key: cord-0856343-qys4jl5j
authors: Ghosh, Sourish; Dellibovi-Ragheb, Teegan A.; Kerviel, Adeline; Pak, Eowyn; Qiu, Qi; Fisher, Matthew; Takvorian, Peter M.; Bleck, Christopher; Hsu, Victor; Fehr, Anthony R.; Perlman, Stanley; Achar, Souraj R.; Straus, Marco R.; Whittaker, Gary R.; de Haan, Cornelis A.M.; Kehrl, John; Altan-Bonnet, Gregoire; Altan-Bonnet, Nihal
title: β-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway
date: 2020-10-27
journal: Cell
DOI: 10.1016/j.cell.2020.10.039
sha: dad6ddfd6127d2c4db3ce101d609ee8f9514d69e
doc_id: 856343
cord_uid: qys4jl5j

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress, rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-Coronavirus results in lysosome deacidification, inactivation of lysosomal degradation enzymes and disruption of antigen presentation pathways. The β−coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

β-Coronaviruses are positive-strand enveloped RNA viruses that comprise one of the four genera of the Coronaviridae family of viruses. β-Coronaviruses infect humans and other mammals with infections resulting in a range of diseases with considerable morbidity and mortality. In late 2019, one member, the SARS-CoV2, originating in bats, spread to humans and caused a world-wide ongoing pandemic (Lu et al., 2020) .

The ability of these viruses to infect many different cell types including those of the pulmonary, cardiovascular, hepatic, gastrointestinal, central nervous and immune systems results in complex multi-organ disease manifestations that can vary from individual to individual (Puelles et al., 2020; Ziegler et al., 2020) . Especially with regards to the immune system, these viruses appear to deregulate the traditional innate and adaptive immune responses to pathogens (Vardhana and Wolchok, 2020) . Currently there is no cure, the antiviral treatment options are few (Williamson et al., 2020) and whether lasting immune responses can be generated to infection by natural means or through vaccine administration remains open to question (Long et al., 2020) .

One of the major reasons for the lack of antiviral therapies is the paucity of knowledge regarding the β-Coronavirus-host cell interface. Once the viral envelope fuses with the plasma membrane and/or endosome membranes, and the viral RNA genome is released into the cytosol, it translates into nonstructural and structural proteins. The nonstructural proteins assemble on ER-derived membranes and replicate the viral RNA (Snijder et al., 2020; Snijder et al., 2006) . While a large amount of molecular detail is known regarding coronavirus entry and replication, very little is known regarding how the newly assembled coronaviruses egress from cells including what cellular pathways they exploit and whether they induce cell lysis (Fung and Liu, 2019; Machamer, 2013; Tooze et al., 1987) .

The egress pathway for all β-Coronaviruses starts with newly synthesized viral genomic RNA, coated with viral N proteins, budding into the lumen of the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) (Cohen et al., 2011; McBride et al., 2007; Perrier et al., 2019; Tooze et al., 1987; Tooze et al., 1988) .

This results in viral particles enveloped with host membranes containing viral M, E and S transmembrane structural proteins (de Haan et al., 1998; Ruch and Machamer, 2012;  J o u r n a l P r e -p r o o f Siu et al., 2008) . Once in the ER/ERGIC, virus particles traffic to the Golgi apparatus and Trans-Golgi Network (TGN) for glycosylation and other post-translational modifications (Fung and Liu, 2018; McBride et al., 2007; Oostra et al., 2006; Tooze et al., 1987) . But after the Golgi/TGN, it has been assumed that coronaviruses use vesicles of the biosynthetic secretory pathway to track to the plasma membrane and egress (Machamer, 2013; Tooze et al., 1987) , similar to other enveloped RNA viruses such as hepatitis C, Dengue, and West Nile (Ravindran et al., 2016; Robinson et al., 2018) .

Here we investigated the egress pathway of β-Coronaviruses and found that rather than the biosynthetic secretory pathway, these viruses use a lysosomal, Arl8bdependent exocytic pathway for release into the extracellular environment. We show that GRP78/BIP an ER-chaperone, that facilitates coronavirus infectivity (Chu et al., 2018; Ha et al., 2020) , is co-released with β-coronaviruses through this pathway. As a consequence of viral exploitation of lysosomal exocytosis, we demonstrate that late endosome/lysosomes are deacidified and lysosomal proteases are inactive.

Significantly we show that this perturbation of lysosome physiology has important functional consequences on the host cell, including disruption of antigen presentation pathways.

We began investigating the mechanism of β-Coronavirus egress using mouse hepatitis virus (MHV), as it is the prototype of the family that can be studied under BSL-2 conditions, with intranasal MHV infections in mice inducing pathogenesis similar to SARS, including acute pneumonia, lung injury as well as hepatic and neurological disease (Channappanavar et al., 2016; De Albuquerque et al., 2006; Khanolkar et al., 2007) .

First, we investigated the kinetics of replication and egress in this model system.

HeLa-ATCC cells stably expressing murine CEACAM1 (HeLa-mCC1a) were infected with MHV-A59 strain virus. Cell lysates and extracellular medium were collected at J o u r n a l P r e -p r o o f different intervals and processed for quantitative PCR (qPCR) determination of viral genomic RNA. The results were plotted as fold change over uninfected cell lysates and extracellular medium ( Figure 1A) . We found that replication rate was highest until 8hr post-infection (pi) and then plateaued. Virus egress was highest from 8hr to 12hr pi before leveling off at 14hr pi ( Figure 1A) . The extracellular genomic RNA (i.e. egressed virus), released between 8hr pi and 14hr pi, was infectious and could be titered ( Figure   S1A ). Notably, viral egress took place in the absence of any cell lysis, as there was no significant change in the permeability of the plasma membrane to the membraneimpermeant cell viability dyes trypan blue and propidium iodide at the time when the virus accumulated extracellularly ( Figure 1B ; Figures S1B and S1C).

β-Coronaviruses are widely thought to use the biosynthetic secretory pathway for egress. Given this we next interrogated the status of the secretory pathway in infected cells and whether this pathway was utilized for MHV egress. Cells were transfected with Gaussia luciferase, a reporter for the biosynthetic secretory pathway (Tannous, 2009) and either infected with MHV or left uninfected. We confirmed that Gaussia luciferase transfection of cells did not block their subsequent infection by MHV ( Figure S1D ).

Extracellular luciferase levels were measured by luminescence and released viral genomes quantified by qPCR ( Figure 1C ). We found that the kinetics of Gaussia luciferase secretion was not significantly altered throughout the MHV egress period, consistent with previous reports (Machamer, 2013; Tooze et al., 1987) .

Given that the secretory pathway remained operational, we next asked if βcoronaviruses utilized it for egress. We treated Gaussia luciferase transfected cells with Brefeldin A (BFA), a small molecule that rapidly shuts down all anterograde biosynthetic secretory traffic from the ER/ERGIC out to the plasma membrane and leads to resorption of the Golgi apparatus back into the ER (Lippincott-Schwartz et al., 1989; Miller et al., 1992) . Both the uninfected/Gaussia luciferase-transfected and the MHVinfected/Gaussia luciferase-transfected cells were treated with/without BFA at 6hr pi, 8hr pi and 10hr pi, and extracellular media were collected at 14hr pi. This resulted in 8hrs, 6hrs and 4hrs of total BFA treatment time respectively. From these collected media, Gaussia luciferase and MHV extracellular genomic RNA levels were simultaneously quantified and plotted ( Figure 1D) . Remarkably, the presence of BFA, J o u r n a l P r e -p r o o f from 6hr pi onwards, did not impact MHV egress: neither quantification of extracellular viral genomic RNA ( Figure 1D ) nor viral titers ( Figure S1A ) of BFA-treated cells showed any significant change relative to untreated infected cells. Note that the secretory pathway was sensitive to BFA in MHV-infected cells as the Golgi apparatus was completely disrupted ( Figure 1E ) and Gaussia luciferase secretion was blocked ( Figure 1D) . Furthermore, the viral egress observed was not due to cell lysis, irrespective of BFA treatment ( Figure S1E) . Thus, MHV could egress even when trafficking through the biosynthetic secretory pathway was blocked. β β β β-Coronaviruses are enriched in late endosomes/lysosomes during egress.

We then investigated the spatiotemporal distribution of MHV during egress to identify which cellular trafficking pathway it exploited. The M protein is the most abundant protein in the envelope of β-Coronaviruses and drives virus assembly, membrane curvature and budding into the ER/ERGIC by oligomerizing with itself and with viral RNA, N, E and S proteins (de Haan and Rottier, 2005; Ruch and Machamer, 2012) . Immunolabeling cells at peak egress (12hr pi) with the J1.3 monoclonal anti-M antibody (Narayanan et al., 2000; Stohlman et al., 1982) , and subsequent immunoelectron microscopy revealed antibody labeling to be concentrated 3-fold more on the envelopes of viral particles compared to membranes elsewhere (ER, ERGIC, Golgi etc.) ( Figure 1F ; Figure S2A ). The J1.3 antibody may recognize free M proteins, but our quantitative analysis of electron micrographs indicated that the antibody mostly detected M within the context of assembled particles. In addition, consistent with recognition of assembled virus particles, MHV(M J1.3 ) antibody labeling colocalized with E and S envelope proteins throughout infection ( Figure 1G; Figures S2B and S2C) .

We infected cells with MHV, washed off virus and fixed the cells at different times post-inoculation. We co-stained the fixed cells with anti-MHV(M J1.3 ), anti-E and other antibodies against organelle resident host proteins. At 6hr pi, consistent with previous reports showing newly synthesized viruses trafficking to the Golgi and TGN at the early stages of infection (Machamer, 2013; Tooze et al., 1987) , MHV(M J1.3 ) labeling was perinuclear ( Figure 1G , 6hr pi) and colocalized with TGN46, Golgin 97 and mannosidase II by immunofluorescence (Figures S2D-S2F ).

J o u r n a l P r e -p r o o f However, at 12hr pi the bulk of the MHV(M J1.3 ) labeling was no longer perinuclear and did not colocalize with these Golgi/TGN markers (Figures S2D-S2F) . Instead it was concentrated in puncta dispersed across the cytoplasm (Figure 1G, 12hr pi) . The late endosome/lysosomal transmembrane protein LAMP1 ( Figure 1H, 12hrpi) and lumenal enzyme cathepsin D ( Figure S2G ) were colocalized with many of the MHV(M J1.3 )labeled puncta. We quantified the colocalization of fluorescence signals in the MHV(M J1.3 ) and LAMP1 channels (Manders et al., 1993) which revealed ~5-fold increase in LAMP1 + / MHV + organelles during the egress period ( Figure 1I ). MHV(M J1.3 ) puncta not with LAMP1 was localized to the ER ( Figure S2H ). In SARS-CoV2 infected Vero E6 cells, M labeling could also be detected within LAMP1 + organelles ( Figure 1J ).

We then fractionated MHV-infected cells at 12hr pi using Nycodenz gradients (Graham et al., 1990) and quantified MHV genomic content within each fraction by qPCR. This revealed viral genomic RNA to be enriched in the LAMP1 + fractions (2 and 3) which correspond to late endosome/lysosomes; and in ERGIC53 + fractions (4 and 5) corresponding to Golgi/ER/ERGIC membranes, where MHV replication takes place ( Figures 1K and 1L; Figures S3A and S3B) . In contrast, a similar fractionation done on poliovirus-infected HeLa cells revealed poliovirus to be enriched in ERGIC53 + /LC3 + fractions but not in the LAMP1 + fractions ( Figure S3C) , a result consistent with poliovirus using ER/Golgi/ERGIC-derived autophagosomes for egress that are blocked from fusing with lysosomes (Chen et al., 2015) .

The LAMP1+ fractions are a mixture of lysosomes and late endosomes which overlap in many membrane and lumenal proteins (Huotari and Helenius, 2011) . One exception is the cation-independent mannose 6-phosphate receptor (CI-MPR) which mainly cycles between late endosomes and Golgi/TGN (Brown et al., 1986) . Indeed, in poliovirus infected cells CI-MPR was detectable throughout fractions 2-5 reflecting this broad distribution ( Figure S3C ). However, in MHV-infected cells it was only detectable in fraction 5 ( Figure S3B ). Consistent with this, imaging of MHV-infected cells coimmunostained with anti-CI-MPR and anti-LAMP1 antibodies revealed a 3-fold decrease in colocalization between CI-MPR and LAMP1 relative to uninfected cells ( Figures S3D and S3E ). This suggests that in infected cells, MHV is largely associated with lysosomes and/or atypical late endosomes, the latter being deficient of CI-MPR.

Significantly, MHV association with the lysosome fractions was not a result of endocytic reuptake of egressed virus. When infected cells were treated with Dyngo-4A, a potent inhibitor of endocytosis (Park et al., 2013) (Figure S3F ) during egress, the quantity of MHV genomic RNA associated with the LAMP1 + fractions remained significant ( Figure 1L ).

Using transmission electron microscopy (TEM) and immuno-electron microscopy (Immuno-EM) methodologies, which have resolution in the few nanometers, we further interrogated the association between β-coronaviruses and lysosomes in MHV and SARS-CoV2 infected cells (Figure 2 ). By TEM, lysosomes can be easily recognized by specific hallmarks such as intralumenal electron dense material and intralumenal membrane swirls (Fawcett, 1966) . TEM of MHV or SARS-CoV2 infected cells during egress stage revealed many such organelles filled with intact viruses of typical 70-90nm particle size. These organelles were likely lysosomes as they displayed all the physiological hallmarks (Figures 2A-2C) . Immuno-EM against native LAMP1 revealed this protein to be on the membrane of these virus-filled organelles ( Figure 2B ) thus further confirming the colocalization between LAMP1 and MHV(M J1.3 ) observed by confocal imaging (Figures 1H-1J ) and the biochemical association between LAMP1 + fractions and MHV genomes (Figures 1K and 1L ).

β β β β-Coronaviruses and ER chaperones are co-released during infection.

We next investigated whether any host proteins co-trafficked with MHV during egress. The KDEL-Receptor, an ER/Golgi cycling-transmembrane protein that is critical for retrieving escaped ER resident proteins from the Golgi apparatus (Munro and Pelham, 1987) and its cargo, the KDEL sequence containing ER lumenal chaperones GRP78/BIP (Figures 3A-3C) and calreticulin (not shown), were found to colocalize with LAMP1 and MHV during peak virus egress. Soluble ER chaperones generally do not escape the ER/Golgi and become secreted (Munro and Pelham, 1987) . However, in MHV-infected cells, these chaperones were co-released with the virus and this was not due to cell lysis as actin was undetectable in the extracellular medium ( Figure 3D ).

Remarkably chaperone release, much like MHV, was not inhibited by BFA when the drug was added during the peak virus egress period (8-14hr pi) ( Figure 3E ). Note that since the BFA molecular target GBF1 is required for coronavirus replication (Verheije et al., 2008) , addition of BFA during peak replication (<8hr pi) inhibited GRP78/BIP release.

Together these data demonstrate that during egress, MHV and ER chaperones are co-trafficked to lysosomal organelles and released from cells through a route bypassing the BFA-sensitive biosynthetic secretory pathway.

β β β β-Coronaviruses and GRP78/BIP use an Arl8b-dependent lysosomal exocytic pathway for egress.

Lysosome exocytosis is a known BFA-insensitive pathway whereby lysosomes traffic to the cell periphery and fuse with the plasma membrane to release their lumenal contents (Laulagnier et al., 2011) . We conjectured that MHV may be exploiting this route for egress. In support, we found that plasma membrane LAMP1 levels were ~2.5 fold higher in infected cells ( Figures 4A and 4B) , implying significant fusion of lysosomes with the plasma membrane.

Cathepsin D (~30kD) is a proteolytic enzyme that is synthesized as pro-cathepsin D (~50kD) and becomes cleaved into the mature form by lysosomal proteases (Samarel et al., 1989) . Collection of extracellular media from MHV-infected cells at peak egress revealed ~2-fold more cathepsin D and ~3-fold more pro-cathepsin D secreted relative to uninfected cells (Figures 4C-4E ).

Cell surface total internal reflection fluorescence (TIRF) imaging of transiently expressed pHluorin-LAMP1-mCherry (Raiborg et al., 2015) also showed ~3-fold more lysosome fusion events in MHV-infected than in uninfected cells ( Figure 4F ). Immuno-EM of infected cells showed LAMP1 + /MHV + organelles just beneath ( Figure 4G , pink arrows) and LAMP1 on ( Figure 4G , white arrows) the plasma membrane.

Arl8b, is a small Arf-like Ras family GTPase that localizes to late endosomes/lysosomes (Boda et al., 2019; Khatter et al., 2015; Michelet et al., 2015; Michelet et al., 2018; Xu et al., 2014) and regulates their movement to the plasma membrane and ultimately their exocytosis (Michelet et al., 2015) . indicates that this pathway is not a major contributor to MHV egress. However late endosomes/MVBs are intermediates in the biogenesis of lysosomes (Huotari and Helenius, 2011) and thus may yet play a role in viral trafficking to lysosomes.

Rab7 is a small GTPase found on lysosomes, late endosomes and MVBs ( Figure 5A ). It plays a critical role in lysosome biogenesis and lysosome maintenance (Bucci et al., 2000; Langemeyer et al., 2018) : depleting Rab7 inhibits maturation of late endosomes/MVBs and leads to reduced lysosome numbers in cells (Vanlandingham and Ceresa, 2009) To investigate the impact of perturbing Rab7 activity on β-coronavirus egress we treated MHV-infected cells from 8 to 14 hr pi with the Rab7 selective competitive nucleotide binding inhibitor CID1067700 (Agola et al., 2012) . After CID1067700 treatment we measured a ~40% decrease in intracellular LAMP1 protein levels by western blot (Figure 5B ) relative to DMSO treated MHV-infected cells. Furthermore, we observed a ~50% decrease in LAMP1 + punctate organelles (per cell) suggesting a decrease in lysosome numbers (Figures 5C and 5D ). These phenotypes are consistent with previous reports of Rab7 depletion (Vanlandingham and Ceresa, 2009 ), but the full lysosome proteome will need to be characterized to determine how CID1067700 impacts lysosomes.

Most significantly while CID1067700 had no effect on cell viability, viral infection or replication ( Figure 5E , intracellular), this compound potently decreased in a dosedependent manner viral egress: by 100-fold at 4µM and by 1000-fold at 40µM ( Figure   5E , extracellular). These data further support the critical role of lysosomal biogenesis and exocytic pathways in regulating the egress of β−coronaviruses and potentially provide a new class of potent therapeutics to impede their spread.

Lysosomes are deacidified and lysosomal enzymes are inactive in β β β β-coronavirus infected cells.

We then assessed the functional consequences of the path of egress taken by βcoronavirus in terms of lysosomal functions. We used LysoTracker Red DND-99, a cell permeable weak base dye that is acidotropic and accumulates in acidified organelles (Sanman et al., 2016) . Indeed, labeling cells with the dye prior to fixing with aldehydes and staining with anti-LAMP1 antibodies (without detergents) revealed near complete localization of the dye fluorescence to LAMP1 + organelles i.e. lysosomes and late endosomes ( Figure 6A ).

12hrs and Vero E6 cells infected with SARS-CoV2 for up to 24hrs were labeled with LysoTracker Red before imaging. We observed a stark decrease in both the

LysoTracker Red fluorescence intensity per puncta and in the number of LysoTracker

Red positive puncta, indicating that both the acidity and number of acidified lysosomes in β-Coronavirus infected cells were decreased compared to uninfected cells ( Figures   6B-6D ). Note that separate LAMP1 staining confirmed that the decrease in Lysotracker

Red positive puncta in β−coronavirus infected cells was not due to a decrease in late endosome/lysosome quantities ( Figure 1H and 1J).

Lysosensor Green DND-189 is a useful dye for quantifying pH in lysosomes as it accumulates in acidified organelles; it has a low pK of 5.2 which renders it nonfluorescent except in highly acidic organelles such as lysosomes; and its fluorescence amplitude changes with pH in a calibratable manner (Brazill et al., 2000) . Using

Lysosensor Green we found that the mean pH of lysosomes in uninfected cells was pH 4.7 (with range between 4.2 to 5.2) and in MHV-infected cells it was 5.7 (with a range between 5.0 to 6.4) a very significant deacidification, one full pH unit higher than in uninfected conditions (Figures 6E).

Lysosomal enzymes are optimized to function in these organelles' highly acidic pH and even small increases of pH can be sufficient to decrease protease activity (Lie and Nixon, 2019; Sanman et al., 2016) . Given our observations above, we quantified the in situ lysosomal enzyme activities using self-quenched enzymatic substrates that are taken up by endocytosis, targeted to lysosomes and turn fluorescent upon enzymatic activity (Humphries and Payne, 2012) . To account for any potential change in endocytic uptake we co-incubated cells with a pH-insensitive fluorophore-coupled dextran, which was endocytosed along with substrate into lysosomes. Mean fluorescence intensity of substrate was quantified in lysosomes of uninfected and MHVinfected cells with similar mean dextran fluorescence intensity. These measurements revealed that, consistent with the observed increased lysosomal pH in MHV-infected cells, lysosome enzyme activities were reduced by ~40% relative to uninfected cells ( Figure 6F ). Furthermore, the increased secretion of pro-cathepsin D from MHVinfected cells ( Figure 4E ) likely reflects this decreased lysosomal enzymatic activity.

In that context, it has also been reported that the SARS-CoV1 open reading frame protein 3A (ORF3a) is a viroporin that localizes to lysosomes, disrupts their acidification (Yue et al., 2018) and contributes to viral egress (Castaño-Rodriguez et al.,

2018; Lu et al., 2006; Yue et al., 2018) . We found that SARS-CoV2 ORF3a (Gordon et al., 2020) was also targeted to lysosomes ( Figure S5A ) and limited LysoTracker Red accumulation within them ( Figure S5B ). Though further investigation will be needed, this data suggests that ORF3a may be responsible for deacidifying lysosomes so that SARS-CoV2 can use them for egress.

Lysosome-dependent antigen cross-presentation pathways are disrupted in β β β β-

Finally, we investigated the functional consequences of the altered lysosomal functions during β-coronaviral infection in terms of antigen processing. Myeloid cells rely on active lysosomal degradation of proteins to produce short peptides that are loaded and presented on class I MHC of cells (Trombetta and Mellman, 2005) . We tested whether the decrease in protein degradation by cells infected with MHV would limit antigen cross-presentation of antigens derived from large proteins while minimally impacting presentation of short peptides. We exposed bone-marrow derived primary macrophages to extracellular chicken Ovalbumin (OVA 1-385 protein) or to an Ovalbuminderived class I MHC-restricted oligopeptide (OVA 257-264 peptide) with or without MHV infection ( Figures 7A and 7B) . First, we measured the endocytosis of fluorescent OVA 1-385 protein by macrophages and found that it was not significantly affected by MHV infection ( Figure 7C ). Then we measured the amount of OVA antigen being presented by macrophages using the H-2Kb/ OVA 257-264 -responsive OT-1 TCR transgenic T cells ( Figure 7D ). We found that coronaviral infection made macrophages induce stronger T cell activation when presenting OVA 257-264 peptide, but weaker T cell activation was measured when cross-presenting OVA 1-385 protein ( Figure 7D ) 

Since the 1960s, intact coronaviruses have been detected in lysosomes at late stages of infection (Ducatelle and Hoorens, 1984) but the significance of these observations remained unexplored. Here we demonstrated that β-coronaviruses egress from infected cells by tracking a path through lysosomal organelles. This is unlike other enveloped RNA viruses whose egress either tracks with the biosynthetic secretory pathway or directly buds out of the plasma membrane (Robinson et al., 2018; Ravindran et al., 2016; Pornillos et al., 2002) . Consistent with previous reports (Machamer, 2013; McBride et al., 2007; Tooze et al., 1987) our experiments show that the secretory pathway remains largely operational in β-coronavirus infected cells with newly assembled virus particles budding into the lumen of the ER/ERGIC and trafficking to the Golgi/TGN in the early stages of infection. But, after reaching the Golgi/TGN, our experiments demonstrate that β-coronaviruses traffic to lysosomes and use exocytic lysosomes, instead of the biosynthetic secretory pathway, to egress (Figures 1-5) .

Work is in progress to delineate the route β-coronaviruses take to reach the lysosomes but least two BFA-insensitive (i.e. non-biosynthetic secretory) trafficking routes are possible (Strous et al., 1993) . First is a direct route from Golgi/TGN to lysosomes via late endosome/MVBs. These organelles are known to mature into lysosomes through a Rab7 GTPase regulated pathway (Stroupe, 2018) . Indeed, when we treated cells with CID1067700, a competitive inhibitor of Rab7 activation, lysosome J o u r n a l P r e -p r o o f numbers appeared reduced and β-coronavirus egress was inhibited by ~3-logs.

CID1067700 has been used in vivo with little toxicity (Lam et al., 2016) . Given this, it will be important to test whether CID1067700 can be an inhibitor of SARS-CoV2 spread in mice and non-human primate models. A second, more circuitous route to the lysosomes, would involve retrograde transport back to the ER/ERGIC. From there, viruses would reach lysosomes, again through late endosome/MVB intermediates or through the little understood process of microphagy where lysosomes directly engulf ER (Chino and Mizushima, 2020) .

We reported here that the KDEL-receptor and its cargo, the KDEL-sequence containing ER chaperones GRP78/BIP and calreticulin (not shown) were selectively cotrafficked with the coronaviruses to lysosomes and co-released with them outside the cell (Figure 3 ). In contrast, other secretory pathway resident proteins such as mannosidase II, TGN46, Golgin 97, ERGIC53 or CI-MPR remained behind and did not co-traffic to lysosomes (Figures S2 and S3 ). In the ER, GRP78/BIP in its role as a chaperone likely binds and helps fold newly synthesized coronavirus proteins.

Surprisingly our findings indicate that this interaction is maintained while coronaviruses egress through exocytic lysosomes. Notably, during entry, GRP78/BIP interaction with SARS/MERS was reported to facilitate these viruses' infectivity (Chu et al., 2018) and the S protein of SARS-CoV2 was postulated to directly bind GRP78/BIP (Ha et al., 2020) . Therefore, one benefit of maintaining this interaction through exocytic lysosomes may be that the coronaviruses will be ready to infect a nearby cell as soon as they egress and not be limited by extracellular GRP78/BIP availability.

Lysosome exocytosis can be regulated by calcium (Rodríguez et al., 1997) .

When intracellular calcium stores were chelated with BAPTA-AM, we measured a ~2log decrease in MHV egress. But replication was also decreased ( Figure S4F ). On the other hand, depleting synaptotagmin VII, considered a calcium-dependent trigger for lysosomal fusion with the plasma membrane (Martinez et al., 2000) resulted in decreased viral egress without significantly impacting replication (Figures S4G and   S4H ). It remains to be further investigated in the context of β-coronavirus egress the precise roles played by calcium, synaptotagmins and others.

Lysosomal proteolytic enzyme activities are central to many critical cellular processes including autophagy, cell motility, cholesterol metabolism, release of cell killing enzymes by T-cells, pathogen degradation by macrophages and self/non-self antigen presentation by all cells. Lysosome acidification is required for lysosomal enzyme stability and enzymatic activity, and even a small increase in pH is sufficient to inhibit these enzymes and stop their critical biological functions (Mindell 2012) . Here we reported a significant deacidification of lysosomes in β-coronavirus infected cells and in conjunction with it a reduction in lysosomal enzyme activity (Figures 6B-F) . Moreover, infected cells were found to secrete greater amounts of lysosomal enzymes to the extracellular environment than uninfected cells. The mechanism of deacidification is currently under investigation. One possibility is that lysosomes become deacidified indirectly due to being loaded with too much cargo (i.e. viruses) and/or perturbations in proton pump or ion channel trafficking (Ballabio and Bonifacino, 2020) . Alternatively, deacidification may be a consequence of the action of specific coronavirus proteins that behave like viroporins. For example, the γ-coronavirus E protein increases Golgi/TGN pH from 6.8 to 7.1 (Ruch and Machamer, 2012; Westerbeck and Machamer, 2019) .

SARS-CoV1/CoV2 and MERS, all express ORF3a which previously (Yue et al., 2018) and here (Figures S5A and S5B ) has been shown to traffic to lysosomes and disrupt their acidification. Importantly, SARS/MERS viruses deficient in ORF3a may be unable to egress (Castaño-Rodriguez et al., 2018; Lu et al., 2006; Yue et al., 2018 )-a phenotype that would be consistent with acidic lysosomes being prohibitive to trafficking viral cargo.

Our findings here indicate that the altered lysosomal function of β-coronavirus- 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nihal Altan-Bonnet (nihal.altan-bonnet@nih.gov).

This study did not generate any new unique resources or reagents. Further information on materials, dataset and protocols should be directed to and will be fulfilled by the Lead Contact, Nihal Altan-Bonnet (nihal.altan-bonnet@nih.gov).

This study did not generate any unique datasets or code.

HeLa-mCC1a cells were cultured in complete (with 10% Fetal Bovine Serum (FBS)) or serum-free Dulbecco's Minimal Essential Medium (DMEM)/high glucose/Penicillin-/Streptomycin (Pen/Strep) and maintained at 37°C. Vero E6 cells were cultured in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% FBS and Pen/Strep and maintained at 37°C.

Primary mouse macrophages were prepared starting from bone marrow precursors harvested from 6-12-week old female C57Bl16 mice (Jackson Labs, Bar Harbor, ME). Femoral aspirates were collected, washed with complete RPMI once, resuspended at 1 million per ml in complete RPMI medium augmented with 1nM recombinant mouse M-CSF (R&D Systems, Minneapolis MN). The single cell suspension was then placed in Fluoroethyl polymer culture bags (Origen Biomedical, Austin TX) to expand and to differentiate. Medium was replaced with complete RPMI augmented with 1nM M-CSF after 3 days of culture. Macrophages were harvested after 7 days of culture, washed in complete RPMI, seeded in 96-flat well plates (10,000 cells per well, with 100µl complete RPMI) and left to adhere overnight for additional experiment.

Complete RPMI consists of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES (pH 7.4), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 µg/ml of penicillin, 100 µg/ml of streptomycin and 50 µM β-mercaptoethanol.

HeLa-mCC1a cells were infected with MHV-A59 for 4hrs, washed, and then kept in either complete (with 10%FBS) or serum-free DMEM/high glucose/Pen/Strep for desired period of time. Vero E6 cells were grown in Millicell EZ 8-well glass slides (Millipore) in infection media (EMEM, 4% FBS (Corning)) to a confluency of 90 -100%. Cells were then infected with the SARS-CoV2 isolate USA-WA1/2020 at a MOI 1 for 24hrs.

TCID50/ml determination Extracellular medium collected from infected cell cultures was serially diluted and dilutions used to inoculate multiple cell cultures for up to 72hr. Cytopathic changes in the cells were tabulated to calculate TCID50/mL values, as described (Leibowitz et al., 2011) . Briefly, supernatant (media) from MHV infected and BFA treated cells were obtained and inoculated to HeLa-mCC1a cells seeded at 4X10 4 cells/well in 96-well format. 11 serial dilution of the media was prepared in DMEM (10 -1 to 10 -11 ) and added in triplicates for statistical significance. Cells were left in 37°C incubator for 72hrs. Thereafter the media was discarded and each well received 100µl of Crystal Violet solution (25% crystal violet supplemented with 20% ethanol in double distilled water) and incubated for 15min. The solution was discarded, each well was washed twice with water and the image of the 96-well plate was captured. Dilution that showed 50% cell death (i.e. where 50% of the cells in dish were left behind, stained with Crystal Violet) was used for calculating TCID50/ml.

All DNA transfections were carried out with Fugene 6 according to manufacturer instructions (Promega Corp.).Briefly, Fugene 6 was incubated with serum-free media for 5min and then mixed with plasmid DNA ( Gaussia Luciferase, pHluorin-LAMP1-mCherry, Arl8b-GFP) in a separate tube and incubated for another 20min. The plasmid DNA / Fugene 6 mixture was pipetted slowly onto the culture of cells. Cells were kept at 37°C and utilized after 18-24hrs.

Brefeldin A (BFA) was prepared as a 5mg/ml stock solution in ethanol and Dyngo-4a was prepared as a 100mM stock in DMSO; both were stored long term at -20°C. BAPTA-AM was prepared fresh (prior to experiment) as a 10mM stock solution in DMSO and not stored. BFA and Dyngo-4a treatments were done in cell culture media; For BAPTA-AM treatment, cells at 8hr pi were switched to calcium-free media supplemented with 2mM EGTA and 30µM BAPTA-AM.

Unless otherwise indicated, cells were fixed in 3.7% paraformaldehyde (PFA)/phosphate buffer solution (PBS) for 10min; blocked in PBS/10%FBS. All primary and secondary antibody incubations were carried out in PBS/10%FBS supplemented with saponin at 0.2% for 1hr at room temperature. Cells were rinsed in PBS and mounted with Fluoromount-G (Invitrogen).

For cell surface LAMP1 staining, cells were pre-chilled at 4°C for 20min and incubated on ice with anti-LAMP1 (R & D) antibody in PBS for 30 min. After rinses with chilled PBS, cells were kept on ice and incubated with appropriate secondary antibody in PBS for 30min. Cells were rinsed with chilled PBS, fixed in 2% PFA for 5min, rinsed and mounted.

All microscopy and image acquisition were performed on the LSM780 confocal microscope (Carl Zeiss USA) with a 63X/1.4NA or 40X/1.3 NA oil objectives. Live cells were imaged on a heated stage. Cells were imaged with 12-bit resolution using pinhole settings optimized for either high resolution imaging or for fluorescence quantification, for the latter the pinhole was kept open such that fluorescence from entire organelle or cell volume could be acquired. Zen software (Carl Zeiss USA) were used for all image analysis including quantification of LAMP1, Cl-MPR, Lysotracker Red DND-99 etc. positive organelles.

pHluorin-LAMP1-mCherry (gift of Harald Stanmark, University of Oslo, Norway) transfected cells were plated on coverglass chambers (Nunc Lab-Tek II, ThermoFisher). Cells were left uninfected or infected with MHV. After 4hr, cells were washed with phenol red free DMEM/10%FBS/25mMHepes pH 7.3 and kept in this media. After 10hr of infection, cells were placed on the heated ELYRA.PS1 microscope stage. Fluorescent fusion events at the plasma membrane (facing the coverslip) were imaged using the ELYRA.PS1 in its TIRF setting mode (488nm laser excitation and 505-550BP filter emission to image the pHluorin; 565 excitation and 575LP to image the mCherry) with an incident angle providing an evanescent field <100nm. Time series, with no delay in between frames, was collected for a total of 3min. Appearance of punctate pHluorin fluorescence above background were counted as fusion events, quantified and plotted.

HeLa-mCC1a cells were infected with MHV and fixed in 4% formaldehyde and 0.1% glutaraldehyde in 1x PHEM buffer for 90 min. Cryo-sectioning and immunolabelling were performed as described elsewhere (Griffiths, 1993; Tokuyasu, 1973) . In brief, ultrathin sections (55-70 nm) from gelatin-embedded and frozen cell pellets were obtained using an FC7/UC7-ultramicrotome (Leica, Vienna, Austria). Immunogold labelling was carried out on thawed sections with anti-GFP (2.5 mg/ml, rabbit, Rockland, 600-401-215), J1.3 (1:50, mouse), anti-LAMP1 (1:20, rabbit) antibodies. Mouse primary antibodies were detected with polyclonal rabbit anti-mouse immunoglobulin Gs (0.5 mg/ml, Rockland, 610-40120). All samples were incubated with 5 or 10 nm protein A gold (1:50, UMC Utrecht University, Utrecht, Netherlands), as described (Griffiths, 1993) , and stained/embedded in 4% uranyl acetate / 2% methylcellulose mixture (ratio 1:9) (Slot and Geuze, 2007; Tokuyasu, 1980) . Sections were examined with a JEM-1200EX (JEOL USA) transmission electron microscope (accelerating voltage 80 keV) equipped with a bottom-mounted AMT 6-megapixel digital camera (Advanced Microscopy Techniques Corp).

Vero E6 cells infected with SARS-CoV-2 and HeLa-mCC1a cells infected with MHV were fixed in 4% formaldehyde and 0.1% glutaraldehyde in 1x PHEM buffer for 12hr.

Cryo-sectioning were performed as described elsewhere (Griffiths, 1993; Tokuyasu, 1973) . In brief, ultrathin sections (55-70 nm) from gelatin-embedded and frozen cell pellets were obtained using an FC7/UC7-ultramicrotome (Leica, Vienna, Austria) and stained/embedded in 4% uranyl acetate / 2% methylcellulose mixture (ratio 1:9) (Slot and Geuze, 2007; Tokuyasu, 1980) . Sections were examined with a JEM-1200EX (JEOL USA) transmission electron microscope (accelerating voltage 80 keV) equipped with a bottom-mounted AMT 6-megapixel digital camera (Advanced Microscopy Techniques Corp).

Rather than a Pearson correlation coefficient, we calculated colocalization coefficients as recommended in Manders EMM, Verbeek FJ, Aten JA (1993) (Manders et al., 1993) and computed the weighted colocalization coefficients WCC for the LAMP1 (lysosome) and protein M (MHV particle) channels. Using the colocalization toolbox from the ZEN software, we defined four quadrants for high/low fluorescence in each channel. Then we compute

where:

, is the fluorescence of pixel high in both LAMP-1 and protein M fluorescence , is the total fluorescence for the LAMP-1 channel.

WCC varies between 0 and 1, with 0 corresponding to none of the lysosome pixel colocalizing with the M protein, and 1 corresponding to a perfect overlap where each lysosome pixel has a high M protein fluorescence. Calculation of colocalization coefficients for LAMP1 and CI-MPR was carried out as described above.

Uninfected and MHV/SARS-Cov2-infected cells were incubated with Lysotracker Red DND-99 (100nM) or Lysosensor Green DND-189 (1µM) according to manufacturer (ThermoFisher Scientific) instructions. For Lysosensor Green pH measurements, Step 1: cells were imaged with 458nm Argon laser excitation and 500-550nm bandpass emission filters.

Step 2: the cells in step 1 were treated sequentially with potassium buffers of known pH containing 10µM Nigericin. The images at each buffer condition were collected using the same image acquisition settings as in Step 1 to generate a standard pH curve. This standard curve was then used to convert the fluorescence values collected in step 1 to pH values.

Uninfected and MHV-infected cells were incubated with Alexa-555 10kD dextran (1mg/ml) and Lysosome-Specific Self-Quenched Substrate (Abcam Cat No. ab234622) at manufacturers recommended dosage for 1hr before they were fixed with 4% PFA at room temperature for 15min. Cells were mounted with Fluoromount G (Invitrogen)

containing DAPI and imaged with Zeiss LSM780 Confocal Laser Scanning microscope. Images were analyzed using Zen software. Mean fluorescence intensity of substrate was quantified in lysosomes with similar mean dextran fluorescence intensity in uninfected and MHV-infected cells.

Trypan blue staining for plasma membrane permeability/cell viability After the incubation period cells were harvested from individual treatment groups and resuspended in 1ml of 1X PBS. A 1:1 mixture of cell suspension and 0.4% Trypan Blue stain (Invitrogen, Eugene, OR, Catalogue No. T10282) was made and incubated for 3min at room temperature. 10µl of the mixture was added to Countess cell counting chamber slide (Invitrogen, Eugene, OR, Catalogue No. 100078809 ) and quantified cell viability in Countess Automated Cell Counter (Invitrogen, Eugene, OR, Catalogue No. C10227) . In order to image the Trypan blue staining, cells were seeded in 4-well coverglass bottom chamber slides and incubated overnight before being inoculated with virus. One group received 300nM of staurosporine, an apoptosis inducer, used as a positive control for comparing cell viability for uninfected and MHV-infected cells. At 14hr pi media was replaced with 100µl of PBS and 0.4% Trypan Blue was added in a 1:1 ratio followed by incubation for ~3 min at room temperature. PBS was removed and cells were imaged by DIC.

After the incubation period cells were harvested from individual treatment groups and washed in 200µl of 1X PBS. 50µl of Trypsin Versene was then added to each well, and the plate was incubated for 10 min at 37°C. 150µl of FACS buffer was then added and cells were spun down and washed once in 200µl FACS buffer. Cells were then resuspended in 75µl of propidium iodide (PI) in FACS buffer (final concentration = 10µg/ml), incubated for 5min at room temperature and immediately run on a Fortessa Flow Cytometer: fluorescence of PI was acquired in the PE-CF594 channel, forward scattering (FSC) was also acquired. Live/Dead cells were then counted post-acquisition using FlowJo: Live cells were defined as FSC+PI-and dead cells were defined as FSC-PI+ (see Supplementary Figure 1B ). Each timepoint was set up in quadruplicate.

Gaussia Luciferase Assay was performed using the Pierce Gaussia Luciferase Glow assay kit (Thermo Scientific, USA, Catalog No. 16160). Fugene 6 was used to transfect HeLa-mCC1a cells with Gaussia luciferase plasmid and after mock or MHV infection and with and without BFA treatment, supernatants were collected and analyzed for Gaussia Luciferase activity as dictated by manufacturer instructions. Briefly, 15µl of the media from the treated wells was added to a black opaque 96-well plate and to that added 50µl of the working solution. After a 10min incubation detected the glow luminescence in a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT, USA).

Lysosome isolation was adapted from Graham et al., 1990 (Graham et al., 1990 . Cells were grown in 150cm 2 dishes and divided into 3 groups: uninfected, MHV and MHV + Dyngo-4a. Cells were infected with MHV; washed at 4hr pi with 1X PBS. 30µM of Dyngo-4a was added to one group at 6hr pi. Cells were harvested at 12hr pi from all 3 groups. Medium was removed, cells were washed with ice-cold PBS, scraped on ice, resuspended in 1mL of ice-cold TNE buffer (Tris NaCl EDTA buffer, Quality Biological, Gaithesburg, MD, ; DNase, RNase, and Protease tested) and collected in a 2mL Eppendorf tube. After a quick freeze-thaw, cells were lysed on ice using a 28G syringe (15 strokes) and centrifuged 10min at 800g at 4°C to pellet nuclei and residual non-lysed cells. The supernatant was harvested and centrifuged 15min at 20,000g at 4°C. The obtained pellet was resuspended in 1mL TNE buffer and centrifuged once more 15min at 20,000g at 4°C. After a final resuspension in 1mL TNE buffer, it was loaded on top of a 25%-40% Nycodenz (Fisher Scientific, USA, Catalog No. AN1002423) discontinuous gradient and centrifuged for 2hr at 100,000xg at 4°C using a SW40 Ti rotor (Beckman Coulter, Indianapolis, IN, Catalog No. 331301) . Fractions were harvested from the top; aliquots of each fraction were set aside for Western blot analysis with organelle markers; the rest was processed for qPCR analysis of genomic MHV RNA. 10026938) and run in a Trans-Blot Turbo system as per the user manual. Following transfer blots were blocked using 5% non-fat milk blocking followed by probing with primary antibodies diluted in 5% Bovine Serum Albumin (BSA) and overnight incubation. Blots were thereafter washed with 1X TBS buffer (Bio-Rad, USA, Catalogue No. 1706435) supplemented with Maumee, OH, . Further re-probed with corresponding HRP conjugated secondary antibodies and incubated for 1hr followed by washing steps. Blots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, Catalogue No. 34580) and imaged in Amersham Imager 600 (GE Healthcare Biosciences, Pistcataway, NJ). All densitometric quantification was done in Amersham Imager 600 software.

Serum free extracellular media from cells was collected and concentrated in a spin concentrator followed by TCA precipitation, acetone wash and air dry. Samples were resuspended in SDS-PAGE loading buffer with β-ME, boiled for 5min and ran on 4-20% gradient acrylamide gels (Bio Rad, USA) before being transferred onto nitrocellulose for Western blotting with antibodies against targets of interest (cathepsin D, BIP/GRP78, actin etc.).

J o u r n a l P r e -p r o o f Therapeutics) and incubated for up to 72hrs. They were infected with MHV for 4hr, before washing off the virus and switching to serum free media. At 12-14hr pi, intracellular and extracellular virus was quantified by qPCR. For intracellular RNA levels, cells were lysed with RNA lysis buffer (Zymo Research); for intracellular protein level quantifications cells were scraped and lysed in cell lysis buffer (Invitrogen) containing protease inhibitors. For Rab27 depletion, siRNA was purchased from Ambion. In all siRNA treatments, both intracellular and extracellular proteins were TCA precipitated from serum-free media, acetone washed, air dried and suspended in Laemmli gel loading buffer before SDS-PAGE/Western analysis.

Bone-marrow derived macrophages were prepared from femoral aspirates cultured for 7 days in complete RPMI medium with 1nM M-CSF in Fluoroethyl polymer culture bags (Origen). 2.104 cells were harvested and plated on plastic (96-flat-well plate), let to adhere for 2hrs, then exposed to MHV (or not) for 24hrs. Macrophages were then infected with SIINFEKL peptides or with chicken ovalbumin at varied concentrations for 2hrs, then washed with complete RPMI. C57Bl6 Rag1-/-OT-1 TCR Transgenic mouse splenocytes were then harvested, cleared of their red blood cells by ACK lysis, added and spun onto macrophages (105 cells per well), and incubated for 6hrs. Cell cultures were then harvested using a 15min trypsin-versene treatment, washed and antibodystained for flow cytometry (see panel below) with DAPI added just before acquisition. Cells were analyzed using a 5-laser FORTESSA flow cytometer (BD Bioscience) as well 

Chicken ovalbumin was fluorescently labeled with the Alexa647 dye using a bioconjugation kit (ThermoFisher). Bone-marrow derived macrophages were prepared as described in the previous paragraph. 2.104 cells were harvested, and plated on plastic (96-flat-well plate), let to adhere for 2hrs, then exposed to MHV (or not) for 24hrs. Macrophages were then infected with varying concentrations of fluorescentlylabeled chicken ovalbumin for 2hrs, harvested with a 15min exposure to a solution of trypsin-versene-EDTA mixture (Lonza), washed with FACS buffer (PBS/4%FBS/0.1%Sodium Azide) and immediately analyzed by flow cytometry.

HeLa-mCC1a cells were plated and infected (or not) with MHV for 24hrs. Cells were lifted up using a trypsin-versene solution, washed with PBS, incubated for 1min at room temperature with a 0.1M solution of Glycine in PBS (pH adjusted to 2.4) for acid stripping, or with PBS for control, then washed with complete RPMI twice. KIR3DL1reporter Jurkat cells (Garcia-Beltran et al., 2016) were cultured in complete RPMI and harvested by aspiration. 5.104 HeLa-mCC1a cells were washed with Jurkat cell culture medium then resuspended with 5.104 Jurkat cells, spun at 100g for 15sec and incubated at 37°C for 15min (some wells received only Jurkat cells or Jurkat cells and 1µMol of phorbol 12-myristate 13-acetate for negative and positive controls, respectively). Cells were then immediately resuspended with ice-cold 2% PFA for 15min, permeabilized with ice-cold 90% Methanol for 15min, washed with FACS buffer and stained for phospho-ERK (E10 clone, Cell Signaling Technology) and anti-mouse secondary antibody (Jackson Immunochemicals). A more detailed protocol can be found in Vogel et al., (Vogel et al., 2016) . Cells were analyzed using a 5-laser FORTESSA flow cytometer (BD Bioscience). Data were processed using FlowJo (TreeStar) and a custom-written Python pipeline (python.org). The levels of open HLA-F conformers were quantified by monitoring Jurkat cell activation (itself estimated geometric mean of phosphor-ERK staining in FSCint Jurkat cells).

All graphs were plotted and unpaired two-tailed Student-t Test was performed using the GraphPad Prism 8 software or the SciPy Statistics library in Python. p values were considered significant for p< 0.05 unless otherwise indicated and denoted as * where p<0.05; ** where p<0.01; where *** p<0.0002; **** where p<0.00001; and ns=not significant.

• β-Coronaviruses do not use the biosynthetic secretory pathway to egress. • β-Coronaviruses traffic to lysosomes; egress by Arl8b-dependent lysosomal exocytosis.

• Lysosomes are deacidified and proteolytic enzymes inactive in infected cells.

• Antigen processing and presentation is perturbed in β -Coronavirus infection.

Altan-Bonnet et al. provide evidence that β-Coronaviruses do not use the biosynthetic secretory pathway typically used by enveloped viruses to leave infected cells. Instead, these viruses traffic to lysosomes for unconventional egress by Arl8b-dependent lysosomal exocytosis. Their non-lytic release results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation. 

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MHC class II presentation is controlled by the lysosomal small GTPase, Arl8b

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Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth

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Immunochemistry on ultrathin frozen sections

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells

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Cell biology of antigen processing in vitro and in vivo

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Authors thank Mary Carrington (NCI, Bethesda MD), Rosa Puertollano (NHLBI, Bethesda MD), Jyoti Jaiswal (Children's National Medical Center, Washington DC), Norma Andrews (University of Maryland, College Park MD) and Graham Brogden (University of Veterinary Medicine, Hannover, Germany) for critical discussion and reagents. PMT was supported by NIH R01 A1091985-05; SP by NIH R01 NS36592 and AF by F32-AI113973; VH by NIH R37GM058615; GW by NIH R01AI35270; all others by intramural NIH and NCI funds. 

The authors declare no competing interests.J o u r n a l P r e -p r o o f 100