ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

Abstract

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.

Betacoronavirus Assembly: Clues and Perspectives for Elucidating SARS-CoV-2 Particle Formation and Egress

“…When the mutation is engineered elsewhere in the tail, the location is preserved ( 36 ). This is coherent with MHV and SARS-CoV-2 E being located in the ERGIC and the Golgi apparatus, both in cis and in medial cisternae ( 35 , 37 ). Surprisingly, another recent study located SARS-CoV-2 E at the ER, when E was tagged at the C terminus and expressed alone ( 38 ).…”

“…In a transfected-cell model, E and N were also found to interact with each other through their C termini, and the deletion of a part of E’s C terminus impacted assembly and the E-N interaction. Even though E and N might interact in the wild-type virus, the purpose of this interaction is not clear yet ( 51 ), but it may be to enhance the incorporation of RNPs, confirming the crucial role of E in assembly ( 37 ). A mutation of SARS-CoV-1 with a deletion of E showed aberrant virions, described as vesicles containing dense and granular material, with totally different features from those of a wild-type virion ( 52 ).…”

“…First, as extremities are very important for interactions with other structural proteins involved in virus assembly, tagging the C terminus would in all probability have led to a mislocated protein ( 36 ). Thus, internal tagging seems more adequate to conserve the integrity and location of the E protein ( 37 ). In addition, E interacts not only with structural viral proteins but also with host proteins such as PALS1, a tight junction-associated protein, or syntenin, which impacts subcellular trafficking, even though the latter is debated ( 37 , 39 , 40 ).…”

“…Thus, internal tagging seems more adequate to conserve the integrity and location of the E protein ( 37 ). In addition, E interacts not only with structural viral proteins but also with host proteins such as PALS1, a tight junction-associated protein, or syntenin, which impacts subcellular trafficking, even though the latter is debated ( 37 , 39 , 40 ). Expressing it alone could thus compromise its correct location.…”

“…Among its numerous functions, the E protein of coronaviruses is able to self-oligomerize to create a pentameric ion channel, making this protein a viroporin ( 37 , 39 , 41 – 43 ). However, it is still debated whether this ion channel is specific for Ca 2+ , Na + , K + , or H + ( 41 , 42 ) and if ion flux would trigger the release of the viral particles.…”