The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

hepatitis virus ͉ interferon regulatory factor 3 ͉ interferon-beta ͉ melanoma differentiation associated gene 5 ͉ mitochondrial antiviral signaling protein

The tumour suppressor p53 induces apoptosis or cell-cycle arrest in response to genotoxic and other stresses. In unstressed cells, the anti-proliferative effects of p53 are restrained by mouse double minute 2 (Mdm2), a ubiquitin ligase (E3) that promotes p53 ubiquitination and degradation. Mdm2 also mediates its own degradation through auto-ubiquitination. It is unclear how the cis- and trans-E3 activities of Mdm2, which have opposing effects on cell fate, are differentially regulated. Here, we show that death domain-associated protein (Daxx) is required for Mdm2 stability. Downregulation of Daxx decreases Mdm2 levels, whereas overexpression of Daxx strongly stabilizes Mdm2. Daxx simultaneously binds to Mdm2 and the deubiquitinase Hausp, and it mediates the stabilizing effect of Hausp on Mdm2. In addition, Daxx enhances the intrinsic E3 activity of Mdm2 towards p53. On DNA damage, Daxx dissociates from Mdm2, which correlates with Mdm2 self-degradation. These findings reveal that Daxx modulates the function of Mdm2 at multiple levels and suggest that the disruption of the Mdm2-Daxx interaction may be important for p53 activation in response to DNA damage.

The pathogenesis of bovine viral diarrhea virus (BVDV) infections is complex and only partly understood. It remains controversial whether interferon is produced in cells infected with cytopathic(cp) BVDVs which do not persist in vivo. We show here that a cpBVDV (NADL strain) does not induce interferon responses in cell culture and blocks induction of interferon-stimulated genes by a super-infecting paramyxovirus. cpBVDV infection causes a marked loss of interferon regulatory factor 3 (IRF-3), a cellular transcription factor that controls interferon synthesis. This is attributed to expression of Npro, but not its protease activity. Npro interacts with IRF-3, prior to its activation by virus-induced phosphorylation, resulting in polyubiquitination and subsequent proteasomal degradation of IRF-3. Thermal inactivation of the E1 ubiquitin-activating enzyme prevents Npro-induced IRF-3 loss. These data suggest that inhibition of interferon production is a shared feature of both ncp and cpBVDVs and provide new insights regarding IRF-3 regulation in pestivirus pathogenesis.

The pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas is associated with independent chromosomal translocations that lead to the upregulation of either BCL10 or MALT1 or the generation of a fusion protein, cIAP2-MALT1. While both BCL10 and MALT1 are critically involved in antigen receptormediated NF-κB activation, the role of cIAP2 is not clear. Here we show that cIAP2 is a ubiquitin ligase (E3) of BCL10 and targets it for degradation, inhibiting antigen receptor-mediated cytokine production. cIAP2-MALT1 lacks E3 activity, and concomitantly, the BCL10 protein is stabilized in MALT lymphomas harboring this fusion. Furthermore, BCL10 and cIAP2-MALT1 synergistically activate NF-κB. These results reveal cIAP2 as an inhibitor of antigenic signaling and implicate its dysfunction in MALT lymphomas. IntroductionInhibitor of apoptosis proteins (IAPs) are a family of evolutionarily conserved proteins that regulate apoptosis and other cellular processes (1, 2). IAPs contain 1-3 characteristic baculoviral IAP repeat (BIR) motifs, which can directly bind to and inhibit caspases, the central executioner proteases of apoptosis (3). This inhibitory effect of IAPs on caspases is counteracted during apoptosis by death inducers such as mammalian Smac/DIABLO (4-6). However, some IAPs, including cellular IAP1 (cIAP1) and cIAP2, are poor inhibitors of caspases, and they may have important functions in addition to or instead of inhibiting apoptosis. cIAPs and several other IAPs also contain a RING domain, which possesses ubiquitin ligase (E3) activity that may mediate the degradation of themselves and proapoptotic proteins such as Smac/DIABLO (7-9). cIAP1 targets the antiapoptotic molecule TRAF2 for degradation and thus plays a role in promoting apoptosis (10).cIAP2 is implicated in the mucosa-associated lymphoid tissue (MALT) lymphomas; this presents a definitive example of the involvement of IAPs in human diseases. MALT lymphomas are the most common type of human lymphomas that arise in extranodal sites. They affect virtually every organ in the human body; the majority occur in the stomach (11,12). The pathogenesis of MALT lymphomas is specifically associated with at least 3 independent chromosomal translocations. Two translocations, t(1;14)(q22;q32) and t(14;18)(q32;q21), result in the upregulation of BCL10 (an adaptor protein with a caspase recruitment domain, or CARD) and MALT1 (a paracaspase related to caspases), respectively, because of the juxtaposition of the corresponding genes with the Ig heavy chain locus (13-17). The third and most common translocation, t(11;18)(q21;q21), generates a fusion protein between cIAP2 and MALT1 (18)(19)(20). MALT lymphomas with these distinct translocations present no apparent difference in their clinical and pathological features, indicating that there may be a unifying molecular mechanism for the tumors. Indeed, both BCL10 and MALT1 play a critical role in antigen receptor-mediated lymphocyte proliferation and signaling to NF-κB, which controls the expression of genes critica...

Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to endoplasmic reticulum (ER) stress. However, the mechanisms of p53 destabilization by ER stress are not known. We demonstrate here that the E3 ubiquitin-ligase Hdm2 is essential for the nucleocytoplasmic transport and proteasome-dependent degradation of p53 in ER-stressed cells. We also demonstrate that p53 phosphorylation at S315 and S376 is required for its nuclear export and degradation by Hdm2 without interfering with the ubiquitylation process. Furthermore, we show that p53 destabilization in unstressed cells utilizes the cooperative action of Hdm2 and glycogen synthase kinase 3␤, a process that is enhanced in cells exposed to ER stress. In contrast to other stress pathways that stabilize p53, our findings further substantiate a negative role of ER stress in p53 activation with important implications for the function of the tumor suppressor in cells with a dysfunctional ER.The p53 gene encodes for a protein whose loss of function is associated with the majority of human cancers (17). The p53 protein primarily functions as a transcription factor and mediates several biological effects including growth arrest, senescence, and apoptosis in response to diverse forms of stress (50). In the absence of stress, p53 is a short-lived protein whose activity is maintained at low levels. Upon exposure to a variety of stresses, p53 becomes stabilized and accumulates in the nucleus, where it resumes its transcriptional function. The levels and localization of p53 are tightly controlled through several posttranslational mechanisms, including protein stability, phosphorylation, and subcellular localization (3,42,46). Although several factors influence p53 function, the Hdm2 (human Mdm2) protein plays an essential role in regulating p53 protein levels in unstressed cells. Hdm2 is a nucleoplasmic and nucleolar RING-finger protein that promotes p53 nuclear export and degradation through its specific E3 ubiquitin ligase activity (2, 40). Other studies have revealed various Hdm2-independent pathways that impinge on p53 turnover, including c-Jun N-terminal kinase (10), COP1 (9), and Pirh2 (32). However, little is known about how and in what cellular context these pathways act.The current model places both p53 and Hdm2 in an autoregulatory feedback loop where p53 induces the transcription of Hdm2 gene. The Hdm2 protein then binds to and ubiquitylates p53 in the nucleus, a process that allows the nuclear export and cytoplasmic proteasome-dependent degradation of the tumor suppressor (40, 56). The importance of this autoregulatory loop was demonstrated when the lethality of mdm2-null mice was rescued by the deletion of the p53 gene (22, 41). In this model, Hdm2 does not physically shuttle p53 out of the nucleus since the nuclear export sequence (NES) of Hdm2 is not necessary for p53 degradation, as opposed to its RING domain, which is important for p53 ubiquitylation and degradation (5,11,43). It has also been proposed that low Hdm2 l...

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2␣ kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2␣ kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2␣ kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2␣ kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2␣ kinases and p53 with implications in cell proliferation and tumorigenesis.The tumor suppressor p53 is a transcription factor mutated in ϳ50% of human cancers (1). In normal cells, p53 plays a pivotal role in controlling cell cycle, apoptosis, and DNA repair in response to various forms of genotoxic stress (2, 3). The regulation of p53 is complex and occurs mainly at the post-translational level (4). This is mediated by various post-translational modifications, such as phosphorylation and acetylation, which contribute to its stabilization and activation (5). The stability of p53 is regulated by its interaction with Hdm2 (human Mdm2), an E3-ubiquitin ligase that acts as an antagonist limiting p53 tumor suppressor function (6). Both p53 and Hdm2 are in an autoregulatory feedback loop in which p53 induces Hdm2 expression at the transcriptional level. The Hdm2 protein then binds to and ubiquitinates p53 in the nucleus, a process that allows the nuclear export and the cytoplasmic proteasome-dependent degradation of the tumor suppressor (6). In addition to Hdm2, other ubiquitin ligases, such as COP1 (7) and Pirh2 (8), have been shown to disrupt p53 stability. However, compared with Hdm2, little is currently known about how these ligases act on p53 (9).The majority of stress responses that activate p53 require its nuclear accumulation and function (10). This is mediated mainly through inactivation of the Hdm2-dependent degradation pathway as well as through interactions with nuclear proteins that promote post-translational modifications of p53 leading to its stabilization and activation (10). The current interest in p53 is underscored by the tremendous therapeutic benefits of its reactivation in cancer cells. Small molecules or peptides that restore the function of mutant p53 proteins have a great anti-tumor potential by enhancing the apoptotic sensitivity of tumor cells (11-13). Because p53 activity is influenced by many factors, targeting of proteins that regulate p53 function may...