Autophagy receptors as viral targets.

Activation of autophagy is part of the innate immune response during viral infections. Autophagy involves the sequestration of endogenous or foreign components from the cytosol within double-membraned vesicles and the delivery of their content to the lysosomes for degradation. As part of innate immune responses, this autophagic elimination of foreign components is selective and requires specialized cargo receptors that function as links between a tagged foreign component and the autophagic machinery. Pathogens have evolved ways to evade their autophagic degradation to promote their replication, and recent research has shown autophagic receptors to be an important and perhaps previously overlooked target of viral autophagy inhibition. This is a brief summary of the recent progress in knowledge of virus-host interaction in the context of autophagy receptors.

Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase family.

Autophagy is an important component of the innate immune response that restricts infection by different types of pathogens. Viruses have developed multiple strategies to avoid autophagy to complete their replication cycle and promote spreading to new hosts. Here, we report that the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autophagy by inhibiting the activity of the autophagy receptor SQSTM1/p62. We found that all the homologs bind to and deubiquitinate SQSTM1/p62 but with variable efficiency, which correlates with their capacity to prevent the colocalization of light chain 3 (LC3) with SQSTM1/p62 aggregates and promote the accumulation of a model autophagy substrate. The findings highlight important differences in the strategies by which herpesviruses interfere with selective autophagy.

The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy.

Macroautophagy/autophagy plays an important role in the control of viral infections and viruses have evolved multiple strategies to interfere with autophagy to avoid destruction and promote their own replication and spread. Here we report that the deubiquitinase encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein, BPLF1, regulates selective autophagy. Mass spectrometry analysis identified several vesicular traffic and autophagy related proteins as BPLF1 interactors and potential substrates, suggesting that the viral protein targets this cellular defense during productive infection. Direct binding of BPLF1 to the autophagy receptor SQSTM1/p62 (sequestosome 1) was confirmed by co-immunoprecipitation of transfected BPLF1 and by in vitro affinity isolation of bacterially expressed proteins. Expression of the catalytically active BPLF1 was associated with decreased SQSTM1/p62 ubiquitination and failure to recruit LC3 to SQSTM1/p62-positive aggregates. Selective autophagy was inhibited as illustrated by the accumulation of large protein aggregates in BPLF1-positive cells co-transfected with an aggregate-prone HTT (huntingtin)-Q109 construct, and by a slower autophagy-dependent clearance of protein aggregates upon transfection of BPLF1 in cells expressing a tetracycline-regulated HTT-Q103. The inhibition of aggregate clearance was restored by overexpression of a SQSTM1/p62[E409A,K420R] mutant that does not require ubiquitination of Lys420 for cargo loading. These findings highlight a previously unrecognized role of the viral deubiquitinase in the regulation of selective autophagy, which may promote infection and the production of infectious virus.Abbreviations: BPLF1, BamH1 fragment left open reading frame-1; EBV, Epstein-Barr virus; GFP, green fluorescent protein; HTT, huntingtin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PB1, Phox and Bem1 domain; PE, phosphatidylethanolamine; SQSTM1/p62, sequestosome 1; UBA, ubiquitin-associated domain.

Interaction With 14-3-3 Correlates With Inactivation of the RIG-I Signalosome by Herpesvirus Ubiquitin Deconjugases.

The hijacking of cellular function through expression of proteins that interfere with the activity of cellular enzymes and regulatory complexes is a common strategy used by viruses to remodel the cell environment in favor of their own replication and spread. Here we report that the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), target an early step of the IFN signaling cascade that involves the formation of a trimolecular complex with the ubiquitin ligase TRIM25 and the 14-3-3 molecular scaffold. Different from other homologs, the HSV-1 encoded enzyme fails to interact with 14-3-3, which correlates with failure to promote the autoubiquitination and sequestration of TRIM25 in cytoplasmic aggregates, and inability to block the activation and nuclear translocation of the IRF3 transcription factor. These findings highlight a key role for 14-3-3 molecular scaffolds in the regulation of innate immune response to herpesvirus infections and points to a possible target for the development of a new type of antivirals with applications in a broad spectrum of human diseases.

14-3-3 scaffold proteins mediate the inactivation of trim25 and inhibition of the type I interferon response by herpesvirus deconjugases.

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 (DUB) 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.

A novel mechanism for regulation of the type I IFN response by herpesvirus deconjugases.

Upon infection, viral nucleic acids are recognized by germline-encoded pattern-recognition receptors (PRRs), and cytosolic retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) that initiate signaling pathways resulting in the production of type I IFN and pro-inflammatory cytokines. Binding of RIG-I to viral nucleic acids triggers the formation of the RIG-I signalosome where RIG-I is ubiquitinated by the TRIM25 ligase and, with the help of 14-3-3 scaffolds, further translocated to mitochondrial anti-viral signalling proteins (MAVS). Subsequent ubiquitination-mediated events trigger transcriptional activation of the effectors of innate immunity. We have found a new mechanism by which herpesviruses interfere with this signalling pathway to favour the establishment of latency and promote virus replication. The cysteine protease encoded in the conserved N-terminal domain of the herpesvirus large tegument protein binds to 14-3-3 proteins and forms a tri-molecular complex with TRIM25, promoting the activation and autoubiquitination of the ligase. RIG-I is recruited to the complex but its ubiquitination is drastically reduced, which effectively inactivates downstream signalling and blocks the type I IFN response.

Herpesvirus deconjugases inhibit the IFN response by promoting TRIM25 autoubiquitination and functional inactivation of the RIG-I signalosome.

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.

Roles for RAB24 in autophagy and disease.

Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.

RAB24 facilitates clearance of autophagic compartments during basal conditions.

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.

Ultrastructural relationship of the phagophore with surrounding organelles.

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.

3D tomography reveals connections between the phagophore and endoplasmic reticulum.

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 known whether these two membranes are directly connected. To investigate the relationship of the phagophore membrane and the ER, we used electron microscopic tomography of serum and amino acid starved normal rat kidney cells. The cells were fixed in glutaraldehyde and reduced osmium tetroxide and embedded in Epon. Dual axis tilt image series were acquired from two successive 250-nm sections. To analyze the three-dimensional (3D) morphology of phagophores and the associated rough ER, 3D tomograms were used to model the ER and phagophore membranes. The tomographic reconstructions revealed connections between the phagophore/autophagosome membrane and the closely located ER cisternae, especially with the ER located inside the autophagosome. The connections were typically formed by narrow extensions from the phagophore/autophagosome to the ER. This finding has potential implications on the origin of autophagosome membranes, and on the mechanism of phagophore membrane extension. In addition, we observed lipid droplets in very close contact with the phagophores/autophagosomes.

Monitoring autophagy by electron microscopy in Mammalian cells.

Electron microscopy remains one of the most accurate methods for the detection of autophagy and quantification of autophagic accumulation. Compared to fluorescence microscopy, the resolution of transmission electron microscopy is superior. In this chapter we describe the fine structure of early and late autophagic compartments in mammalian cells. Instructions are given for the preparation of samples for conventional electron microscopy using three different protocols suitable for cultured cells and animal tissues. We also introduce tomography as a tool to study the three-dimensional morphology of autophagic organelles and show the morphology of a phagophore as an example. Finally, we describe a protocol for the quantification of autophagic compartments by electron microscopy and point counting.