OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses

Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

Serine/threonine phosphorylation of the T cell adaptor proteins SLP76 and GADS by HPK1 induces their release from signaling microclusters and subsequent termination of the T cell response

Analyse comparative des interactions entre l'ARN polymérase des virus influenza A et le système ubiquitine-protéasome de la cellule hôte

On estime que 10%-20% de la population mondiale est infectée chaque année par des virus influenza A (IAV) saisonniers, causant 250 à 500 000 morts. De plus ces virus présentent des risques de pandémie, et sont à ce titre un problème de santé publique majeur. Le cycle viral est dépendant de la capacité du virus à manipuler le protéome cellulaire. Par ailleurs, le système ubiquitine-protéasome (SUP) cellulaire est impliqué dans de nombreux processus de régulation cellulaires par l'induction de la dégradation de protéines, ou par la modification de leur activation ou de leur localisation sub-cellulaire. Le SUP est une cible privilégiée des virus lors de l'infection. Des études récentes indiquent qu'un réseau d'interactions entre les protéines virales des IAV et les protéines du SUP pourrait contribuer à la réplication virale et l’échappement du virus face au système immunitaire. Cependant ces interactions restent encore mal connues. Nous avons construit une banque contenant 570 facteurs du SUP, ce qui représente environ 60% des facteurs SUP humains connus. Puis nous avons mis au point une méthodologie permettant de réaliser un crible comparatif des interactions entre cette banque SUP et cinq PB2 provenant de souches de virus influenza A de virulence différentes chez l’homme : deux souches saisonnières circulant actuellement dans la population humaine (H1N1pdm09 et H3N2), deux souches hautement pathogènes chez l’homme (H7N9 et H1N1-1918) et une souche de laboratoire (H1N1-WSN). Cette première phase de cartographie a permis de sélectionner 42 facteurs du SUP interagissant avec au moins une des protéines PB2 étudiées. Par ailleurs, l’analyse des similarités de profils d’interaction PB2/UPS des souches étudiées a permis de mettre en évidence une corrélation avec le temps de circulation de chaque souche dans la population humaine. Nous avons ensuite caractérisé le rôle fonctionnel des partenaires de PB2 dans le cycle viral par des expériences de déplétion transitoire de l’expression des facteurs cellulaires par siARN, et validé 36 des 42 facteurs testés. La très grande quantité de facteurs identifiés impliqués dans le cycle viral démontre la qualité de la méthodologie développée pour l’identification de ces interacteurs. Parmi ces facteurs, nous avons étudié plus en détail le rôle de trois deubiquitinases (DUBs) dans l’infection. Nous avons montré que les DUBs sont impliquées dans les phases précoces et tardives du cycle viral. De plus, avec des collègues de Hong Kong nous avons mis en évidence que la DUB OTUB1 est impliquée dans la réponse cellulaire à l’infection produisant des cytokines, et probablement dans l’assemblage des nouveaux virions. Nous avons identifié que la DUB OTUD6A est également impliquée dans les phases tardives du cycle viral. A l’inverse PAN2 qui fait partie des complexes de poly-d’adénylation est impliqué dans les phases précoces. Nous poursuivons nos études afin d’élucider le rôle de ces DUBs dans l’infection par IAV.An estimated 10%-20% of the world's population is affected each year by seasonal epidemic influenza, causing about 250,000 to 500,000 fatal cases. The pandemic risk reinforces the trait of influenza A virus (IAV) infection as a public health issue. The virus life cycle critically relies on its ability to manipulate the host proteome. Besides, the ubiquitin-proteasome system (UPS) is involved in many regulatory processes in mammalian cells by inducing protein degradation, mediating protein activation or shaping their sub-cellular localisation. Therefore, UPS is a prime target hijacked by viruses. Recent evidence indicates that an intricate regulatory network involving viral proteins and the cellular UPS is likely to contribute to viral replication and immune evasion of influenza A viruses. However, usurpation of the host UPS by IAV is far from being comprehensively deciphered. To gain better understanding, we assessed the interplay between the human UPS and the PB2 subunit of the influenza A virus polymerase through a global proteomic profiling approach. For that purpose, an UPS-dedicated library of 590 human cDNAs, comprising 63% of the whole human UPS, was constituted and characterised. In an initial screen, UPS factors were challenged using a high-throughput split luciferase assay for interaction with the PB2 protein from 5 influenza A strains of different pathogenicity in human. A total of 80 UPS factors emerged as potential PB2 partners, of which 42 were validated as high-confidence PB2 partners for at least one of the strains. Further comparison of interaction profiles of the 5 PB2 with the UPS by hierarchical clustering revealed an interaction dendrogram fitting with the circulation time in the human population.Functional importance of interactors was tested by siRNA-mediated knock down experiments using luciferase tagged recombinant IAV viruses. Depletion of 36 out of the 42 tested UPS factors showed an effect on the infection with all or a subset of IAV strains, underlying the strong functional output of the developed methodology. Among these factors three deubiquitinases (DUBs) were further studied to decipher their involvement in IAV viral cycle. We have shown that they are involved in early and late stage of the infection and began to draw their function in viral cycle. We demonstrated with our colleagues in Hong-Kong that OTUB1 is involved in the host cytokine response and most probably in virus assembly. OTUD6A was also shown to be implicated in late stages of the infection but we still don't know its exact role. Contrariwise, the inactive DUB PAN2, which is part of poly-deadenylation complexes, is implicated in early phase of IAV infection, but surprisingly apparently not through viral mRNA regulation. More work is on-going to precise by which mechanisms these DUBs are implicated in IAV infection

Résolution de la structure de l’ARN polymérase dépendante de l’ARN trimérique des virus influenza : son impact sur notre compréhension des interactions de la polymérase avec les facteurs viraux et cellulaires

International audienceInfluenza viruses are segmented negative-sense RNA viruses whose RNA dependant RNA polymerase (RdRp) multiple activities are central for the viral life cycle. The RdRp is composed of three subunits, PB1, PB2 and PA. It binds to the extremities of each vRNA segments encapsidated with multiple copies of the Nucleoprotein (NP), altogether constituting the viral ribonucleoproteins (vRNPs). The RdRp performs both vRNA transcription and replication in the context of vRNP in the nuclei of infected cells. The temporal regulation of RdRp-associated activities is essential for the successful completion of the virus life cycle, but its understanding has been limited by the lack of structural information about the polymerase complex. The atomic-resolution of polymerase complexes from influenza virus type A, type B and type C came out in the past two years. We compile here the data provided by the near-concomitant resolution of several influenza polymerase crystal structures. We will highlight how structural information can contribute to our understanding of the interactions between the RdRp and viral or host factors

Interactions avec le système ubiquitine : un prérequis de l’adaptation des virus influenza à l’homme ?

International audienceLes virus influenza de types A, B, et C sont les agents étiologiques de la grippe. Parmi les virus de type A, les virus de sous-types H1N1 pdm09 1 et H3N2 sont responsables d’épidémies de grippe saisonnières. Ils sont la cause de 260 000 à 650 000 décès par an dans le monde (source de l’Organisation mondiale de la santé, janvier 2018). D’autres virus influenza A peuvent représenter un risque d’émergence virale zoonotique, comme l’a démontrée la pandémie de 2009 provoquée par un virus H1N1 d’origine porcine [...

Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases

International audienceThe multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets

Influenza A virus co-opts ERI1 exonuclease bound to histone mRNA to promote viral transcription

International audienceCellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus

Acceptable symbiont cell size differs among cnidarian species and may limit symbiont diversity

Reef-building corals form symbiotic relationships with dinoflagellates of the genus Symbiodinium. Symbiodinium are genetically and physiologically diverse, and corals may be able to adapt to different environments by altering their dominant Symbiodinium phylotype. Notably, each coral species associates only with specific Symbiodinium phylotypes, and consequently the diversity of symbionts available to the host is limited by the species specificity. Currently, it is widely presumed that species specificity is determined by the combination of cell-surface molecules on the host and symbiont. Here we show experimental evidence supporting a new model to explain at least part of the specificity in coral-Symbiodinium symbiosis. Using the laboratory model Aiptasia-Symbiodinium system, we found that symbiont infectivity is related to cell size; larger Symbiodinium phylotypes are less likely to establish a symbiotic relationship with the host Aiptasia. This size dependency is further supported by experiments where symbionts were replaced by artificial fluorescent microspheres. Finally, experiments using two different coral species demonstrate that our size-dependent-infection model can be expanded to coral-Symbiodinium symbiosis, with the acceptability of large-sized Symbiodinium phylotypes differing between two coral species. Thus the selectivity of the host for symbiont cell size can affect the diversity of symbionts in corals.This work was supported by an Australian Research Council Discovery Project grant (no. DP110102364 to ST) and a Grants-in-Aid for Scientific Research grant from the ministry of Education, Culture, Sports, Science and Technology of Japan (no. 15K14611 to ST)

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment

Summary: The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design. : The mechanisms that regulate intracellular viral envelopment are unknown. Kumar et al. report that MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural 2 protein and subsequently ESCRT recruitment and HCV envelopment. MARCH8 is required for infection with other Flaviviridae family members, thereby representing a potential host target for antiviral strategies. Keywords: ubiquitination, hepatitis C virus, HCV, MARCH8, virus-host interactions, proteomics, assembly, envelopment, intracellular membrane trafficking, ESCR

OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses