls from substrates by deconjugating en- proteins that regulate the Ub-proteasome system, the role of the zymes is another step that pathogens can hijack [96]. Several 26S proteasome during poxvirus infection has only recently been members from the families of cellular metalloproteases and cys- investigated. For example, orthopoxvirus infection is dramatically teine proteases have been defined to be responsible for the Ub affected by a range of proteasome inhibitors, such as MG132, deconjugation in the cell, for instance, Ub-specific proteases (USPs, MG115, lactacystin and Velcade (Barry, personal communication). also referred to as Ub processing proteases, UBPs), Ub C-terminal Treatment of vaccinia virus-infected cells with MG132 or Velcade hydrolases (UCHs), ovarian tumor related proteases/Cezanne results in a failure of viral replication factories to appear within (OTUs), and Josephin domain proteases (JD) [5]. the cytoplasm. This is accompanied by the absence of late viral The I7L protein of vaccinia virus and the adenovirus protease gene expression and DNA replication, whereas, early viral gene Avp (a deubiquitin and deISGlatin enzymes) share the critical res- expression is unaffected. Proteasomal inhibition with MG132 or idues that surround the catalytic triad of proteinases found in the Velcade also has dramatic effects on viral progeny titer, severely Ub pathway for protein degradation, such as the YopJ proteinase blocking viral replication. One possible explanation for the effect in Yersinia pestis [97]. I7L belongs to the peptidase_C57 superfam- of MG132 on poxviral infection is due to lack of available ubiquitin ily. The substrate specificity for I7L has been defined as AG/X, because MG132 depleted the cellular pool of ubiquitin, but the where X in the P10 position has a preference for a small residue mechanism remains to be better defined. The addition of a Ub-acti- like Ala, Ser or Thr [98]. I7L is required for AG/X-specific cleavages vating enzyme (E1) inhibitor had a similar effect on early and late of viral membrane and core proteins, which occur at early and late protein expression. This suggests that a functional cellular Ub-pro- stages of virus assembly, respectively [98]. It remains to be inves- teasome system is required during poxvirus infection. However, tigated whether I7l is a true Ub/Ubl deconjugating enzyme. the precise target(s) needed for productive viral replication are still unknown. 4.2. Poxviruses encoded homologs of ubiquitin

Ubiquitin has been detected in vaccinia virions and suggests 6. Future prospects that ubiquination of viral or host proteins is an important process [99,100]. Proteins with sequence similarity to ubiquitin are present In different ways that share the feature of favoring virus repli- in the genomes of entomopox and canarypox virus. For example, cation, poxviruses encode not only many diverse proteins as the the entomopox virus MSV144 is an example of one such ubiquitin potential components of E3 Ub ligase complexes, but also an homolog. The amino acid identity between MSV144 and eukaryotic impressive set of effectors and inhibitors of various Ub/Ubl ubiquitin is from 83% to 88% [101]. Likewise, CNPV096 from modification pathways. Although progress has been made in canarypox virus is a homolog of cellular ubiquitin as well [102]. understanding the role of poxviral proteins in re-programing host Although it has been suggested that poxvirus virulence and host Ub/Ubl systems, there are many important questions still unan- range are influenced by Ub homologs, the precise roles of these swered. Our understanding of the poxvirus E3 Ub ligases is very proteins needs to be investigated. limited, including identification of the precise substrates of many E3 Ub ligases and their relevant functions. Similarly, the molecular mechanisms relating to SUMOylation and ISGylation during poxvi- 4.3. Poxviral proteins that interfere with host protein Ub-degradation rus life cycles are poorly understood. It will also be important to determine whether the E3 Ub ligases encoded by poxviruses have Besides components of E3 Ub ligase complex and deconjugation the proteasome trafficking function as predicted by their se- enzymes, poxviruses also encode different proteins that inhibit or quences. Given the intricacy of the interplay between poxviruses enhance the host Ub/Ubl system for their own advantage. and their hosts, future research on how the Ub/Ubl systems TRAF6 an E3 Ub ligase that functions to activate TLR signaling become re-configured in the poxvirus life cycle may shed light can be inhibited by several proteins encoded by poxviruses [103]. on novel host cell functions of these key host cell pathways in The TRAF domain of TRAF6 is necessary and required for the inter- the absence of viral infections as well. action with vaccinia A52 protein [104]. By direct binding, A52 dis- rupts the TRAF6-TAB1-containing complex, activates p38 MAP kinase and inhibits TLR3 induced NFjB activation [104,105].Ina Note in proof murine intranasal model, the vaccinia A52 deletion mutant virus caused milder signs of illness and reduced weight loss compared After the submission of this review, two papers (Orthopoxvirus- to the effects of wild-type virus, thus suggesting that the A52 pro- es Require a Functional Ubiquitin-Proteasome System for Produc- tein contributes to virus virulence [104]. Another vaccinia protein, tive Replication. Teale A, Campbell S, Van Buuren N, Magee WC, K7, not only shares the intracellular targets of A52, such as TRAF6 Watmough K, Couturier B, Shipclark R, Barry M. J. Virol. 2008 and IRAK2, but also interacts with DDX3 and plays a role in the December 24. Inhibition of the Ubiquitin-Proteasome System Pre- TBK1/IKKe-mediated IRF activation [106]. vents Vaccinia Virus DNA Replication and Expression of Intermedi- The protein level of p53 plays an important role in apoptosis ate and Late Genes. Satheshkumar PS, Anton LC, Sanz P, Moss B. J. and cell cycle control. The cellular p53 protein levels are tightly Virol. 2009 January 7) were published online. These two papers re- controlled by binding to an E3 Ub ligase, Mdm2, which promotes port that the host cell proteasome is required during vaccinia virus its ubiquitination and subsequent proteolytic degradation [107]. infection. L. Zhang et al. / FEBS Letters 583 (2009) 607–614 613

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