Bioengineered small extracellular vesicles deliver multiple SARS-CoV-2 antigenic fragments and drive a broad immunological response.

The COVID-19 pandemic highlighted the clear risk that zoonotic viruses pose to global health and economies. The scientific community responded by developing several efficacious vaccines which were expedited by the global need for vaccines. The emergence of SARS-CoV-2 breakthrough infections highlights the need for additional vaccine modalities to provide stronger, long-lived protective immunity. Here we report the design and preclinical testing of small extracellular vesicles (sEVs) as a multi-subunit vaccine. Cell lines were engineered to produce sEVs containing either the SARS-CoV-2 Spike receptor-binding domain, or an antigenic region from SARS-CoV-2 Nucleocapsid, or both in combination, and we tested their ability to evoke immune responses in vitro and in vivo. B cells incubated with bioengineered sEVs were potent activators of antigen-specific T cell clones. Mice immunised with sEVs containing both sRBD and Nucleocapsid antigens generated sRBD-specific IgGs, nucleocapsid-specific IgGs, which neutralised SARS-CoV-2 infection. sEV-based vaccines allow multiple antigens to be delivered simultaneously resulting in potent, broad immunity, and provide a quick, cheap, and reliable method to test vaccine candidates.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease.

Alveolar type 2 (AT2) cells maintain lung health by acting as stem cells and producing pulmonary surfactant1-3. AT2 dysfunction underlies many lung diseases including interstitial lung disease (ILD), in which some inherited forms result from mislocalisation of surfactant protein C (SFTPC) variants4,5. Disease modelling and dissection of mechanisms remains challenging due to complexities in deriving and maintaining AT2 cells ex vivo. Here, we describe the development of expandable adult AT2-like organoids derived from human fetal lung which are phenotypically stable, can differentiate into AT1-like cells and are genetically manipulable. We use these organoids to test key effectors of SFTPC maturation identified in a forward genetic screen including the E3 ligase ITCH, demonstrating that their depletion phenocopies the pathological SFTPC redistribution seen for the SFTPC-I73T variant. In summary, we demonstrate the development of a novel alveolar organoid model and use it to identify effectors of SFTPC maturation necessary for AT2 health.

Establishing SARS-CoV-2 membrane protein-specific antibodies as a valuable serological target via high-content microscopy.

The prevalence and strength of serological responses mounted toward SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high-content microscopy to assess antibody responses against full-length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) of unvaccinated individuals with RT-PCR confirmed SARS-CoV-2 infections and 74% (31/42) of individuals infected after being vaccinated developed detectable IgG against the structural protein M, which is higher than previous estimates. Compared with N antibodies, M IgG displayed a shallower time-dependent decay and greater specificity. Sensitivity for SARS-CoV-2 seroprevalence was enhanced when N and M IgG detection was combined. These findings indicate that screening for M seroconversion may be a good approach for detecting additional vaccine breakthrough infections and highlight the potential to use HCM as a rapidly deployable method to identify the most immunogenic targets of newly emergent pathogens.

Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

Ultrastructural insight into SARS-CoV-2 entry and budding in human airway epithelium.

Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Here, we examined human airway epithelium infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistently, extracellular virions were seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion where tomography showed that the viral membrane was continuous with the apical plasma membrane and the nucleocapsids diluted, compared with unfused virus, demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Tomography showed concentration of nucleocapsids round the periphery of profiles strongly suggestive of viral budding into these compartments and this may explain how virions gain their S glycoprotein containing envelope.

Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of 'hourglass' profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Exploiting Connections for Viral Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2-infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

A dysfunctional endolysosomal pathway common to two sub-types of demyelinating Charcot-Marie-Tooth disease.

Autosomal dominant mutations in LITAF are responsible for the rare demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1C (CMT1C). The LITAF protein is expressed in many human cell types and we have investigated the consequences of two different LITAF mutations in primary fibroblasts from CMT1C patients using confocal and electron microscopy. We observed the appearance of vacuolation/enlargement of late endocytic compartments (late endosomes and lysosomes). This vacuolation was also observed after knocking out LITAF from either control human fibroblasts or from the CMT1C patient-derived cells, consistent with it being the result of loss-of-function mutations in the CMT1C fibroblasts. The vacuolation was similar to that previously observed in fibroblasts from CMT4J patients, which have autosomal recessive mutations in FIG4. The FIG4 protein is a component of a phosphoinositide kinase complex that synthesises phosphatidylinositol 3,5-bisphosphate on the limiting membrane of late endosomes. Phosphatidylinositol 3,5-bisphosphate activates the release of lysosomal Ca2+ through the cation channel TRPML1, which is required to maintain the homeostasis of endosomes and lysosomes in mammalian cells. We observed that a small molecule activator of TRPML1, ML-SA1, was able to rescue the vacuolation phenotype of LITAF knockout, FIG4 knockout and CMT1C patient fibroblasts. Our data describe the first cellular phenotype common to two different subtypes of demyelinating CMT and are consistent with LITAF and FIG4 functioning on a common endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes. Although our experiments were on human fibroblasts, they have implications for our understanding of the molecular pathogenesis and approaches to therapy in two subtypes of demyelinating Charcot-Marie-Tooth disease.

OPTN recruitment to a Golgi-proximal compartment regulates immune signalling and cytokine secretion.

Optineurin (OPTN) is a multifunctional protein involved in autophagy and secretion, as well as nuclear factor κB (NF-κB) and IRF3 signalling, and OPTN mutations are associated with several human diseases. Here, we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-κB and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations in OPTN linked to primary open-angle glaucoma (POAG) cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. By using proximity labelling proteomics, we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-κB and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment, leading to altered pro-inflammatory cytokine secretion.

Spastin MIT Domain Disease-Associated Mutations Disrupt Lysosomal Function.

The hereditary spastic paraplegias (HSPs) are genetic motor neuron diseases characterized by progressive degeneration of corticospinal tract axons. Mutations in SPAST, encoding the microtubule-severing ATPase spastin, are the most common causes of HSP. The broad SPAST mutational spectrum indicates a haploinsufficiency pathogenic mechanism in most cases. Most missense mutations cluster in the ATPase domain, where they disrupt the protein's ability to sever microtubules. However, several putative missense mutations in the protein's microtubule interacting and trafficking (MIT) domain have also been described, but the pathogenicity of these mutations has not been verified with functional studies. Spastin promotes endosomal tubule fission, and defects in this lead to lysosomal enzyme mistrafficking and downstream lysosomal abnormalities. We investigated the function of three disease-associated spastin MIT mutants and found that none was able to promote normal endosomal tubule fission, lysosomal enzyme receptor trafficking, or lysosomal morphology. One of the mutations affected recruitment of spastin to endosomes, a property that requires the canonical function of the MIT domain in binding endosomal sorting complex required for transport (ESCRT)-III proteins. However, the other mutants did not affect spastin's endosomal recruitment, raising the possibility of pathologically important non-canonical roles for the MIT domain. In conclusion, we demonstrate that spastin MIT mutants cause functional abnormalities related to the pathogenesis of HSP. These mutations do not directly affect spastin's microtubule-severing capacity, and so we identify a new molecular pathological mechanism by which spastin mutations may cause disease.

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization.

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling.

The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell-cell adhesion.

Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.

Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue by TMEM106B knockdown.

Mutations in the endosome-associated protein CHMP2B cause frontotemporal dementia and lead to lysosomal storage pathology in neurons. We here report that physiological levels of mutant CHMP2B causes reduced numbers and significantly impaired trafficking of endolysosomes within neuronal dendrites, accompanied by increased dendritic branching. Mechanistically, this is due to the stable incorporation of mutant CHMP2B onto neuronal endolysosomes, which we show renders them unable to traffic within dendrites. This defect is due to the inability of mutant CHMP2B to recruit the ATPase VPS4, which is required for release of CHMP2B from endosomal membranes. Strikingly, both impaired trafficking and the increased dendritic branching were rescued by treatment with antisense oligonucleotides targeting the well validated frontotemporal dementia risk factor TMEM106B, which encodes an endolysosomal protein. This indicates that reducing TMEM106B levels can restore endosomal health in frontotemporal dementia. As TMEM106B is a risk factor for frontotemporal dementia caused by both C9orf72 and progranulin mutations, and antisense oligonucleotides are showing promise as therapeutics for neurodegenerative diseases, our data suggests a potential new strategy for treating the wide range of frontotemporal dementias associated with endolysosomal dysfunction.

AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A.

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

Fas Ligand localizes to intraluminal vesicles within NK cell cytolytic granules and is enriched at the immune synapse.

INTRODUCTION: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear. METHODS: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells. RESULTS: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell. CONCLUSIONS: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells.