key: cord-0901135-gjk8kc8g
authors: Li, Fei; Li, Jingyao; Wang, Pei-Hui; Yang, Nanyan; Huang, Junyu; Ou, Jinxin; Xu, Ting; Zhao, Xin; Liu, Taoshu; Huang, Xueying; Wang, Qinghuan; Li, Miao; Yang, Le; Lin, Yunchen; Cai, Ying; Chen, Haisheng; Zhang, Qing
title: SARS-CoV-2 spike promotes inflammation and apoptosis through autophagy by ROS-suppressed PI3K/AKT/mTOR signaling
date: 2021-08-27
journal: Biochim Biophys Acta Mol Basis Dis
DOI: 10.1016/j.bbadis.2021.166260
sha: 2cb233c1beae67c6ba0b22f60743e28eaadebe50
doc_id: 901135
cord_uid: gjk8kc8g

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation. METHODS: SARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses. RESULTS: Angiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.

Since its outbreak in January 2020, the novel coronavirus disease 2019 , caused by the highly contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread worldwide and become a serious public health problem. 1,2 SARS-CoV-2 mainly infects the respiratory tract and lungs and leads to a new type of coronavirus pneumonia. 3, 4 As of August 2021, over 198 million confirmed cases and 4 million deaths had been reported worldwide. The rapid spread and high mortality rate have generated huge concerns to find effective methods to reduce SARS-CoV-2 transmission and develop specific treatment options for its infection. Currently, although many vaccines are being used to prevent the spread of SARS-CoV-2, the therapeutic methods for treating SARS-CoV-2 infections are mainly conventional antiviral drugs. Thus, no specific therapeutic agent is available for the resulting complications, such as dysregulated immune and inflammatory responses.

Therefore, exploring the pathogenic mechanism of SARS-CoV-2 infection is important for developing effective drugs for treating Autophagy, triggered by endoplasmic reticulum stress and the unfolded protein response, is a cellular degradation process in which cytoplasmic materials such as proteins, lipids, and organelles are degraded by lysosomes. 5, 6 Autophagy has been recognized as a cell defense and survival mechanism since cellular components are engulfed by double membrane autophagosomes and transported to lysosomes for degradation to generate nutrients for deprived cells. 7, 8 Alternatively, autophagy can induce irreversible autophagic cell death and autophagy-dependent apoptosis. 9 Excessive autophagy may lead to cell death, and it has been regularly observed in dying cells, suggesting that autophagy may be a mode of cell death or simply a failed attempt to rescue stressed cells from death. 8, 10 Therefore, moderate levels of autophagy can protect against cell death but excessive autophagy may result in autophagic J o u r n a l P r e -p r o o f Journal Pre-proof cell death. [11] [12] [13] However, considering the complexity of autophagy in different physiological and pathological conditions, further quantitative studies are needed to determine the selected boundary threshold of autophagy and its role in cell survival and death. Viral infections frequently cause severe inflammatory responses that may play protective or destructive roles in innate immune responses against viruses and tissue damage. [14] [15] [16] Similarly, SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) infections are all associated with dysregulated immune, inflammatory responses, and multi-organ failure. 17, 18 Clinically, SARS-CoV-2 primarily infects the respiratory tract and causes severe lung injury and inflammatory responses, ultimately leading to severe systemic inflammatory responses. 19, 20 Substantial evidence has revealed that dysregulated host immune responses and inflammation-induced cytokine storms, including the production of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), granulocyte colony-stimulating factor, IL-1β, and IL-7, are associated with worsening clinical outcomes in COVID-19 patients. 21, 22 However, the molecular mechanisms underlying the uncontrolled release of inflammatory cytokines in SARS-CoV-2 infection have not been fully elucidated.

Notably, autophagy performs an important role in the regulation of inflammatory signaling. 23 Some studies have reported that autophagy plays a negative regulatory role in inflammatory responses, whereas a few others have suggested that autophagy can promote the release of inflammatory factors. [24] [25] [26] [27] [28] Thus, autophagy may play a balancing role in supporting inflammatory responses while simultaneously preventing excessive inflammatory responses. 29 As most RNA viral infections, including respiratory syncytial virus, hepatitis C virus, and human immunodeficiency virus type-1 (HIV), can induce autophagy, [30] [31] [32] it is therefore valuable to further explore whether SARS-CoV-2 infection can also cause autophagy, and the regulatory role of autophagy in SARS-CoV-2 infection-induced inflammation.

J o u r n a l P r e -p r o o f Journal Pre-proof

HEK293T, Vero E6, and 16HBE cells were obtained from ATCC, and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA). HMEC-1 cell line was obtained from ATCC, and it was cultured in endothelial cell medium supplemented with 10% FBS and 1% endothelial cell growth factor (ScienCell, USA). The identities of all cell lines were confirmed using short tandem repeat (STR) profiling analysis. LipoFilter (Hanbio, China) was used for transfection according to manufacturer's protocol. HEK293T-hACE2 cells were generated by transfecting HEK293T cells with hACE2-expressing lentiviruses obtained from HEK293T cells co-transfected with pCDH-CMV-hACE2-EF1-Puro, psPAX2, and pMD2.G.

Total cell proteins were extracted and boiled in RIPA buffer (Abcam, USA), and 10 μg protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically blotted onto polyvinylidene fluoride membranes (Millipore, Germany) and probed with anti-ACE2, anti-LC3, anti-p62, anti-Bcl-2, anti-Bax, anti-phospho-mTOR, anti-mTOR, anti-phospho-STAT3, anti-STAT3, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT, anti-AKT, anti-Flag, and anti-GAPDH antibodies (Abcam). After incubating with an HRP-labeled goat anti-rabbit IgG or an HRP-labeled goat anti-mouse IgG, the protein expression levels were visualized using the ECL chemiluminescence reagent (Millipore). Detailed information on the antibodies is summarized in Table S1 . All Western blot bands were evaluated using Gel-Pro Analyzer 4.0 and quantified by normalization to GAPDH. For Co-IP, whole-cell lysates of Vero E6, 16HBE, and HMEC-1 cells transfected with pCDH-CMV-SARS-CoV-2-S-Flag were J o u r n a l P r e -p r o o f Journal Pre-proof successively incubated with ACE2 monoclonal antibodies for 6 h, followed by capturing with protein A/G agarose beads for 16 h. Bound proteins were then washed with lysis buffer, resuspended in protein sample buffer, and analyzed using Western blotting.

Total RNA was extracted from HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC- 

SARS-CoV-2 spike pseudovirions were generated and used to infect cells as described previously. 34 The viral titers were determined using plaque assays as described previously. 35 To transduce target cells with SARS-CoV-2 spike pseudovirions, HEK293T, HEK293T-hACE2, Vero E6, 16HBE, or HMEC-1 cells were seeded into 12-well plates. After adding 10 μg/mL polybrene to the viral dilution, cells were infected with the SARS-CoV-2 spike pseudovirions (multiplicity of infection, MOI=5) for 24 h. The infection efficiency of SARS-CoV-2 spike pseudovirions was viewed using a Nikon Eclipse Ti2-E fluorescence microscope (Nikon, Japan) at an excitation wavelength of 488 nm or it was measured using a luciferase substrate that determines firefly luciferase activity using a microplate reader (TECAN, Germany).

HEK293T-hACE2 and Vero E6 cells were infected with or without SARS-CoV-2 spike pseudovirions (MOI=5) for 48 h. Total RNA was extracted using TRIzol (Invitrogen, USA)

according to the manufacturer's instructions. RNA-seq libraries were constructed, and the transcriptome was sequenced on an Illumina HiSeq 2500 platform by Beijing Biomarker Technology Corporation as our previously report. 36 All the raw data have been deposited under the Gene Expression Omnibus (GEO) accession number GSE169370.

HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells were infected with or without SARS-CoV-2 spike pseudovirions (MOI=5) for 48 h. Cells (1 × 10 5 ) were collected and treated with 0.4% trypan blue probe for 2 min. The number of trypan blue-positive cells was calculated using a hemocytometer (Cellometer, USA).

Culture media were collected from HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and Table S2 .

Intracellular ROS levels were detected as previously described. 36 Briefly, adherent 

For flow cytometry analysis of cell surface ACE2, 100 μL of HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells (1 × 10 6 ) was digested to a single-cell suspension, and incubated with primary rabbit anti-human ACE2 antibodies followed by FITC-conjugated IgG secondary antibodies. Rabbit IgG was used as the isotype control. Cells were then washed thrice with centrifugation at 400g and analyzed using a FC500 flow cytometer J o u r n a l P r e -p r o o f

Immunofluorescence assay was performed as described previously. 36 Briefly, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells were grown on μ-Dish (Ibidi, Germany) and

infected with or without SARS-CoV-2 spike pseudovirions in parallel with addition of 5 mM ROS inhibitor (Sigma, USA) or 10 μM MHY 1485 (MCE, China) treatment. Cells were then fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked in 5% BSA-PBS solution, and incubated with primary anti-human LC3 antibodies. FITC-conjugated IgG secondary antibodies were used for LC-3 labeling. Cell nuclei were stained with Hoechst 33342, and imaged with a 63× objective using an LSM 880 confocal microscope (Zeiss, Germany).

HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells were seeded onto μ-Dish (Ibidi) and

infected with or without SARS-CoV-2 spike pseudovirions in parallel with the addition of an equal volume of DMSO or 3-MA (10 mM) for 24 h. Cells were then fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and subjected to a TUNEL assay using an in situ TUNEL apoptosis detection kit (Abbkine, USA), according to the manufacturer's instructions. Finally, cell nuclei were stained with DAPI and visualized with a 40× objective using an LSM 880 confocal microscope (Zeiss). The apoptotic cells were calculated by counting the total number of TUNEL-stained nuclei (FITC-positive).

Five-week-old wild-type BALB/c mice were maintained in the Laboratory Animal Center of Sun Yat-sen University. All animal studies were approved by the Institutional Animal Care and Use Committees of the Sun Yat-sen University (approval number: SYSU-IACUC-2020-B0778). Mice were sacrificed, and the trachea, lung, and liver were harvested for J o u r n a l P r e -p r o o f immunohistochemical analysis according to standard procedures. 37 The images from tissue sections were obtained and analyzed using a Nikon Eclipse Ti2-E microscope (Nikon).

All results are presented as the mean ± SD of three entirely independent experiments derived from separate transfection and treatment rounds. The differences among groups were analyzed using Student's t-test when only two groups were compared. For comparison between more than two groups, all data were first analyzed for adherence to a normal distribution and then subjected to one-way ANOVA followed by Tukey's post hoc test. *P <0.05, **P <0.01, and ***P <0.001 were considered statistically significant. All data were analysed using SPSS (version 22.0) statistical software.

J o u r n a l P r e -p r o o f 3. Results

ACE2, first identified in 2000, functions as a cellular receptor for the spike protein of coronaviruses to facilitate viral entry into target cells. 38, 39 To investigate the underlying pathogenic mechanisms of SARS-CoV-2, which is considered to be the third most pathogenic coronavirus, 3 (Fig. S1a) , which corresponded to the ACE2 transcripts in Vero E6, 16HBE, and HMEC-1 cells (Fig. S1b) . Importantly, flow cytometry revealed ACE2 expression on the cell surfaces of Vero E6, 16HBE, and HMEC-1 cells (Fig. 1a) . To further verify the binding interaction between SARS-CoV-2 spike protein and ACE2 in ACE2-expressing cells, we transfected the cells with Flag-labeled spike protein.

Co-IP analysis revealed that the spike protein of SARS-CoV-2 co-immunoprecipitated with endogenous ACE2 in Vero E6, 16HBE, and HMEC-1 cells (Fig. 1b) . Meanwhile, ACE2

expression mediated SARS-CoV-2 spike pseudovirions infection in Vero E6, 16HBE, and HMEC-1 cells (Fig. 1c and Fig. S1c) . Notably, immunohistochemistry further proved that ACE2 was expressed in the tracheal and bronchial epithelial cells and vascular endothelial cells of the lungs and liver of mice ( Fig. 1d and Fig. S1d ).

Several viral infections have been reported to activate autophagy, which is beneficial to viral replication. 40, 41 To determine whether autophagy is triggered upon SARS-CoV-2 infection, HEK293T cells were transfected with human ACE2-expressing lentivirus to construct stable HEK293T-hACE2 cells (Fig. 2a and Fig. 2b) . We observed that the autophagosome marker LC3-Ⅱ, proteolytically cleaved and lipidated from LC3, was increased in HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells that were infected with SARS-CoV-2 spike pseudovirions or treated with recombinant spike protein for 24h ( Fig. 2c and Fig. S2a) .

Consistently, SARS-CoV-2 spike pseudovirions infection and recombinant spike protein treatment reduced p62 expression in ACE2-expressing HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells, but not in control HEK293T cells ( Fig. 2c and Fig. S2a) . Subsequently, the cells were transfected with a double-tagged pmCherry-GFP-LC3 plasmid to visualize the progression of autophagy after SARS-CoV-2 spike pseudovirions infection. We found that following SARS-CoV-2 spike pseudovirions infection, both GFP-LC3 and mCherry-LC3

were significantly upregulated in HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells, but not in HEK293T control cells (Fig. 2d-2f) , indicating that SARS-CoV-2 spike induces the occurrence of autophagic responses in infected cells. Additionally, high throughput RNA-Seq analyses of differentially expressed genes (DEGs) revealed a significant overlap in the expression of altered autophagy-regulatory genes in HEK293T-hACE2 and Vero E6 cells upon SARS-CoV-2 spike pseudovirions infection ( Fig. 3a and Fig. 3b) . The expression levels of the majority of autophagy-promoting genes, including ATG4B and MAP1LC3B2, were significantly increased in HEK293T-hACE2 and Vero E6 cells infected with SARS-CoV-2 spike pseudovirions ( Fig. 3a and Fig. 3c) . Conversely, the subset of autophagysuppressing genes was significantly decreased in SARS-CoV-2 spike pseudovirions-treated HEK293T-hACE2 and Vero E6 cells (Fig. 3a and Fig. 3c) . Furthermore, Kyoto

Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene set J o u r n a l P r e -p r o o f enrichment analysis (GSEA) of DEGs revealed that the regulation of autophagy and lysosome-related pathways were significantly enriched in SARS-CoV-2 spike pseudovirionsinfected cells (Fig. 3d and Fig. 3e) .

Meanwhile, flow cytometry analysis demonstrated that SARS-CoV-2 spike pseudovirions and spike protein did not alter apoptosis in HEK293T control cells, which was in striking contrast to the result that late apoptotic (FITC+/7-AAD+) populations were significantly enhanced in SARS-CoV-2 spike pseudovirions-infected and recombinant spike proteintreated HEK293T-hACE2 cells on the stable expression of hACE2 ( Fig. 4a and Fig. S2b ).

Annexin V-FITC staining also confirmed that SARS-CoV-2 spike pseudovirions increased the apoptosis of Vero E6, 16HBE, and HMEC-1 cells (Fig. 4b) .

Consistently, RNA-Seq analysis indicated that SARS-CoV-2 spike pseudovirions upregulated the majority of genes correlated with pro-apoptotic responses, including Bax and ARC, and downregulated the prosurvival gene Bcl-2 in HEK293T-hACE2 and Vero E6 cells ( Fig. 3a and Fig. 3c) , which was further confirmed by Western blot analysis in HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells (Fig. 4c) . KEGG pathway enrichment analysis of DEGs also revealed that the apoptosis-related pathway was significantly enriched in SARS-CoV-2 spike pseudovirionsinfected cells (Fig. 3d) . Moreover, trypan blue staining indicated that SARS-CoV-2 spike pseudovirions infection resulted in a significantly decreased cell viability in ACE2expressing cells (Fig. 4d) . Thus, these findings suggest that SARS-CoV-2 spike may induce autophagy and therefore play a pro-death regulatory role in ACE2-expressing cells.

Many studies have shown that ROS levels are aberrantly upregulated in virus-infected cells, which may be responsible for cell apoptosis. 42, 43 To further investigate the mechanism by J o u r n a l P r e -p r o o f which SARS-CoV-2 spike promotes autophagy and pro-apoptotic responses in ACE2expressing cells, we evaluated the intracellular ROS levels using DCFH-DA probe, and we observed that SARS-CoV-2 spike pseudovirions treatment did not significantly alter the intracellular ROS levels in HEK293T control cells. However, the intracellular ROS levels of HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells were significantly increased after SARS-CoV-2 spike pseudovirions treatment ( Fig. 5a and Fig. 5b) . Notably, consistent with previous results showing that ROS upregulation induced autophagy through PI3K/AKT/mTOR inhibition, 44,45 ROS inhibition induced AKT and mTOR activation in all cells ( Fig. 5c and Fig. 5d) . However, on treatment with SARS-CoV-2 spike pseudovirions, phosphorylation/ activation of mTOR and AKT, rather than STAT3 and ERK1/2 signaling, was markedly inhibited in HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells, but this inhibition was reversed by an ROS inhibitor in ACE2-expressing cells ( Fig. 5c and Fig. 5d) ,

indicating that SARS-CoV-2 spike-induced ROS upregulation inhibited PI3K/AKT/mTOR activation. Since both ROS generation and mTOR suppression are positively associated with enhanced autophagy, 46,47 we treated ACE2-expressing cells with SARS-CoV-2 spike pseudovirions along with an ROS inhibitor or MHY1485, an mTOR selective agonist. Thus, we further validated that SARS-CoV-2 spike pseudovirions increased LC3-Ⅱ levels and decreased p62 expression in ACE2-expressing cells (Fig. 5e) . Nevertheless, it was remarkable that SARS-CoV-2 spike pseudovirions infection-induced LC3-Ⅱ increase and P62 decrease were abolished by an ROS inhibitor and MHY1485 (Fig. 5e) . Meanwhile,

( Fig. 5f) , suggesting that SARS-CoV-2 spike pseudovirions may promote autophagy by upregulating intracellular ROS levels and inhibiting the PI3K/AKT/mTOR pathway.

Furthermore, immunofluorescence confirmed that the ability of SARS-CoV-2 spike J o u r n a l P r e -p r o o f pseudovirions infection to induce an increase in LC3 punctas was considerably reduced in the presence of an ROS inhibitor and MHY1485 (Fig. 5f) . Collectively, these data indicate that SARS-CoV-2 spike inhibits the PI3K/AKT/mTOR pathway via inducing ROS upregulation to promote autophagy.

Respiratory infection caused by SARS-CoV-2 usually result in viral pneumonia and acute respiratory distress syndrome. 48, 49 Meanwhile, SARS-CoV-2 infection can also trigger inflammatory responses, which in turn promote the production of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. 49, 50 Since ACE2 was expressed in 16HBE and

inhibiting the PI3K/AKT/mTOR pathway in ACE2-expressing cells, we sought to investigate whether SARS-CoV-2 spike-induced autophagy was involved in inflammatory responses in bronchial epithelial and microvascular endothelial cells. ELISA data indicated that SARS-CoV-2 spike pseudovirions treatment significantly promoted the production of proinflammatory cytokines, IL-6, IL-8, and TNF-α, in HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells. However, treatment with 3-methyladenine (3-MA), an autophagy inhibitor, led to a significant inhibition of SARS-CoV-2 spike pseudovirions-induced production of pro-inflammatory cytokines IL-6, IL-8, and TNF-α in theses ACE2-expressing cells (Fig. 6a-6c ). These results were further confirmed by performing GSEA and RT-PCR analyses of IL-6, IL-8, and TNF-α transcripts ( Fig. 3f and Fig. 6d) . Similarly, RNA-Seq analyses also showed that SARS-CoV-2 spike pseudovirions infection enhanced the expression of proinflammatory cytokines, including TNF and IL6ST, in infected cells (Fig. 3a and Fig. 3c) .

Notably, inflammation is strongly linked to apoptosis, and it can induce apoptosis to promote J o u r n a l P r e -p r o o f cell death. 51 Autophagy can inhibit apoptosis to maintain cell survival. Alternatively, autophagy can also work with apoptosis to induce cell death. 52, 53 To investigate the regulatory effects of SARS-CoV-2 spike-induced autophagy on apoptosis, we treated SARS-CoV-2 spike pseudovirions-treated cells with 3-MA, and we found that SARS-CoV-2 spike pseudovirions upregulated and downregulated Bax and Bcl-2 expression, respectively.

However, this modulation was reversed by 3-MA treatment ( Fig. 6e and Fig. 6f) . Annexin V-FITC/7-AAD double staining and TUNEL staining also verified that SARS-CoV-2 spike pseudovirions-induced apoptosis (the percentage of TUNEL-positive cells) was reversed by 3-MA ( Fig. 6g and Fig. S3) . Altogether, these findings indicate that SARS-CoV-2 spikeinduced autophagy via the ROS-suppressed PI3K/AKT/mTOR axis promotes inflammation and apoptosis in infected cells, which highlights the pathogenic regulatory role of autophagy in SARS-CoV-2 infection (Fig. 6h) . Several RNA viruses can induce autophagic responses in infected cells, which are closely related to viral replication and pathogenesis. 32, 41, 59 Viruses have learned to manipulate the autophagic pathway, exploiting autophagosomes to facilitate viral replication. [60] [61] [62] Meanwhile, some studies have demonstrated that some viral proteins block the fusion of J o u r n a l P r e -p r o o f autophagosomes with lysosomes, thus inhibiting autophagic processes. 63, 64 In viral infections, a balanced mechanism of self-interest may exist, that is, pathogenic proteins, such as spike proteins, trigger autophagy, whereas other accessory proteins carried by the virus may prevent the final step of autophagy and lysosome fusion, thus utilizing autophagy to accumulate viral components. Previous studies suggested that the accessory protein ORF3a encoded by SARS-CoV-2 inhibits the formation of autophagolysosomes by blocking the assembly of the SNARE complex mediated by the HOPS complex. 65 In the present study, we found that SARS-CoV-2 spike proteins induced autophagy through the ROS-suppressed PI3K/AKT/mTOR pathway in infected cells ( Fig. 2 and Fig. 5) , implying that spike protein may induce autophagy initiation and progression, whereas accessory protein ORF3a block the fusion of autophagosomes with lysosomes in SARS-CoV-2-infected cells. However, considering that the current research only focuses on the regulation of autophagic processes by specific proteins of SARS-CoV-2, further studies are required to determine whether multiple proteins of SARS-CoV-2, such as ORF3a and spike proteins, have a balanced mechanism to promote the accumulation of autophagosomes, which is conducive to viral replication.

As microvascular endothelial cells play a crucial role in maintaining body homeostasis, viral infection via ACE-2 receptor usually leads to inflammation and causes vascular dysfunction through microvascular thrombosis, which in turn results in organ failure. 66, 67 In this study, we reported that ACE2 was expressed in human microvascular endothelial cells and that ACE2mediated SARS-CoV-2 spike pseudovirions infection enhanced the inflammatory responses and apoptosis of microvascular endothelial cells ( Fig. 1 and Fig. 6) , indicating that SARS-CoV-2 infection may lead to an extensive apoptosis of microvascular endothelial cells and vascular leakage, eventually causing organ dysfunction. Most importantly, although ACE2 structure is different between humans and mice and cannot mediate SARS-CoV-2 infection in J o u r n a l P r e -p r o o f mice, the tissue specificity of ACE2 in humans and mice may be similar. 68, 69 Using immunohistochemistry, we demonstrated that ACE2 was specifically enriched in epithelial cells of the trachea and pulmonary bronchus and vascular endothelial cells of the lungs and liver ( Fig. 1d and Fig. S1d ). This implies that SARS-CoV-2 infection not only causes an inflammation of the respiratory system but also affects the functions of many organs by destroying microvascular endothelial cells.

ROS are formed by an incomplete one-electron reduction of oxygen and mainly encompass a range of small, short-lived, and highly reactive oxygen-containing molecules, including oxygen anions, free radicals, and peroxides. 70, 71 A majority of viral infections cause an increase in ROS levels in infected cells, and an increasing number of evidence suggests the role of ROS in the pathogenesis of viral infections as a factor for endothelial damage and inflammation. [72] [73] [74] Consistent with previous studies, we found that SARS-CoV-2 spike upregulated intracellular ROS levels and promoted the inflammatory responses in infected cells by inducing autophagy (Fig. 5 and Fig. 6 

This study is the first to reveal that SARS-CoV-2 spike induces autophagy through the 

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

Association of Blood Glucose Control and Outcomes in Patients with COVID-19 and Pre-existing Type 2 Diabetes

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

A Novel Coronavirus from Patients with Pneumonia in China

Itraconazole inhibits the Hedgehog signaling pathway thereby inducing autophagy-mediated apoptosis of colon cancer cells

Rapamycin delays disease onset and prevents PrP plaque deposition in a mouse model of Gerstmann-Straussler-Scheinker disease

Regulation of autophagy by kinases. Cancers (Basel)

Targeting autophagy in cancer

Autophagy in cell death: an innocent convict?

Autophagy protects gastric mucosal epithelial cells from ethanol-induced oxidative damage via mTOR signaling pathway

alpha-hederin induces autophagic cell death in colorectal cancer cells through reactive oxygen species dependent AMPK/mTOR signaling pathway activation

Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Determinants of host susceptibility to murine respiratory syncytial virus (RSV) disease identify a role for the innate immunity scavenger receptor MARCO gene in human infants

Extracellular vesicles from CLEC2-activated platelets enhance dengue virus-induced lethality via CLEC5A/TLR2

COVID-19: consider cytokine storm syndromes and immunosuppression

Statin use is associated with lower disease severity in COVID-19 infection

A parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase 2/3, clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe SARS-CoV-2 infection: the Pyridostigmine In Severe COvid-19 (PISCO) trial protocol

Cytokine Storms: Understanding COVID-19

COVID-19 as an Acute Inflammatory Disease

Regulation of innate immune responses by autophagy-related proteins

TNFAIP3-DEPTOR complex regulates inflammasome secretion through autophagy in ankylosing spondylitis monocytes

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice

Autophagy Supports Breast Cancer Stem Cell Maintenance by Regulating IL6 Secretion

Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta

Translocation of interleukin-1beta into a vesicle intermediate in autophagy-mediated secretion

Autophagy balances inflammation in innate immunity

Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis

Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy

Selective cell death of latently HIV-infected CD4(+) T cells mediated by autosis inducing nanopeptides

Down-regulation of IL-6, IL-8, TNF-alpha and IL-1beta by glucosamine in HaCaT cells, but not in the presence of TNF-alpha

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling

EGFR-rich extracellular vesicles derived from highly metastatic nasopharyngeal carcinoma cells accelerate tumour metastasis through PI3K/AKT pathway-suppressed ROS

CXCL16 Deficiency Attenuates Renal Injury and Fibrosis in Salt-Sensitive Hypertension

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Avian metapneumovirus subgroup C induces autophagy through the ATF6 UPR pathway

Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication

HPV16 E6 and E7 proteins induce a chronic oxidative stress response via NOX2 that causes genomic instability and increased susceptibility to DNA damage in head and neck cancer cells

EV71 virus reduces Nrf2 activation to promote production of reactive oxygen species in infected cells

Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin

ROS-Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A-Sonodynamic Therapy

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease

A pneumonia outbreak associated with a new coronavirus of probable bat origin

COVID-19 and cardiovascular disease: from basic mechanisms to clinical perspectives

The trinity of COVID-19: immunity, inflammation and intervention

Arctigenin Treatment Protects against Brain Damage through an Anti-Inflammatory and Anti-Apoptotic Mechanism after Needle Insertion

Life and death partners: apoptosis, autophagy and the cross-talk between them

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Lung transcriptome of a COVID-19 patient and systems biology predictions suggest impaired surfactant production which may be druggable by surfactant therapy

Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients

Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease

COVID-19 and the cardiovascular system

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells

Autophagy proteins promote hepatitis C virus replication

Autophagy and viruses: adversaries or allies?

Coronavirus NSP6 restricts autophagosome expansion

Autophagy induced by avian reovirus enhances viral replication in chickens at the early stage of infection

Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

Autophagy is involved in influenza A virus replication

ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation

The vascular endothelium: the cornerstone of organ dysfunction in severe SARS-CoV-2 infection

Endothelial cell dysfunction: a major player in SARS-CoV-2 infection (COVID-19)?

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Generation of a Broadly Useful Model for COVID-19

How mitochondria produce reactive oxygen species

Lectin-induced oxidative stress in human platelets

Antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung inflammation

Oxygen radicals, nitric oxide, and peroxynitrite: Redox pathways in molecular medicine

Interactions and Molecular Regulatory Mechanisms

Effect of Waterlogging-Induced Autophagy on Programmed Cell Death in Arabidopsis Roots

Eupatilin induces human renal cancer cell apoptosis via ROS-mediated MAPK and PI3K/AKT signaling pathways

Mitochondrial dysfunction in rheumatoid arthritis: A comprehensive analysis by integrating gene expression, protein-protein interactions and gene ontology data

Gangliosides induce autophagic cell death in astrocytes

A surface-layer protein from Lactobacillus acidophilus NCFM induces autophagic death in HCT116 cells requiring ROS-mediated modulation of mTOR and JNK signaling pathways

Cell viabilities of HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells after 24 h of treatment with SARS-CoV-2 spike pseudovirions were determined with trypan blue staining. SCV-2-S, SARS-CoV-2 spike pseudovirions. All results were collected in three independent experiments

Figure 5. SARS-CoV-2 spike promotes autophagy through the ROS-suppressed

Intracellular ROS levels in HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells treated with or without SARS-CoV-2 spike pseudovirions using (a) fluorescence microscopy (Scale bar, 200 μm) and (b) fluorescence microplate reader. (c) Western blot analysis of phospho-mTOR, mTOR

Vero E6, 16HBE, and HMEC-1 cells treated with or without SARS-CoV-2 spike pseudovirions. (d) Western blot analysis of phospho-mTOR, mTOR, phospho-AKT, AKT, phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2 in SARS-CoV-2-S pseudovirionstreated, ROS-inhibited, and SARS-CoV-2 spike pseudovirions and ROS inhibitor (5 mM)-cotreated HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells. GAPDH was used as the loading control. phospho-mTOR/GAPDH

or MHY 1485 (10 μM). GAPDH was used as the loading control. LC3-Ⅱ/GAPDH, p62/GAPDH, Bcl-2/GAPDH, and Bax/GAPDH densitometric J o u r n a l P r e -p r o o f ratios were recorded. (f) LC3 expression using confocal microscopy in SARS-CoV-2 spike pseudovirions-treated HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells pretreated with DMSO, ROS inhibitor (5 mM), or MHY 1485 (10 μM). Protein expression was determined using LC3-specific antibodies followed by FITC-labeled secondary antibodies (green)

SCV-2-S, SARS-CoV-2 spike pseudovirions

SARS-CoV-2 spike induces inflammation and apoptosis through enhanced autophagy. (a-c) The protein expression levels of IL-6, IL-8, and TNF-α in SARS-CoV-2 spike pseudovirions-infected HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells pretreated with DMSO or 3-MA (10 mM) were quantified using ELISA

Semiquantitative RT-PCR analysis of IL-6, IL-8, and TNF-α expression in SARS-CoV-2 spike pseudovirions-infected HEK293T, HEK293T-hACE2

(e, f) Western blot analysis of Bcl-2 and Bax in SARS-CoV-2 spike pseudovirions-treated, 3-MA-treated, and SARS-CoV-2 spike pseudovirions and 3-MA (10 mM)-treated HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells

Bax/GAPDH densitometric ratios were recorded. (g) Apoptotic cells were determined using

mM)-treated, and SARS-CoV-2 spike pseudovirions and 3-MA (10 mM)-treated HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells. (h) Schematic illustrating the mechanism of SARS-CoV-2 spike to promote inflammation and apoptosis in infected cells

Fei Li planned and carried out experiments, analysed data, and prepared the manuscript

Yunchen Lin, Ying Cai, and Haisheng Chen performed the research and analysed data. Qing Zhang supervised the research

P r e -p r o o f SARS-CoV-2 spike promotes inflammation and apoptosis through autophagy by ROSsuppressed PI3K/AKT/mTOR signaling Highlights

SARS-CoV-2 spike triggers autophagy and apoptosis in ACE2-expressing cells

SARS-CoV-2 spike induces autophagy through ROS-suppressed PI3K/AKT/mTOR pathway

SARS-CoV-2 spike-induced autophagy promotes inflammatory responses and apoptosis

All data generated or analyzed during this study are included in this article. Additional raw data may be available from the corresponding author for reasonable reasons. The RNA-seq raw data have been deposited under the GEO accession number GSE169370.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:J o u r n a l P r e -p r o o f