The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C PRO ) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses. INTRODUCTIONPicornaviruses include important human pathogens such as rhinovirus, poliovirus and hepatitis A virus, as well as significant insect, plant and agricultural pathogens, such as foot-and-mouth disease virus (Whitton et al., 2005). Encephalomyocarditis virus (EMCV) is the prototype of the cardiovirus subgroup of picornaviruses. Its genome consists of a single-strand of messenger-sense RNA. Viral proteins are expressed from a large open reading frame encoding a giant precursor polyprotein (approx. 255 kDa), which is processed through a series of proteolytic cleavages to produce both capsid and non-structural proteins (Palmenberg, 1990). The majority of the processing reactions are carried out by the 3C protease (3C PRO ), which acts both inter-and intramolecularly to produce polyprotein precursors as well as the mature 3C PRO polypeptide (Palmenberg et al., 1979). Cleavage of the polyprotein normally occurs between glutamine-glycine or glutamine-serine amino acid pairs, which are usually flanked by proline residues (Palmenberg et al., 1984). Besides its commitment to the processing of the viral polyprotein, picornaviral 3C PRO can also facilitate virus replication by altering fundamental cellular processes such as transcription and translation of host cell mRNA (Barral et al., 2007;Ehrenfeld, 1982;Kuyumcu-Martinez et al., 2004; Neznanov et al., 2005;Shen et al., 1996;Yalamanchili et al., 1996Yalamanchili et al., , 1997. Because the regulated proteolysis mediated by 3C PRO is a critical feature in both the processing of picornaviral polyprotein and the inhibition of cellular defences, 3C PRO constitutes an important target for host antiviral innate pathways. Consistent with this, the degradation of EMCV 3C PRO by the ubiquitin/26S proteasome system has been reported (Losick et al., 2003;Schlax et al., 2007). Although the precise ro...

RNA helicase-like receptors MDA-5 but not RIG-I has been shown to be essential for triggering innate immune responses against picornaviruses. However, virus-host co-evolution has selected for viruses capable of replicating despite host cells antiviral defences. In this report, we demonstrate that RIG-I is degraded during encephalomyocarditis virus (EMCV) infection. This effect is mediated by both the viral-encoded 3C protease and caspase proteinase. In addition, we show that RIG-I overexpression confers IFN-beta promoter activation during EMCV infection, in MDA-5 knockout (MDA-5(-/-)) mouse embryo fibroblasts. This induction is followed by a strong inhibition reflecting the ability of EMCV to disrupt RIG-I signalling. Taken together, our data strongly suggest that during evolution RIG-I has been involved for triggering innate immune response to picornavirus infections.

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The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3␣, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.

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