The β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae , responsible for endoplasmic reticulum–to–Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein–1 (ARFRP1/AP-1)–dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

The beta-coronavirus SARS-CoV-2 is the causative agent of the current global COVID-19 pandemic. Coronaviruses are enveloped RNA viruses. Assembly and budding of coronavirus particles occur at the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC), with the structural proteins Nucleocapsid, Spike, Membrane and Envelope facilitating budding and release of virions into the secretory pathway lumen. This allows viral release which can occur through delivery of virus particles to deacidified lysosomes and subsequent lysosomal secretion. Coronaviral Envelope proteins are necessary for coronavirus assembly, play important roles in replication and can form oligomeric cation channels. Whilst synthesised in the ER, the mechanism by which Envelope achieves its steady state localisation to the ERGIC remains unclear. Here, we used fluorescent reporters to illuminate the Envelope protein from SARS-CoV-2. We discovered that internal tagging of this protein is necessary to preserve the functionality of a C-terminal ER-export motif and to allow localisation of Envelope to the ERGIC. Using this non-disruptive form of tagging, we used proximity biotinylation to define the vicinal proteome of wild type and ER-restricted versions of Envelope. We show that both Envelope and the presence of its ER-export motif contribute to the packaging of nucleocapsid into virus like particles. Finally, using our labelled versions of Envelope, we discovered that a minor pool of this protein is delivered to lysosomes. We show that lysosomal Envelope is oligomeric and can contribute to pH neutralisation in these organelles.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER–endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

Movie 1–GFP-SNX1 dynamics in mock and spastin siRNA depleted MRC5 cells. MRC5 cells stably expressing GFP-SNX1 were treated with either mock or spastin siRNA and imaged using live-cell spinning disk microscopy to visualise endosomal tubule fission dynamics. Imaging was performed at 400ms per frame for 3 minutes. The large left image shows an overview of a typical mock cell, and the right image shows an overview of a typical spastin siRNA depleted cell. The insets in each show a zoom of the region highlighted by the white box in each overview image respectively. Note the difference in time for tubule fission in the mock and spastin siRNA treated cell. Scale bars for both overview images represent 10µm. 1 minute of GFP-SNX1 dynamics is shown in total. The video is displayed at 15 frames per second.

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

The Role of Spastin in Endosomal Tubule Fission: Hereditary Spastic Paraplegias (HSP) are a group of diseases that are characterised by a genetically inherited axonopathy of cortical neurones. Of the fifty-nine genes associated with HSP, mutations in SPG4, encoding the protein Spastin, are the most common cause of HSP, accounting for 40% of familial HSP cases. Spastin exists as two main isoforms; a short M87 isoform and a M1 isoform that possess an additional hydrophobic region allowing it to locate to the endoplasmic reticulum (ER). Both M1 and M87 Spastin possess a Microtubule Binding Domain (MTBD) and an AAA-ATPase domain allowing the severing of microtubules, and a MIT domain that allows their recruitment to endosomes by interaction with ESCRT-III proteins IST1 and CHMP1B. Upon depletion of Spastin or the mutation of these domains, endosomal defects are observed including the elongation of recycling tubules that typically sort cargo away from lysosomal degradation. Intriguingly, this phenotype could be rescued by siRNA-resistant M1 Spastin that localises both at endosomes and at the ER. However the discovery that ER-endosome contacts are required for tubule fission led to the hypothesis that Spastin may be present at these contacts to facilitate endosome fission. Here, I show by fixed/live-cell microscopy that this is true, with Spastin or IST1 depletion impairing tubule fission, despite ER-endosome contacts still able to form. This suggests that Spastin’s endosomal recruitment by IST1 is required for ER-mediated endosomal tubule fission.