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Viruses and the 26S proteasome: hacking into destruction
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Viruses and the 26S proteasome: hacking into destruction Lawrence Banks, David Pim and Miranda Thomas International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy
The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus–host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases. In the quest for successful infection, viruses are faced with many obstacles, including host immune-surveillance and blocks to the cell-cycle machinery, and hence to viral replication. This might ultimately include host-induced suicidal apoptotic cell death and restriction of the infection to a single cell, thereby protecting the rest of the organism from further viral spread. To overcome these host responses, viruses across a wide spectrum of host and tissue tropisms have evolved many similar strategies. Often, the mechanisms employed are different but the ultimate effects are the same: productive viral infection. One of the common obstacles facing a virus is the overwhelming stoichiometric imbalance of the host target proteins over those encoded by the virus. To overcome this, many viruses have evolved mechanisms whereby their cellular target-proteins are directed to the 26S proteasome and then subjected to proteolytic degradation. Although this is often achieved in different ways, the viruses invariably act at the point of substrate recognition. This means that the targeting of the cellular proteins is a highly specific process and almost always involves an E3 ubiquitin ligase (Fig. 1). This ubiquitin ligase can be directly encoded by the virus, or could be a host ligase that has been redirected to a substrate that it would not normally recognize, alternatively the normal function of a host-encoded ligase could be stimulated. The first example of a viral protein using the proteasome machinery to direct the degradation of a cellular protein was provided by the human papillomavirus (HPV) E6 protein and its stimulation of the ubiquitination and degradation of the cellular tumour-suppressor protein Corresponding author: Lawrence Banks ([email protected]).
p53. Since then, many different viruses have been shown to direct the ubiquitination of diverse cellular proteins, mainly for degradation at the 26S proteasome (for examples, see Table 1). These include avoidance of host immune surveillance processes by human herpes virus 8 (HHV8), which is responsible for Kaposi’s Sarcoma. In this instance, the virus encodes two transmembrane proteins – modulator of immune recognition (MIR)-1 and MIR2, which represent a novel class of membrane-bound E3 ubiquitin ligases. Both MIR1 and MIR2 ubiquitinate major histocompatibility complex-I chains leading to their endolysosomal degradation, whereas MIR2 also induces the ubiquitination of the B7.2 cell-surface protein, which is a co-stimulator of T-cell activation [1]. Others examples include vesicular stomatitis virus and rabies virus, the matrix proteins of which interact directly with the Nedd4 cellular ubiquitin-ligase – a process that appears to be required for efficient viral egress – although how this takes place is still unclear [2]. Interestingly, Epstein – Barr virus also makes use of Nedd4. In this case, the viral latent membrane protein 2a redirects Nedd4 activity to a number of B-cell tyrosine kinases, which are subsequently targeted to the proteasome. This activity alters B-cell-receptor signalling and is believed to contribute to the establishment of a latent viral infection [3]. Likewise, the adenovirus penton-base protein, which is required for cell entry, also interacts with Nedd4 [4]. The amazing level of evolutionarily conserved use of the proteasome as a means of successfully completing the viral life-cycle has even been implicated in some plant viruses: regulation of tobacco mosaic virus movement protein is believed to be proteasome mediated, and plays a role in regulating viral spread [5]. Ubiquitination of substrate proteins Before discussing specific cases, it is worth briefly reviewing the ubiquitin– proteasome pathway. As shown in Fig. 1, the E3 ubiquitin ligases are involved in the specific recognition of substrates for ubiquitination. Eukaryotic organisms encode very few, sometimes only one, E1 ubiquitin-activating enzyme, which binds to the C terminus of ubiquitin (Ub) through a thioester linkage. The ubiquitin is then transferred, again through a thioester linkage, to a ubiquitin-conjugating enzyme (Ubc or E2), of which . 50 are known in humans. The ubiquitin is then transferred to the target protein by E3 ubiquitin-protein ligases, which interact with the E2 and with specific protein substrates. This allows the formation
http://tibs.trends.com 0968-0004/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0968-0004(03)00141-5
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Fig. 1. The ubiquitin proteasome pathway and common points of viral entry. In the majority of cases the virus intervenes at the step of the ubiquitin-protein ligase (E3). This can be virally encoded, or can be a cellular ligase that is either redirected or stimulated by another viral protein. The heavily ubiquitinated target protein is then rapidly degraded at the proteasome.
of isopeptide bonds between the C terminus of Ub and lysine residues either on the target protein or on the last Ub of an existing poly-Ub chain. Multi-Ub chains act as potent targeting signals for protein degradation in the http://tibs.trends.com
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proteasome [6]. The number of known E3 ligases is increasing almost daily. They provide specificity, both temporally and spatially, for the labelling of proteins for destruction by the proteasome and, as such, are one of the targets of choice for viral intervention in the activities of the host cell. Here, we focus on some well-defined systems that provide three different examples of how viral proteins direct their cellular substrates to the proteasome: herpes simplex virus (HSV)-1 infected cell protein 0 (ICP0); adenovirus E4orf6– E1B 55K and HPV E6.
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HSV-1 ICP0: a single component viral ubiquitin ligase The HSV-1 protein ICP0 is expressed early during infection and is one of the first viral proteins to be expressed upon reactivation from latency. ICP0 acts in synergy with the major viral transactivator ICP4 to induce the expression of the viral genes. This activity of ICP0 is thought to depend upon its localization to the nuclear structures known as ND10s or PODs [7]. The viral genomes also localize to ND10s, where virus replication compartments develop [8]. The presence of ICP0 results in the degradation of two major ND10 components, promyelocytic leukemia antigen (PML) and Sp100 [9,10] thereby disrupting the ND10. The RING (really interesting new gene)-finger domain found at the N terminus of the protein has been shown to be required for all these activities [11] and we should briefly consider this type of domain. The majority of E3 ubiquitin-protein ligases have either a HECT [homologous to E6-associated protein (E6AP) carboxy terminus] domain (see E6AP below) or a RINGfinger domain. RING fingers have the consensus sequence Cx2Cx9 – 39Cx1 – 3Hx2 – 3[C/H]x2Cx4 – 48Cx2C, in which the C and H residues are zinc binding. RING fingers facilitate the transfer of ubiquitin or ubiquitin-like molecules from the E2 enzyme onto a range of heterologous substrates and, indeed, onto themselves. Proteins containing RING fingers form the largest known class of E3 ligases and appear to be involved in many cellular processes [12]. The ICP0 protein, or a fragment containing only the N terminus and the RING finger (Fig. 2a), has been shown to induce the accumulation of poly-Ub chains in the presence of an E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzymes UbcH5a and UbcH6 [13]. The interaction appears to be specific because a number of related E2 proteins showed no poly-Ub accumulation in the presence of ICP0. Interestingly, these two E2 proteins were originally identified as being involved in the E6– E6AP-induced degradation of p53, albeit with different efficiency [14], suggesting that HSV and HPV have both evolved to interact with a common cellular pathway. In this context it is worth noting that, although the two viruses are very different, they both infect similar mucosal epithelia and so might be expected to encounter a similar cellular environment. The question that obviously arises is what does the virus gain from inducing the degradation of these cellular proteins? Numerous mutational studies have shown that more cells become committed to productive infection, particularly at low doses of input virus, when the ICP0 is fully functional and it would appear that ICP0 activity circumvents a cellular silencing activity that drives the
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Table 1. Examples of how viruses use the cellular ubiquitin pathway to target cellular proteins during the viral life cyclea Factor Function
Virus
Host
Viral
Target proteins
Refs
Entry Transcription/reactivation
Adenovirus HSV
Nedd4 family –
Penton-base protein ICP0
[4] [15 –17]
HPV Coxsackie HCMV HPV Adenovirus KSHV HCMV EBV Paramyxovirus Myxomavirus Ebola Retrovirus Rhabdovirus
Unknown Unknown Unknown E6AP SCF complex – ERAD complex Nedd4 SCF complex – Nedd4 BUL1 (Nedd4-like) Nedd4
E7 Unknown pp71 E6 E4orf6 MIR1, MIR2 US2, US11 LMP2A V protein M153R VP40L domain ?Gag M protein
Unknown PML, Sp100, CENP-A and C/DNA-PK catalytic subunit pRb, p107, p130 cyclin D1 pRb, p107, p130 p53, Bak p53 MHC components MHC-1 heavy chains Lyn, Syk STAT proteins CD4 Unknown Unknown Unknown
Apoptosis avoidance Immune avoidance
Virus release/budding
[71] [73] [74] [40,54] [32] [1] [75] [3] [76] [77] [78] [79] [2]
a Abbreviations: CENP, centromere protein; DNA-PK, DNA-dependent protein kinase; E6AP, E6-associated protein; EBV, Epstein– Barr virus; ERAD, endoplasmic reticulumassociated degradation; HCMV, human cytomegalovirus; HPV, human papillomavirus; HSV, herpes simplex virus; ICP0, infected cell protein 0; KSHV, Kaposi’s sarcomaassociated herpes virus; LMP2A, latent membrane protein 2A; MHC, major histocompatibility complex; MIR, modulator of immune recognition; PML, promyelocytic leukemia antigen; SCF, skp1– cdc53– F-box complex.
virus towards a latent infection [11]. Many genes that are silenced are closely linked to transcriptionally silent regions of heterochromatin such as centromeres and telomeres, and the heterochromatin protein HP1 is found interacting with Sp100 in ND10s and interphase centromeres [11]. Because the known ICP0 targets include PML, Sp100, the catalytic subunit of DNA-PK [15], and two centromere proteins CENP-A and CENP-C [16,17], the following model can be proposed: upon virus infection, the DNA uncoats and localizes to ND10s, which have been suggested to be ‘stores’ of compounds and factors required for transcription and replication. ICP0-induced degradation of PML and Sp100 then disrupts the heterochromatin, thus, ‘mugging the storekeeper’ and making the useful contents of ND10 available for viral transcription and replication. Mutations resulting in the functional loss of the RING finger of ICP0 have a devastating effect on most activities of the protein and upon viral infection [18]. However, mutations in other regions of the protein also result in reduced activation of viral gene expression [18]. The C-terminal half of the protein contains a nuclear localization signal (NLS) and a binding site for a ubiquitinspecific protease USP7 [19]. Interestingly, USP7 is also involved in the de-ubiquitination and stabilization of p53 [20]. Hence, sequestration of this protein by ICP0 might also be a means of overcoming p53 activity. ICP0 can bind and stabilize cyclin D3 and appears to relocalize it to the ND10. This causes formation of an active complex of cyclin D3 and cyclin-dependent kinase 4 (cdk4), which, in turn, phosphorylates the pRb tumour suppressor, causing release of E2F and promotion of G1 progression [21]. This provides an interesting parallel with certain g-herpes viruses, such as HHV8 and herpesvirus saimiri, which encode their own D-type cyclins [22,23]. The C-terminal region of ICP0 also contains a second ubiquitin ligase, which does not have a RING finger and which appears to act through the E2 cdc34 (UbcH3) [24]. This is the major E2 that acts through the skp1 – cdc53 – F-box http://tibs.trends.com
(SCF) complex. One of the functions of this complex is to promote the ubiquitination and degradation of cyclin D1 [25]. As it has been reported that ICP0 can stabilize D1 without binding to it [26], this might suggest that this E3-ligase domain is a pseudo-E3, sequestering cdc34 from the SCF complex. The D cyclins are normally involved in the promotion of cell cycle through G1/S phase transition, thus, continued high levels of these proteins would probably keep the host cell in a state of replicative readiness, that is, an ideal environment for the replication of the viral genome. Thus, it would appear that ICP0 interacts in two ways with the 26S proteasome: the RING finger acts as an E3 to direct the degradation of components of ND10 and centromeres, thereby aiding viral transcription. Simultaneously, the C-terminal domain appears to act as a pseudo-E3, competitively inhibiting the proteasomal degradation of cyclin D1, thus, promoting a cellular environment that is conducive to viral DNA replication. Adenovirus E4orf6 –E1B55k –cullin complex: a multi-component ligase A major obstacle to the replication of many different viruses is the p53 tumour suppressor, and numerous strategies have been employed to overcome its function. As noted above, HSV-1 ICP0 can potentially inactivate p53 through association with USP7, whilst HPV uses the E6 – E6AP complex. In adenovirus it was thought that the viral E1B 55 kDa (E1B 55K) protein alone was sufficient to inactivate p53 by blocking its transcriptional activity [27]. However, recent studies have shown a more complex pattern of interaction whereby p53 is also targeted for proteasome-mediated degradation. In adenovirus-infected and transformed cells, p53 levels are reduced [28]. The association of both E1B 55K [29] and the E4orf6 protein with p53 [30] suggested that these proteins might be responsible. E1B 55K and E4orf6 bind to p53 near its N and C termini, respectively [31,30], and it was shown that they also led to a proteasome-dependent reduction in p53 half-life [32]. Immunoprecipitation of Ad5
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(a) Ub Ub Ub
Ub
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Rbx1
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(c) Ub
Ub
Ub
E1
Ubc H7
Ub Ub Ub HECT
Ub Ub Ub
26s Proteasome E6AP
p53
E6 Ti BS
Fig. 2. Three examples of viral ubiquitin-ligase complexes. (a) The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) ubiquitin ligase directs the degradation of promyelocytic leukemia antigen (PML) oncogenic domain (POD) components PML and Sp100 through direct interactions with the substrates and the E2 enzymes (UbcH5a/H6) via its RING(really interesting new gene)-finger domain. The C-terminal half of ICP0 also interacts with the E2 enzyme cdc34 (UbcH3), which is involved in regulating cyclin D1 levels. Because ICP0 stabilizes cyclin D, this might be acting as a pseudo E3. (b) The adenovirus E1b 55K– E4orf6– cullin ubiquitin-ligase complex directs the degradation of the p53 tumour-suppressor protein. E1B55K and E4orf6 both directly bind to the p53 tumour suppressor. Through interactions with Elongins B and C E4orf6 recruits cullin 5 and E2. Thus, the E1B 55K –E4orf6 provide specificity and can be seen as being functionally analogous to the skp1– cdc53– F-box (SCF) complex. (c) The human papillomavirus (HPV) E6–E6-associated protein (E6AP) ubiquitin-ligase complex directs the degradation of the p53 tumour-suppressor protein. E6 interacts directly with p53 and the E6AP ubiquitin ligase, thereby redirecting E6AP to p53 together with the E2 enzyme UbcH7 family members.
E4orf6 from cells co-precipitates a group of cellular proteins [33] that have been identified [34] as the previously described 84 kDa cullin 5, 19 kDa elongin B, 16 kDa Rbx1 and 14 kDa elongin C. Further studies showed that E4orf6 binds directly to the Elongin BC complex [34,35]. Elongin BC is a component of the large von Hippel– Lindau (VHL) tumour-suppressor complex, which functions as an E3 ubiquitin ligase [36]. The VHL component acts as a substrate recognition motif [37], whereas the Elongin BC complex links substrates to a module containing Rbx1 and cullin 2, which, in turn, activates an E2 ubiquitin-conjugating enzyme [37]. The striking similarity between VHL and mammalian SCF ubiquitin ligase complexes [38] suggested that the complex between E4orf6, Elongin BC and a cullin family member might form an E3 ligase that could be recruited by E1B 55K to target p53 for proteasome-mediated degradation. http://tibs.trends.com
Because cullin activity is dependent on its modification by NEDD8 ([39] and references therein), a temperaturesensitive cell line with a mutation in NEDD8 was then used to confirm that NEDD8 modification of cullin 5 was indeed required for E1b – E4orf6-induced degradation of p53 [34]; Fig. 2b shows the structure of the complex. Taken together, these studies demonstrate a complex pattern of events whereby adenovirus overcomes the inhibitory potential of p53 at the transcriptional level and at the level of protein turnover. This highlights the need to overcome p53 functions that are independent of its ability to activate gene expression, and it seems most likely that these are the pro-apoptotic activities of p53. HPV E6–E6AP: a bi-partite ubiquitin-protein ligase E6AP is the prototype HECT-domain-containing E3 ligase, which was originally identified in association with the E6
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proteins of oncogenic HPV-16 and HPV-18 in the ubiquitination of p53 [40]. It is a monomeric 100 kDa polypeptide with a C-terminal ubiquitin-protein ligase HECT domain, and its loss or mutation is associated with Angelman syndrome – a serious developmental disease, characteristics of which include a variety of behavioural disorders [41]. Unlike other E3 ligases, HECT-domain proteins form a thioester linkage with the ubiquitin moiety, as part of the process of transferring it from the E2 conjugating enzyme to the bound substrate protein. The presence of a cysteine residue (C820 in E6AP) is required for this activity and is strictly conserved in HECT-domain proteins [42,43]. The N-terminal domains of HECT proteins are very divergent, and this is thought to largely define their target-protein specificity and might possibly affect their interaction with specific E2 enzymes [43]. E6AP has been shown to interact with a number of E2s, including UbcH5 and UbcH6 (as mentioned above) plus UbcH7 and UbcH8 [44]. Specificity in the binding of E2s to the HECT domain determines the preferred E2 [45], and this might be affected by the binding of E6 or other similar targetdetermining molecules. E6 binds to the E6AP molecule through a linear helical domain between residues 391 and 408 of E6AP, one side of the helix has a hydrophobic patch formed by the motif Glu-Leu-Leu/Val-Gly, which is essential for E6 binding [46]. E6 simultaneously binds to p53, allowing the HECT domain to transfer ubiquitin from the bound E2, via a thioester intermediate, to the p53 molecule (Fig. 2c). It is clear that the binding of E6 to E6AP alters its substrate specificity because E6AP is not thought to normally recognize p53 as a target for ubiquitination. This has been shown by several studies in which interfering with E6AP activity by using antisense oligonucleotides [47], dominant-negative mutants [48] and competing peptides [49] only affects p53 levels in E6-expressing cells. In the absence of E6, p53 levels are also regulated by the proteasome using mouse double minute 2 (MDM2) as the specific ubiquitin ligase [50]. This is a very different interaction because MDM2 is a RING-finger protein but, in E6-expressing cells, the E6 – E6AP completely takes over the control of p53 levels [51]. Interestingly, p53 has to be phosphorylated on Thr155 for it to be susceptible to E6 – E6AP-induced degradation [52], indicating that the precise timing of p53 degradation and/or localization of p53 must be tightly controlled to allow a productive viral life cycle. Again, the parallels between adenovirus and HPV abolition of p53 function are striking, with identical results, yet attained by very different mechanisms. Although p53 is the best known of the E6 – E6AP targets, others have also been described including the apoptosis-promoting Bak protein as well as E6AP itself [53], which might represent a feedback control mechanism. In the case of Bak, it is clear that it is targeted by E6AP in the absence of E6, but that the presence of E6 enhances this activity [54]. Many other cellular proteins are directed for degradation by E6–E6AP, including E6TP1 [55], Scribble [56] and the DNA-repair protein O6-methylguanine DNA methyltransferase [57]. At present, it is not http://tibs.trends.com
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clear whether E6AP is involved in the normal regulation of these proteins in the uninfected cell. Neither is it clear what effect E6 – E6AP has upon E6-independent E6AP targets, such as activated Src [58] and the Rad23 homologue HHR23A [59]. E6AP-independent targets of HPV E6 Despite the fundamental importance of the E6– E6AP interaction for a wide range of biological activities of E6, it is becoming clear that other functions of E6 are required for its contribution to malignancy. Indeed, the ability of E6 to bring about cell transformation and induce tumours in transgenic animals requires activities other than the E6 – E6AP interaction [60,61]. Interestingly, a recently identified subset of E6 target proteins containing PDZ domains might be responsible for E6-induced malignancy. The first of these proteins to be described – the discs large (Dlg) protein [62] – is targeted by E6 for ubiquitindependent degradation [63]. Dlg belongs to a family of PDZ domain-containing proteins known as membrane-associated guanylate kinases (MAGUKs), several other members of which are also directed for degradation by E6. These include membrane-associated guanylate kinase with inverted orientation (MAGI)-1 [64], MAGI-2 and MAGI-3, [65] and hScrib [56]. These proteins are found typically, but not exclusively, at the membrane– cytoskeletal interfaces of cells, mediating interactions between molecules involved in cell – cell, cell – substratum contact, receptor clustering, cell polarity and signal transduction [66]. Many of the proteins have been assigned the roles of potential tumour-suppressor proteins, with Drosophila Dlg and Scribble acting in concert to regulate cell polarity and cell growth [67]. Not surprisingly, there is now thought to be a strong correlation between the ability of E6 to direct the degradation of these proteins and its contribution to malignant progression. The E6 proteins from the high-risk cancer-associated mucosal HPVs (HPV-16 and HPV-18) interact with MAGUKs through a C-terminal four-amino-acid consensus PDZ-binding motif, and direct their degradation by recruiting a cellular E3 ubiquitin ligase. Interestingly, this binding motif is absent in E6 proteins that are derived from the low-risk benign HPV types (HPV-6 and HPV-11). In the case of hScrib, E6 – AP has been implicated in the degradation [56]. However, for Dlg and the MAGI proteins, there is mounting evidence indicating that a different cellular ligase is involved. Thus, mutants of HPV-18 E6, which cannot induce p53 degradation, can still induce Dlg degradation, and E6 also degrades Dlg in systems that lack E6AP [68]. Furthermore, chimaeric E6 molecules based on an HPV-11 or HPV-6 E6 backbone, which cannot interact with E6AP, nonetheless target Dlg for proteasomemediated degradation when provided with a PDZ-binding motif [69]. Finally, it should also be remembered that the kinetics with which these different substrates of E6 are degraded are wildly different [64,65,68]. Intrestingly, the ability of HPV to direct cellular proteins for proteasome-mediated degradation is not restricted to the E6 protein. The E7 protein encoded by cancer-associated HPV types is responsible for driving cells into an artificial S phase, rendering them capable of
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E3 ligase?
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p130
pRB
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E3 ligase?
growth arrest apoptosis
HPV
Metastasis Proliferation
Keratinocyte
Immortalization
DNA damage
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Loss of cell contact
DNA damage
DNA damage Distal organs Ti BS
Fig. 3. The contribution of the human papillomavirus (HPV) E6 and E7 oncoproteins to malignant transformation by directing their substrates for proteasome-mediated degradation. HPV infects keratinocytes that have exited the cell cycle. The E7 protein, through interaction with the proteasome machinery, targets the ‘pocket-protein’ family of proteins for degradation, resulting in increased DNA replication and proliferation. This unscheduled replication results in a p53 response that is targeted by the E6 –E6-associated protein (E6AP) ubiquitin-ligase complex. At later stages of malignancy, the ability of E6 to direct the degradation of a family of membrane-associated guanylate kinase (MAGUK) proteins probably contributes to the metastatic progression. Throughout this process, the continued proliferation and abolition of normal detectors of DNA damage (p53) results in the accumulation of genetic damage within the transformed cells.
replicating the viral DNA [70]. This activity requires the ability of E7 to interact with a family of cell-cycle regulators including the pRb tumour suppressor and other members of the pocket protein family. In all cases, E7 efficiently directs the degradation of these substrates at the proteasome [71]. It should be clear that this has striking parallels to the activity of the HSV ICP0, which stabilizes D-type cyclins and results in the release of free of E2F from pRb. This underlines the essential similarity in the cellular obstacles to a successful viral life cycle. At present, the ligase employed by E7 is unknown, although E7 itself has been reported to directly interact with different components of the proteasome machinery [72]. Elucidating this particular pathway remains a major goal within HPV research in the coming few years. Concluding remarks The use of the proteasome by a plethora of different viral proteins emphasizes the central importance of this cellular pathway for many successful viral infections. However, in rare cases, this perturbation of normal cellular regulatory pathways initiates events that, ultimately, result in cell transformation and malignancy. This is most notable with the high-risk HPVs – infection with which can result in human malignancy, including cervical cancer. As can be seen from Fig. 3, the targeting of cellular proteins to the proteasome via interactions with cellular ubiquitin ligases is central to this whole process. HPV E7 targets the pocket protein family and induces cell proliferation. Under normal circumstances, p53 would then induce growth arrest and/or apoptosis, however, the E6 – E6AP ubiquitinligase complex effectively abolishes this response. Finally, http://tibs.trends.com
at later stages of disease progression, the degradation of the MAGUK family of proteins by E6 probably results in metastasis and death of the host. Designing strategies to block the viral perturbation of these cellular ligases are major goals in cancer therapy and in antiviral therapeutics as a whole. Acknowledgements The list of viruses that encounter this pathway is growing daily and we apologize to those colleagues whose work has not been cited owing space limitations. We gratefully acknowledge research support provided by the Associazione Italiana per la Ricerca sul Cancro.
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proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins. Oncogene 18, 935 – 941 Everett, R.D. (2000) ICP0, a regulator of herpes simplex virus during lytic and latent infection. Bioessays 22, 761– 770 Joazeiro, C.A. and Weissman, A.M. (2000) RING finger proteins: mediators of ubiquitin ligase activity. Cell 102, 549 – 552 Boutell, C. et al. (2002) Herpes simplex virus type 1 immediate-early protein ICP0 and its isolated RING finger domain act as ubiquitin E3 ligases in vitro. J. Virol. 76, 841– 850 Nuber, U. et al. (1996) Cloning of human Ub-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterisation of their interaction with E6-AP and RSP5. J. Biol. Chem. 271, 2795 – 2800 Parkinson, J. et al. (1999) Herpes simplex virus type 1 immediate-early protein Vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase. J. Virol. 73, 650 – 657 Everett, R.D. et al. (1999) Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110. EMBO J. 18, 1526 – 1538 Lomonte, P. et al. (2001) Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0. J. Biol. Chem. 276, 5829– 5835 Everett, R.D. et al. (1999) The ability of Herpes simplex virus type 1 immediate-early protein Vmw110 to bind to a ubiquitin-specific protease contributes to its roles in the activation of gene expression and stimulation of virus replication. J. Virol. 73, 417– 426 Everett, R.D. et al. (1997) A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J. 16, 566 – 577 Li, M. et al. (2002) De-ubiquitination of p53 by HAUSP is an important pathway for p53 stabilisation. Nature 416, 648– 653 Van Sant, C. et al. (2001) Role of Cyclin D3 in the biology of Herpes simplex virus 1 ICP0. J. Virol. 75, 1888– 1898 Nicholas, J. et al. (1992) Herpesvirus saimiri encodes homologues of G protein-coupled receptor and cyclins. Nature 355, 362 – 365 Chang, Y. et al. (1996) Cyclin encoded by KS herpesvirus. Nature 382, 410 Van Sant, C. et al. (2001) The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity. Proc. Natl. Acad. Sci. U. S. A. 98, 8815– 8820 Yu, Z.K. et al. (1998) Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21 (CIP1/WAF1) and cyclin D proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 11324 – 11329 Maul, G.G. and Everett, R.D. (1994) The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 protein ICP0. J. Gen. Virol. 75, 1223 – 1233 Yew, P.R. and Berk, A.J. (1992) Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature 357, 82 – 85 Moore, M. et al. (1996) Oncogenic potential of the adenovirus E4orf6 protein. Proc. Natl. Acad. Sci. U. S. A. 93, 11295– 11301 Sarnow, P. et al. (1982) Adenovirus E1B 58 kD tumor antigen and SV40 large tumour antigen are physically associated with the same 54 kD cellular protein in transformed cells. Cell 28, 387 – 394 Dobner, T. et al. (1996) Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science 272, 1470– 1473 Kao, C.C. et al. (1990) Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins. Virology 179, 806– 814 Steegenga, W.T. et al. (1998) The large E1B protein together with the E4orf6 protein target p53 for active degradation in adenovirus infected cells. Oncogene 16, 349 – 357 Boivin, D. et al. (1999) Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5. J. Virol. 73, 1245– 1253 Querido, E. et al. (2001) degradation of p53 by adenovirus E4orf6 and E1B55k proteins occurs via a novel mechanism involving a Cullincontaining complex. Genes Dev. 15, 3104– 3117 Harada, J.N. et al. (2002) Analysis of the adenovirus E1B-55k-anchored
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proteome reveals its link to ubiquitination machinery. J. Virol. 76, 9194–9206 Lisztwan, J. et al. (1999) The von Hippel– Lindau tumour suppressor protein is a component of an E3 ubiquitin-protein ligase activity. Genes Dev. 13, 1822 – 1833 Kamura, T. et al. (2000) Activation of HIF1a ubiquitination by a reconstituted von Hippel – Lindau (VHL) tumour suppressor complex. Proc. Natl. Acad. Sci. U. S. A. 97, 10430 – 10435 Patton, E.E. et al. (1998) Combinatorial control in ubiquitin-dependent proteolysis: Don’t skp the F-box hypothesis. Trends Genet. 14, 236 – 243 Ohh, M. et al. (2002) An intact NEDD8 pathway is required for Cullindependent ubiquitylation in mammalian cells. EMBO Rep. 3, 177 – 182 Thomas, M. et al. (1999) The role of the E6 – p53 interaction in the molecular pathogenesis of the HPV. Oncogene 18, 7690– 7700 Clayton-Smith, J. and Laan, L. (2003) Angelman syndrome: a review of the clinical and genetic aspects. J. Med. Genet. 40, 87 – 95 Scheffner, M. et al. (1995) Protein ubiquitination involving an E1 – E2– E3 enzyme ubiquitin thioester cascade. Nature 373, 81 – 83 Huibregtse, J.M. et al. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. U. S. A. 92, 2563 – 2567 Huang, L. et al. (1999) Structure of an E6AP – UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321– 1326 Kumar, S. et al. (1997) Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity. J. Biol. Chem. 272, 13548 – 13554 Elston, R.C. et al. (1998) The identification of a conserved binding motif within human papillomavirus type 16 E6 binding peptides, E6AP and E6BP. J. Gen. Virol. 79, 371 – 374 Beer-Romero, P. et al. (1997) Antisense targeting of E6-AP elevates p53 in HPV-infected cells but not in normal cells. Oncogene 14, 595– 602 Talis, A.L. et al. (1998) The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells. J. Biol. Chem. 273, 6439 – 6445 Butz, K. et al. (2000) Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein. Proc. Natl. Acad. Sci. U. S. A. 97, 6693 – 6697 Honda, R. et al. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumour suppressor p53. FEBS Lett. 420, 25 – 27 Hengstermann, A. et al. (2001) Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells. Proc. Natl. Acad. Sci. U. S. A. 98, 1218– 1223 Bech-Otschir, D. et al. (2001) COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J. 20, 1630– 1639 Kao, W.H. et al. (2000) Human papillomavirus type 16 E6 induces selfubiquitination of the E6-AP ubiquitin protein ligase. J. Virol. 74, 6408– 6417 Thomas, M. and Banks, L. (1998) Inhibition of Bak-induced apoptosis by HPV-18 E6. Oncogene 17, 2943– 2954 Gao, Q. et al. (2002) Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase. Cancer Res. 62, 3315– 3321 Nakagawa, S. and Huibregtse, J.M. (2000) Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol. Cell. Biol. 20, 8244 – 8253 Srivenugopal, K.S. and Ali-Osman, F. (2002) The DNA repair protein, O(6)-methylguanine-DNA methyltransferase is a proteolytic target for the E6 human papillomavirus oncoprotein. Oncogene 21, 5940 – 5945 Oda, H. et al. (1999) Regulation of the Src family tyrosine kinase Blk through E6AP-mediated degradation. Proc. Natl. Acad. Sci. U. S. A. 96, 9557 – 9562 Kumar, S. et al. (1999) Identification of HHR23A as a substrate for E6associated protein-mediated ubiquitination. J. Biol. Chem. 274, 18785 – 18792 Pim, D. et al. (1994) Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo and immortalisation of primary BMK cells. Oncogene 9, 1869– 1876 Nguyen, M. et al. (2002) A mutant of human papillomavirus type 16 E6
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deficient in binding a-helix partners displays reduced oncogenic potential in vivo. J. Virol. 76, 13039 – 13048 Lee, S.S. et al. (1997) Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein. Proc. Natl. Acad. Sci. U. S. A. 94, 6670–6675 Gardiol, D. et al. (1999) Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation. Oncogene 18, 5487– 5496 Glaunsinger, B. et al. (2000) Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19, 1093 – 1098 Thomas, M. et al. (2002) Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation. Oncogene 21, 5088 – 5096 Caruana, G. (2002) Genetic studies define MAGUK proteins as regulators of epithelial cell polarity. Int. J. Dev. Biol. 46, 511 – 518 Bilder, D. et al. (2000) Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113 – 116 Pim, D. et al. (2000) HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase. Oncogene 19, 719– 725 Pim, D. et al. (2002) Chimaeric HPV E6 proteins allow dissection of the proteolytic pathways regulating different E6 cellular target proteins. Oncogene 21, 8140– 8148 Mu¨nger, K. et al. (2001) Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene 20, 7888– 7898 Boyer, S.N. et al. (1996) E7 protein of human papillomavirus-16
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induces degradation of retinoblastoma protein through the ubiquitinproteasome pathway. Cancer Res. 56, 4620 – 4624 Berezutskaya, E. and Bagchi, S. (1997) The human papillomavirus E7 oncoprotein functionally interacts with the S4 subunit of the 26S proteasome. J. Biol. Chem. 272, 30135 – 30140 Luo, H. et al. (2003) Ubiquitin-dependent proteolysis of Cyclin D1 is associated with coxsackievirus-induced cell growth arrest. J. Virol. 77, 1–9 Kalejta, R.F. et al. (2003) Human cytomegalovirus pp71 stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors. Mol. Cell. Biol. 23, 1885– 1895 van der Wal, F.J. et al. (2002) The HCMV gene products US2 and US11 target MHC class I molecules for degradation in the cytosol. Curr. Top. Microbiol. Immunol. 269, 37 – 55 Gotoh, B. et al. (2002) Paramyxovirus strategies for evading the interferon response. Rev. Med. Virol. 12, 337 – 357 Mansouri, M. et al. (2003) The PHD/LAP-domain protein M153R of myxomavirus is a ubiquitin ligase that induces the rapid internalisation and lysosomal destruction of CD4. J. Virol. 77, 1427 – 1440 Licata, J.M. et al. (2003) Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4. J. Virol. 77, 1812– 1819 Yasuda, J. et al. (2002) Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep. 3, 636 – 640
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Viruses and the 26S proteasome: hacking into destruction Lawrence Banks, David Pim and Miranda Thomas International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy
The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus–host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases. In the quest for successful infection, viruses are faced with many obstacles, including host immune-surveillance and blocks to the cell-cycle machinery, and hence to viral replication. This might ultimately include host-induced suicidal apoptotic cell death and restriction of the infection to a single cell, thereby protecting the rest of the organism from further viral spread. To overcome these host responses, viruses across a wide spectrum of host and tissue tropisms have evolved many similar strategies. Often, the mechanisms employed are different but the ultimate effects are the same: productive viral infection. One of the common obstacles facing a virus is the overwhelming stoichiometric imbalance of the host target proteins over those encoded by the virus. To overcome this, many viruses have evolved mechanisms whereby their cellular target-proteins are directed to the 26S proteasome and then subjected to proteolytic degradation. Although this is often achieved in different ways, the viruses invariably act at the point of substrate recognition. This means that the targeting of the cellular proteins is a highly specific process and almost always involves an E3 ubiquitin ligase (Fig. 1). This ubiquitin ligase can be directly encoded by the virus, or could be a host ligase that has been redirected to a substrate that it would not normally recognize, alternatively the normal function of a host-encoded ligase could be stimulated. The first example of a viral protein using the proteasome machinery to direct the degradation of a cellular protein was provided by the human papillomavirus (HPV) E6 protein and its stimulation of the ubiquitination and degradation of the cellular tumour-suppressor protein Corresponding author: Lawrence Banks ([email protected]).
p53. Since then, many different viruses have been shown to direct the ubiquitination of diverse cellular proteins, mainly for degradation at the 26S proteasome (for examples, see Table 1). These include avoidance of host immune surveillance processes by human herpes virus 8 (HHV8), which is responsible for Kaposi’s Sarcoma. In this instance, the virus encodes two transmembrane proteins – modulator of immune recognition (MIR)-1 and MIR2, which represent a novel class of membrane-bound E3 ubiquitin ligases. Both MIR1 and MIR2 ubiquitinate major histocompatibility complex-I chains leading to their endolysosomal degradation, whereas MIR2 also induces the ubiquitination of the B7.2 cell-surface protein, which is a co-stimulator of T-cell activation [1]. Others examples include vesicular stomatitis virus and rabies virus, the matrix proteins of which interact directly with the Nedd4 cellular ubiquitin-ligase – a process that appears to be required for efficient viral egress – although how this takes place is still unclear [2]. Interestingly, Epstein – Barr virus also makes use of Nedd4. In this case, the viral latent membrane protein 2a redirects Nedd4 activity to a number of B-cell tyrosine kinases, which are subsequently targeted to the proteasome. This activity alters B-cell-receptor signalling and is believed to contribute to the establishment of a latent viral infection [3]. Likewise, the adenovirus penton-base protein, which is required for cell entry, also interacts with Nedd4 [4]. The amazing level of evolutionarily conserved use of the proteasome as a means of successfully completing the viral life-cycle has even been implicated in some plant viruses: regulation of tobacco mosaic virus movement protein is believed to be proteasome mediated, and plays a role in regulating viral spread [5]. Ubiquitination of substrate proteins Before discussing specific cases, it is worth briefly reviewing the ubiquitin– proteasome pathway. As shown in Fig. 1, the E3 ubiquitin ligases are involved in the specific recognition of substrates for ubiquitination. Eukaryotic organisms encode very few, sometimes only one, E1 ubiquitin-activating enzyme, which binds to the C terminus of ubiquitin (Ub) through a thioester linkage. The ubiquitin is then transferred, again through a thioester linkage, to a ubiquitin-conjugating enzyme (Ubc or E2), of which . 50 are known in humans. The ubiquitin is then transferred to the target protein by E3 ubiquitin-protein ligases, which interact with the E2 and with specific protein substrates. This allows the formation
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O +
C Ub HO
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E2
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C S
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Viral pr otein
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Ub
ATP
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Pi + ADP
Ti BS
Fig. 1. The ubiquitin proteasome pathway and common points of viral entry. In the majority of cases the virus intervenes at the step of the ubiquitin-protein ligase (E3). This can be virally encoded, or can be a cellular ligase that is either redirected or stimulated by another viral protein. The heavily ubiquitinated target protein is then rapidly degraded at the proteasome.
of isopeptide bonds between the C terminus of Ub and lysine residues either on the target protein or on the last Ub of an existing poly-Ub chain. Multi-Ub chains act as potent targeting signals for protein degradation in the http://tibs.trends.com
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proteasome [6]. The number of known E3 ligases is increasing almost daily. They provide specificity, both temporally and spatially, for the labelling of proteins for destruction by the proteasome and, as such, are one of the targets of choice for viral intervention in the activities of the host cell. Here, we focus on some well-defined systems that provide three different examples of how viral proteins direct their cellular substrates to the proteasome: herpes simplex virus (HSV)-1 infected cell protein 0 (ICP0); adenovirus E4orf6– E1B 55K and HPV E6.
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HSV-1 ICP0: a single component viral ubiquitin ligase The HSV-1 protein ICP0 is expressed early during infection and is one of the first viral proteins to be expressed upon reactivation from latency. ICP0 acts in synergy with the major viral transactivator ICP4 to induce the expression of the viral genes. This activity of ICP0 is thought to depend upon its localization to the nuclear structures known as ND10s or PODs [7]. The viral genomes also localize to ND10s, where virus replication compartments develop [8]. The presence of ICP0 results in the degradation of two major ND10 components, promyelocytic leukemia antigen (PML) and Sp100 [9,10] thereby disrupting the ND10. The RING (really interesting new gene)-finger domain found at the N terminus of the protein has been shown to be required for all these activities [11] and we should briefly consider this type of domain. The majority of E3 ubiquitin-protein ligases have either a HECT [homologous to E6-associated protein (E6AP) carboxy terminus] domain (see E6AP below) or a RINGfinger domain. RING fingers have the consensus sequence Cx2Cx9 – 39Cx1 – 3Hx2 – 3[C/H]x2Cx4 – 48Cx2C, in which the C and H residues are zinc binding. RING fingers facilitate the transfer of ubiquitin or ubiquitin-like molecules from the E2 enzyme onto a range of heterologous substrates and, indeed, onto themselves. Proteins containing RING fingers form the largest known class of E3 ligases and appear to be involved in many cellular processes [12]. The ICP0 protein, or a fragment containing only the N terminus and the RING finger (Fig. 2a), has been shown to induce the accumulation of poly-Ub chains in the presence of an E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzymes UbcH5a and UbcH6 [13]. The interaction appears to be specific because a number of related E2 proteins showed no poly-Ub accumulation in the presence of ICP0. Interestingly, these two E2 proteins were originally identified as being involved in the E6– E6AP-induced degradation of p53, albeit with different efficiency [14], suggesting that HSV and HPV have both evolved to interact with a common cellular pathway. In this context it is worth noting that, although the two viruses are very different, they both infect similar mucosal epithelia and so might be expected to encounter a similar cellular environment. The question that obviously arises is what does the virus gain from inducing the degradation of these cellular proteins? Numerous mutational studies have shown that more cells become committed to productive infection, particularly at low doses of input virus, when the ICP0 is fully functional and it would appear that ICP0 activity circumvents a cellular silencing activity that drives the
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Table 1. Examples of how viruses use the cellular ubiquitin pathway to target cellular proteins during the viral life cyclea Factor Function
Virus
Host
Viral
Target proteins
Refs
Entry Transcription/reactivation
Adenovirus HSV
Nedd4 family –
Penton-base protein ICP0
[4] [15 –17]
HPV Coxsackie HCMV HPV Adenovirus KSHV HCMV EBV Paramyxovirus Myxomavirus Ebola Retrovirus Rhabdovirus
Unknown Unknown Unknown E6AP SCF complex – ERAD complex Nedd4 SCF complex – Nedd4 BUL1 (Nedd4-like) Nedd4
E7 Unknown pp71 E6 E4orf6 MIR1, MIR2 US2, US11 LMP2A V protein M153R VP40L domain ?Gag M protein
Unknown PML, Sp100, CENP-A and C/DNA-PK catalytic subunit pRb, p107, p130 cyclin D1 pRb, p107, p130 p53, Bak p53 MHC components MHC-1 heavy chains Lyn, Syk STAT proteins CD4 Unknown Unknown Unknown
Apoptosis avoidance Immune avoidance
Virus release/budding
[71] [73] [74] [40,54] [32] [1] [75] [3] [76] [77] [78] [79] [2]
a Abbreviations: CENP, centromere protein; DNA-PK, DNA-dependent protein kinase; E6AP, E6-associated protein; EBV, Epstein– Barr virus; ERAD, endoplasmic reticulumassociated degradation; HCMV, human cytomegalovirus; HPV, human papillomavirus; HSV, herpes simplex virus; ICP0, infected cell protein 0; KSHV, Kaposi’s sarcomaassociated herpes virus; LMP2A, latent membrane protein 2A; MHC, major histocompatibility complex; MIR, modulator of immune recognition; PML, promyelocytic leukemia antigen; SCF, skp1– cdc53– F-box complex.
virus towards a latent infection [11]. Many genes that are silenced are closely linked to transcriptionally silent regions of heterochromatin such as centromeres and telomeres, and the heterochromatin protein HP1 is found interacting with Sp100 in ND10s and interphase centromeres [11]. Because the known ICP0 targets include PML, Sp100, the catalytic subunit of DNA-PK [15], and two centromere proteins CENP-A and CENP-C [16,17], the following model can be proposed: upon virus infection, the DNA uncoats and localizes to ND10s, which have been suggested to be ‘stores’ of compounds and factors required for transcription and replication. ICP0-induced degradation of PML and Sp100 then disrupts the heterochromatin, thus, ‘mugging the storekeeper’ and making the useful contents of ND10 available for viral transcription and replication. Mutations resulting in the functional loss of the RING finger of ICP0 have a devastating effect on most activities of the protein and upon viral infection [18]. However, mutations in other regions of the protein also result in reduced activation of viral gene expression [18]. The C-terminal half of the protein contains a nuclear localization signal (NLS) and a binding site for a ubiquitinspecific protease USP7 [19]. Interestingly, USP7 is also involved in the de-ubiquitination and stabilization of p53 [20]. Hence, sequestration of this protein by ICP0 might also be a means of overcoming p53 activity. ICP0 can bind and stabilize cyclin D3 and appears to relocalize it to the ND10. This causes formation of an active complex of cyclin D3 and cyclin-dependent kinase 4 (cdk4), which, in turn, phosphorylates the pRb tumour suppressor, causing release of E2F and promotion of G1 progression [21]. This provides an interesting parallel with certain g-herpes viruses, such as HHV8 and herpesvirus saimiri, which encode their own D-type cyclins [22,23]. The C-terminal region of ICP0 also contains a second ubiquitin ligase, which does not have a RING finger and which appears to act through the E2 cdc34 (UbcH3) [24]. This is the major E2 that acts through the skp1 – cdc53 – F-box http://tibs.trends.com
(SCF) complex. One of the functions of this complex is to promote the ubiquitination and degradation of cyclin D1 [25]. As it has been reported that ICP0 can stabilize D1 without binding to it [26], this might suggest that this E3-ligase domain is a pseudo-E3, sequestering cdc34 from the SCF complex. The D cyclins are normally involved in the promotion of cell cycle through G1/S phase transition, thus, continued high levels of these proteins would probably keep the host cell in a state of replicative readiness, that is, an ideal environment for the replication of the viral genome. Thus, it would appear that ICP0 interacts in two ways with the 26S proteasome: the RING finger acts as an E3 to direct the degradation of components of ND10 and centromeres, thereby aiding viral transcription. Simultaneously, the C-terminal domain appears to act as a pseudo-E3, competitively inhibiting the proteasomal degradation of cyclin D1, thus, promoting a cellular environment that is conducive to viral DNA replication. Adenovirus E4orf6 –E1B55k –cullin complex: a multi-component ligase A major obstacle to the replication of many different viruses is the p53 tumour suppressor, and numerous strategies have been employed to overcome its function. As noted above, HSV-1 ICP0 can potentially inactivate p53 through association with USP7, whilst HPV uses the E6 – E6AP complex. In adenovirus it was thought that the viral E1B 55 kDa (E1B 55K) protein alone was sufficient to inactivate p53 by blocking its transcriptional activity [27]. However, recent studies have shown a more complex pattern of interaction whereby p53 is also targeted for proteasome-mediated degradation. In adenovirus-infected and transformed cells, p53 levels are reduced [28]. The association of both E1B 55K [29] and the E4orf6 protein with p53 [30] suggested that these proteins might be responsible. E1B 55K and E4orf6 bind to p53 near its N and C termini, respectively [31,30], and it was shown that they also led to a proteasome-dependent reduction in p53 half-life [32]. Immunoprecipitation of Ad5
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(a) Ub Ub Ub
Ub
E1
Ubc H5a /H6
PML/ Sp100
RING
Ub
Ub Ub Ub
26s Proteasome
HUL-1 ICPO Cdc34
Ub Ub
Ub Ub Ub
(b) Ub Ub
Ub Ub Ub 26s Proteasome
E1B 55K p53 Ub
Ub
Ub
E4orf6
E2 E1
Rbx1
C B Elongins Cullin 5
(c) Ub
Ub
Ub
E1
Ubc H7
Ub Ub Ub HECT
Ub Ub Ub
26s Proteasome E6AP
p53
E6 Ti BS
Fig. 2. Three examples of viral ubiquitin-ligase complexes. (a) The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) ubiquitin ligase directs the degradation of promyelocytic leukemia antigen (PML) oncogenic domain (POD) components PML and Sp100 through direct interactions with the substrates and the E2 enzymes (UbcH5a/H6) via its RING(really interesting new gene)-finger domain. The C-terminal half of ICP0 also interacts with the E2 enzyme cdc34 (UbcH3), which is involved in regulating cyclin D1 levels. Because ICP0 stabilizes cyclin D, this might be acting as a pseudo E3. (b) The adenovirus E1b 55K– E4orf6– cullin ubiquitin-ligase complex directs the degradation of the p53 tumour-suppressor protein. E1B55K and E4orf6 both directly bind to the p53 tumour suppressor. Through interactions with Elongins B and C E4orf6 recruits cullin 5 and E2. Thus, the E1B 55K –E4orf6 provide specificity and can be seen as being functionally analogous to the skp1– cdc53– F-box (SCF) complex. (c) The human papillomavirus (HPV) E6–E6-associated protein (E6AP) ubiquitin-ligase complex directs the degradation of the p53 tumour-suppressor protein. E6 interacts directly with p53 and the E6AP ubiquitin ligase, thereby redirecting E6AP to p53 together with the E2 enzyme UbcH7 family members.
E4orf6 from cells co-precipitates a group of cellular proteins [33] that have been identified [34] as the previously described 84 kDa cullin 5, 19 kDa elongin B, 16 kDa Rbx1 and 14 kDa elongin C. Further studies showed that E4orf6 binds directly to the Elongin BC complex [34,35]. Elongin BC is a component of the large von Hippel– Lindau (VHL) tumour-suppressor complex, which functions as an E3 ubiquitin ligase [36]. The VHL component acts as a substrate recognition motif [37], whereas the Elongin BC complex links substrates to a module containing Rbx1 and cullin 2, which, in turn, activates an E2 ubiquitin-conjugating enzyme [37]. The striking similarity between VHL and mammalian SCF ubiquitin ligase complexes [38] suggested that the complex between E4orf6, Elongin BC and a cullin family member might form an E3 ligase that could be recruited by E1B 55K to target p53 for proteasome-mediated degradation. http://tibs.trends.com
Because cullin activity is dependent on its modification by NEDD8 ([39] and references therein), a temperaturesensitive cell line with a mutation in NEDD8 was then used to confirm that NEDD8 modification of cullin 5 was indeed required for E1b – E4orf6-induced degradation of p53 [34]; Fig. 2b shows the structure of the complex. Taken together, these studies demonstrate a complex pattern of events whereby adenovirus overcomes the inhibitory potential of p53 at the transcriptional level and at the level of protein turnover. This highlights the need to overcome p53 functions that are independent of its ability to activate gene expression, and it seems most likely that these are the pro-apoptotic activities of p53. HPV E6–E6AP: a bi-partite ubiquitin-protein ligase E6AP is the prototype HECT-domain-containing E3 ligase, which was originally identified in association with the E6
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proteins of oncogenic HPV-16 and HPV-18 in the ubiquitination of p53 [40]. It is a monomeric 100 kDa polypeptide with a C-terminal ubiquitin-protein ligase HECT domain, and its loss or mutation is associated with Angelman syndrome – a serious developmental disease, characteristics of which include a variety of behavioural disorders [41]. Unlike other E3 ligases, HECT-domain proteins form a thioester linkage with the ubiquitin moiety, as part of the process of transferring it from the E2 conjugating enzyme to the bound substrate protein. The presence of a cysteine residue (C820 in E6AP) is required for this activity and is strictly conserved in HECT-domain proteins [42,43]. The N-terminal domains of HECT proteins are very divergent, and this is thought to largely define their target-protein specificity and might possibly affect their interaction with specific E2 enzymes [43]. E6AP has been shown to interact with a number of E2s, including UbcH5 and UbcH6 (as mentioned above) plus UbcH7 and UbcH8 [44]. Specificity in the binding of E2s to the HECT domain determines the preferred E2 [45], and this might be affected by the binding of E6 or other similar targetdetermining molecules. E6 binds to the E6AP molecule through a linear helical domain between residues 391 and 408 of E6AP, one side of the helix has a hydrophobic patch formed by the motif Glu-Leu-Leu/Val-Gly, which is essential for E6 binding [46]. E6 simultaneously binds to p53, allowing the HECT domain to transfer ubiquitin from the bound E2, via a thioester intermediate, to the p53 molecule (Fig. 2c). It is clear that the binding of E6 to E6AP alters its substrate specificity because E6AP is not thought to normally recognize p53 as a target for ubiquitination. This has been shown by several studies in which interfering with E6AP activity by using antisense oligonucleotides [47], dominant-negative mutants [48] and competing peptides [49] only affects p53 levels in E6-expressing cells. In the absence of E6, p53 levels are also regulated by the proteasome using mouse double minute 2 (MDM2) as the specific ubiquitin ligase [50]. This is a very different interaction because MDM2 is a RING-finger protein but, in E6-expressing cells, the E6 – E6AP completely takes over the control of p53 levels [51]. Interestingly, p53 has to be phosphorylated on Thr155 for it to be susceptible to E6 – E6AP-induced degradation [52], indicating that the precise timing of p53 degradation and/or localization of p53 must be tightly controlled to allow a productive viral life cycle. Again, the parallels between adenovirus and HPV abolition of p53 function are striking, with identical results, yet attained by very different mechanisms. Although p53 is the best known of the E6 – E6AP targets, others have also been described including the apoptosis-promoting Bak protein as well as E6AP itself [53], which might represent a feedback control mechanism. In the case of Bak, it is clear that it is targeted by E6AP in the absence of E6, but that the presence of E6 enhances this activity [54]. Many other cellular proteins are directed for degradation by E6–E6AP, including E6TP1 [55], Scribble [56] and the DNA-repair protein O6-methylguanine DNA methyltransferase [57]. At present, it is not http://tibs.trends.com
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clear whether E6AP is involved in the normal regulation of these proteins in the uninfected cell. Neither is it clear what effect E6 – E6AP has upon E6-independent E6AP targets, such as activated Src [58] and the Rad23 homologue HHR23A [59]. E6AP-independent targets of HPV E6 Despite the fundamental importance of the E6– E6AP interaction for a wide range of biological activities of E6, it is becoming clear that other functions of E6 are required for its contribution to malignancy. Indeed, the ability of E6 to bring about cell transformation and induce tumours in transgenic animals requires activities other than the E6 – E6AP interaction [60,61]. Interestingly, a recently identified subset of E6 target proteins containing PDZ domains might be responsible for E6-induced malignancy. The first of these proteins to be described – the discs large (Dlg) protein [62] – is targeted by E6 for ubiquitindependent degradation [63]. Dlg belongs to a family of PDZ domain-containing proteins known as membrane-associated guanylate kinases (MAGUKs), several other members of which are also directed for degradation by E6. These include membrane-associated guanylate kinase with inverted orientation (MAGI)-1 [64], MAGI-2 and MAGI-3, [65] and hScrib [56]. These proteins are found typically, but not exclusively, at the membrane– cytoskeletal interfaces of cells, mediating interactions between molecules involved in cell – cell, cell – substratum contact, receptor clustering, cell polarity and signal transduction [66]. Many of the proteins have been assigned the roles of potential tumour-suppressor proteins, with Drosophila Dlg and Scribble acting in concert to regulate cell polarity and cell growth [67]. Not surprisingly, there is now thought to be a strong correlation between the ability of E6 to direct the degradation of these proteins and its contribution to malignant progression. The E6 proteins from the high-risk cancer-associated mucosal HPVs (HPV-16 and HPV-18) interact with MAGUKs through a C-terminal four-amino-acid consensus PDZ-binding motif, and direct their degradation by recruiting a cellular E3 ubiquitin ligase. Interestingly, this binding motif is absent in E6 proteins that are derived from the low-risk benign HPV types (HPV-6 and HPV-11). In the case of hScrib, E6 – AP has been implicated in the degradation [56]. However, for Dlg and the MAGI proteins, there is mounting evidence indicating that a different cellular ligase is involved. Thus, mutants of HPV-18 E6, which cannot induce p53 degradation, can still induce Dlg degradation, and E6 also degrades Dlg in systems that lack E6AP [68]. Furthermore, chimaeric E6 molecules based on an HPV-11 or HPV-6 E6 backbone, which cannot interact with E6AP, nonetheless target Dlg for proteasomemediated degradation when provided with a PDZ-binding motif [69]. Finally, it should also be remembered that the kinetics with which these different substrates of E6 are degraded are wildly different [64,65,68]. Intrestingly, the ability of HPV to direct cellular proteins for proteasome-mediated degradation is not restricted to the E6 protein. The E7 protein encoded by cancer-associated HPV types is responsible for driving cells into an artificial S phase, rendering them capable of
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growth arrest apoptosis
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Fig. 3. The contribution of the human papillomavirus (HPV) E6 and E7 oncoproteins to malignant transformation by directing their substrates for proteasome-mediated degradation. HPV infects keratinocytes that have exited the cell cycle. The E7 protein, through interaction with the proteasome machinery, targets the ‘pocket-protein’ family of proteins for degradation, resulting in increased DNA replication and proliferation. This unscheduled replication results in a p53 response that is targeted by the E6 –E6-associated protein (E6AP) ubiquitin-ligase complex. At later stages of malignancy, the ability of E6 to direct the degradation of a family of membrane-associated guanylate kinase (MAGUK) proteins probably contributes to the metastatic progression. Throughout this process, the continued proliferation and abolition of normal detectors of DNA damage (p53) results in the accumulation of genetic damage within the transformed cells.
replicating the viral DNA [70]. This activity requires the ability of E7 to interact with a family of cell-cycle regulators including the pRb tumour suppressor and other members of the pocket protein family. In all cases, E7 efficiently directs the degradation of these substrates at the proteasome [71]. It should be clear that this has striking parallels to the activity of the HSV ICP0, which stabilizes D-type cyclins and results in the release of free of E2F from pRb. This underlines the essential similarity in the cellular obstacles to a successful viral life cycle. At present, the ligase employed by E7 is unknown, although E7 itself has been reported to directly interact with different components of the proteasome machinery [72]. Elucidating this particular pathway remains a major goal within HPV research in the coming few years. Concluding remarks The use of the proteasome by a plethora of different viral proteins emphasizes the central importance of this cellular pathway for many successful viral infections. However, in rare cases, this perturbation of normal cellular regulatory pathways initiates events that, ultimately, result in cell transformation and malignancy. This is most notable with the high-risk HPVs – infection with which can result in human malignancy, including cervical cancer. As can be seen from Fig. 3, the targeting of cellular proteins to the proteasome via interactions with cellular ubiquitin ligases is central to this whole process. HPV E7 targets the pocket protein family and induces cell proliferation. Under normal circumstances, p53 would then induce growth arrest and/or apoptosis, however, the E6 – E6AP ubiquitinligase complex effectively abolishes this response. Finally, http://tibs.trends.com
at later stages of disease progression, the degradation of the MAGUK family of proteins by E6 probably results in metastasis and death of the host. Designing strategies to block the viral perturbation of these cellular ligases are major goals in cancer therapy and in antiviral therapeutics as a whole. Acknowledgements The list of viruses that encounter this pathway is growing daily and we apologize to those colleagues whose work has not been cited owing space limitations. We gratefully acknowledge research support provided by the Associazione Italiana per la Ricerca sul Cancro.
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