EC5S Ubiquitin Complex Is Recruited by KSHV Latent Antigen LANA for Degradation of the VHL and p53 Tumor Suppressors

Cai, Qiliang; Knight, Jason S.; Verma, Suhbash C.; Zald, Philip; Robertson, Erle S.

doi:10.1371/journal.ppat.0020116

Cited by 179 publications

(212 citation statements)

References 64 publications

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“…The existence of multiple, mutually inclusive complexes can explain why targeted approaches such as coimmunoprecipitation and overexpression-based transcriptional experiments that are conducted in cells lacking the KSHV episome consistently find evidence for p53:LANA interactions (7,10,23,83,97,100), whereas phenotypic analyses of PEL or less sensitive, mass spectrometry (MS)-MS-based screens (4, 84) of PEL could not find evidence for a prominent p53:LANA complex. Further support for multiple independent complexes comes from our half-life analysis.…”

Section: Discussionmentioning

confidence: 99%

Distinct p53, p53:LANA, and LANA Complexes in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas

Chen

Hilton

Staudt

et al. 2010

J Virol

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The role of p53 in primary effusion lymphoma (PEL) is complicated. The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds p53. Despite this interaction, we had found that p53 was functional in PEL, i.e., able to induce apoptosis in response to DNA damage (C. E. Petre, S. H. Sin, and D. P. Dittmer, J. Virol. 81:1912-1922, 2007), and that hdm2 was overexpressed. To further elucidate the relationship between LANA, p53, and hdm2, we purified LANA complexes from PEL by column chromatography. This confirmed that LANA bound p53. However, the LANA:p53 complexes were a minority compared to hdm2:p53 and p53:p53 complexes. The half-life of p53 was not extended, which is in contrast to the half-life of simian virus 40 T antigen-transformed cells. p53:p53, LANA:p53, and LANA:LANA complexes coexisted in PEL, and each protein was able to bind to its cognate DNA element. These data suggest that under normal conditions, p53 is inactive in PEL, thus allowing for exponential growth, but that this inactivation is driven by the relative stoichiometries of LANA, hdm2, and p53. If p53 is activated by DNA damage or nutlin-3a, the complex falls apart easily, and p53 exercises its role as guardian of the genome.Kaposi's sarcoma-associated herpesvirus (KSHV) is found in Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and tumors from patients with the plasmablastic variant of multicentric Castleman disease (MCD) (reviewed in reference 15). The latency-associated nuclear antigen (LANA) is encoded by open reading frame 73 (ORF 73) of the viral genome. It is expressed in every KSHV-infected cell during the latent as well as lytic phase of the viral life cycle. LANA is required for replication and maintenance of the viral DNA during latent infection (2, 13). Experimental abrogation of LANA expression through small interfering RNA (siRNA) or genomic knockout leads to loss of KSHV from latently infected cells, genetically demonstrating that LANA is necessary for maintenance of latency (28,103). LANA is a large protein composed of 1,162 amino acids (KSHV M type, reference sequence NC_003409). It has many known biochemical activities, and many more have yet to be determined. One way to think about LANA is as a nuclear scaffolding protein for viral DNA replication and transcription, analogous to the large T antigen of the polyomaviruses.We hypothesized that there exist multiple biochemically distinct LANA complexes in KSHV-infected PEL, as both LANA and its many potential interaction partners are present at physiological molar ratios to each other. Biochemical analysis of purified native complexes in PEL allowed us to establish that not all of LANA was part of a LANA:p53 complex and that not all of p53 was bound to LANA.The C terminus of the LANA protein has sequence-specific DNA binding activity. LANA can bind to a 20-bp repeated cis element in the terminal repeats (TRs) of the viral genome (3,26,27,45,50,78,85,101). We previously identified a similar cis element that was present as a single c...

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Section: Discussionmentioning

confidence: 99%

Distinct p53, p53:LANA, and LANA Complexes in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas

Chen

Hilton

Staudt

et al. 2010

J Virol

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“…IFN-induced HIF 1α stabilization, as VHL loss, could cooperate in organizing host defenses and have cellular consequences that in some cases can inhibit viral replication (49,50). However, in other instances of persistent viral infection, HIF-1 activation can lead to the activation of the proangiogenic program and oncogenic transformation as a consequence (11,(51)(52)(53)(54). Therefore, considering the primary role of HIF 1α in immune defense, it is not surprising that cells have evolved auxillary mechanisms to rapidly sustain the stabilization of HIF 1α, for instance through the degradation of VHL protein.…”

Section: E G F P P E G F P -S O C S 1 B O X P E G F P -S O C S 3 B O mentioning

confidence: 99%

“…Several viruses encode for their own substrate receptors possessing the BC-box motif and are thus able to interact with EloB/ C cellular adaptor, thereby modifying the cellular ubiquitin E3 ligase complex and their target proteins selection (10)(11)(12)(13)(14)(15)(16)(17). We have demonstrated that the CELO (Chicken Embryo Lethal Orphan) Adenovirus early protein Gam1 is one of such BC boxcontaining proteins that is able to associate with both Cullin2 and Cullin5 to reconstitute active E3 ligase complexes and target the SUMO E1 enzyme (SAE1/SAE2 heterodimer) for degradation (18,19).…”

mentioning

confidence: 99%

BC-box protein domain-related mechanism for VHL protein degradation

Pozzebon

Varadaraj

Mattoscio

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasomedependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.ubiquitylation | oncoviral proteins | hypoxia U biquitylation is a posttranslational modification that involves the tagging of protein substrates with ubiquitin moieties. Ubiquitin transfer requires the coordinated and subsequent activities of the ubiquitin-activating E1 enzyme, ubiquitin-conjugating E2 enzymes, and ubiquitin E3 ligases. E3 ligases show substrate specificity and can be grouped into three families based on their different E2-docking domains. CRLs (Cullin RING Ligases) are the largest subfamily of RING domain ligases (1). They display the same modular structure: a scaffold subunit (Cullin), a RING subunit, an adaptor, and a substrate-receptor subunit to recruit the specific protein target to be ubiquitylated (2). Each Cullin associates with a specific adaptor subunit, except for Cullin2 and Cullin5, which both interact with the Elongin B and Elongin C (hereafter EloB/C) heterodimer (3). The EloB/C heterodimer is specifically bound by substrate receptors that contain a minimal consensus sequence (called BC-box motif) (4-6) usually included in the SOCS (Suppressor of Cytokine Signaling) domain of several substrate-receptor subunits (7,8). The consensus sequence and the function of the BC-box motif were first described for SOCS proteins (5, 8, 9), a family of downstream effectors of cytokine signaling cascade, involved in the negative feedback regulation of this pathway.Several viruses encode for their own substrate receptors possessing the BC-box motif and are thus able to interact with EloB/ C cellular adaptor, thereby modifying the cellular ubiquitin E3 ligase complex and their target proteins selection (10-17). We have demonstrated that the CELO (Chicken Embryo Lethal Orphan) Adenovirus early protein Gam1 is one of such BC boxcontaining proteins that is able to associate with both Cullin2 and Cullin5 to reconstitute active E3 ligase complexes and tar...

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“…Two previous reports have suggested that upon exogenous expression, LANA interferes with p53 and inhibits its transcriptional activity (26,27). More recently, LANA was also suggested to function as a component of the EC5S ubiquitin complex targeting p53 for degradation (28). However, these findings have not progressed toward a mechanistic explanation of how the virus overcomes normal cellular barriers or provided opportunities for therapy.…”

Section: Introductionmentioning

confidence: 99%

Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas

Sarek¹,

Kurki²,

Enbäck³

et al. 2007

J. Clin. Invest.

133

135

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Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent for primary effusion lymphoma (PEL), a nonHodgkin type lymphoma manifesting as an effusion malignancy in the affected individual. Although KSHV has been recognized as a tumor virus for over a decade, the pathways for its tumorigenic conversion are incompletely understood, which has greatly hampered the development of efficient therapies for KSHV-induced malignancies like PEL and Kaposi's sarcoma. There are no current therapies effective against the aggressive, KSHV-induced PEL. Here we demonstrate that activation of the p53 pathway using murine double minute 2 (MDM2) inhibitor Nutlin-3a conveyed specific and highly potent activation of PEL cell killing. Our results demonstrated that the KSHV latency-associated nuclear antigen (LANA) bound to both p53 and MDM2 and that the MDM2 inhibitor Nutlin-3a disrupted the p53-MDM2-LANA complex and selectively induced massive apoptosis in PEL cells. Together with our results indicating that KSHV-infection activated DNA damage signaling, these findings contribute to the specificity of the cytotoxic effects of Nutlin-3a in KSHV-infected cells. Moreover, we showed that Nutlin-3a had striking antitumor activity in vivo in a mouse xenograft model. Our results therefore present new options for exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment modality for this virally induced lymphoma.

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EC5S Ubiquitin Complex Is Recruited by KSHV Latent Antigen LANA for Degradation of the VHL and p53 Tumor Suppressors

Cited by 179 publications

References 64 publications

Distinct p53, p53:LANA, and LANA Complexes in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas

Distinct p53, p53:LANA, and LANA Complexes in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas

BC-box protein domain-related mechanism for VHL protein degradation

Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas

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