Autophagosomes have been reported to form in the vicinity of the endoplasmic reticulum (ER). In many cases, the phagophore membrane is observed between two cisternae of rough ER, but it is not know...

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.

The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. The proteins are expressed during the productive virus cycle and are incorporated into infectious virus particles, being delivered to the target cells upon primary infection. Members of this viral enzyme family were shown to regulate different aspects of the virus life cycle and the innate anti-viral response. However, only few substrates have been identified and the mechanisms of these effects remain largely unknown. In order to gain insights on the substrates and signaling pathways targeted by the viral enzymes, we have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with the Epstein-Barr virus encoded homologue BPLF1. Several members of the 14-3-3-family of scaffold proteins were found amongst the top hits of the BPLF1 interactome, suggesting that, through this interaction, BPLF1 may regulate a variety of cellular signaling pathways. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a tri-molecular complex including, in addition to 14-3-3, the ubiquitin ligase TRIM25 that participates in the innate immune response via ubiquitination of cytosolic pattern recognition receptor, RIG-I. The involvement of BPLF1 in the regulation of this signaling pathway was confirmed by inhibition of the type-I IFN responses in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that the active viral enzyme promotes the dimerization and autoubiquitination of TRIM25. Upon triggering of the IFN response, RIG-I is recruited to the complex but ubiquitination is severely impaired, which functionally inactivates the RIG-I signalosome. The capacity to bind to and functionally inactivate the RIG-I signalosome is shared by the homologues encoded by other human herpesviruses. The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity that was shown to regulate different aspects of the virus life cycle including virus replication, the assembly of infectious virus particles and the host innate anti-viral response. However, only few substrates have been identified, and the mechanisms of these effects remain largely unknown. We have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with Epstein-Barr virus (EBV) encoded homologue BPLF1. Several members of the 14-3-3 family of scaffold proteins were found amongst the top hits of the BPLF1 interactome. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a tri-molecular complex that includes 14-3-3 and the ubiquitin ligase TRIM25. The 14-3-3:TRIM25 complex participates in the innate immune response via Lys-63-linked ubiquitination of cytosolic pattern recognition receptor RIG-I. The capacity of BPLF1 to regulate this signaling pathway was confirmed by inhibition of the type-I IFN in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that BPLF1 promotes the dimerization and autoubiquitination of TRIM25. RIG-I is recruited to the complex upon triggering of the IFN response but ubiquitination is severely impaired, which functionally inactivated the RIG-I signalosome. The capacity to promote TRIM25 autoubiquitination and inhibit the ubiquitination of RIG-I is shared by the homologues encoded by other human herpesviruses, providing a new insight on the mechanism by which these viruses halt the host innate immune antiviral response.

The 14-3-3 molecular scaffolds promote type I interferon (IFN) responses by stabilizing the interaction of RIG-I with the TRIM25 ligase. Viruses have evolved unique strategies to halt this cellular response to support their replication and spread. Here, we report that the ubiquitin deconjugase encoded in the N-terminus of the Epstein-Barr virus (EBV) large tegument protein BPLF1 harnesses 14-3-3 molecules to promote TRIM25 autoubiquitination and sequestration of the ligase into inactive protein aggregates. Catalytically inactive BPLF1 induced K48-linked autoubiquitination and degradation of TRIM25 while the ligase was mono- or di-ubiquitinated in the presence of the active viral enzyme and formed cytosolic aggregates decorated by the autophagy receptor p62/SQSTM1. Aggregate formation and the inhibition of IFN response were abolished by mutations of solvent exposed residues in helix-2 of BPLF1 that prevented binding to 14-3-3 while preserving both catalytic activity and binding to TRIM25. 14-3-3 interacted with the Coiled-Coil (CC) domain of TRIM25 in in vitro pulldown, while BPLF1 interacted with both the CC and B-box domains, suggesting that 14-3-3 positions BPLF1 at the ends of the CC dimer, close to known autoubiquitination sites. Our findings provide a molecular understanding of the mechanism by which a viral deubiquitinase inhibits the IFN response and emphasize the role of 14-3-3 proteins in modulating antiviral defenses. We have performed a molecular characterization of the mechanism by which the ubiquitin deconjugases encoded in the N-terminal domain of the herpesvirus large tegument proteins inhibit the type I IFN response. Starting from our previous finding that BPLF1, the Epstein-Barr virus (EBV) encoded member of the viral DUB family, induces the formation of a trimolecular complex including TRIM25 and 14-3-3 we now show that the complex promotes both the autoubiquitination and deubiquitination of TRIM25, which leads to sequestration of the ligase into protein aggregates decorated by the autophagy receptor p62/SQSTM1. Using mutants of a conserved putative protein-protein interaction motif in helix-2 of BPLF1 we show that binding to 14-3-3 is essential for this effect and for inhibition of the IFN response. Using 14-3-3 binding mutants in co-immunoprecipitation assays, we found that both BPLF1 and TRIM25 interact with the substrate binding groove of 14-3-3, suggesting that 14-3-3 serves as scaffold for the formation of the trimolecular complex. In vitro pulldown assays using TRIM25 subdomains and bacterially expressed BPLF1 and 14-3-3 suggest that 14-3-3 and BPLF1 interact with the tip of the coiled-coil domain, positioning the viral DUB close to a known autoubiquitination site in TRIM25. We used our findings to build a model of the trimeric complex based on available crystal structures and protein docking algorithms. The model provides a first characterization of the molecular interactions involved in the inhibition of TRIM25 by the viral DUB and has interesting implications for the regulation of TRIM25 activity.

RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.