key: cord-0748892-kfn2q7wt authors: Rosa-Fernandes, Livia; Lazari, Lucas C.; da Silva, Janaina Macedo; de Morais Gomes, Vinicius; Machado, Rafael Rahal Guaragna; dos Santos, Ancely Ferreira; Araujo, Danielle Bastos; Coutinho, João Vitor Paccini; Arini, Gabriel Santos; Angeli, Claudia B.; de Souza, Edmarcia E.; Wrenger, Carsten; Marinho, Claudio R. F.; Oliveira, Danielle B. L.; Durigon, Edison L.; Labriola, Leticia; Palmisano, Giuseppe title: SARS-CoV-2 activates ER stress and Unfolded protein response date: 2021-06-21 journal: bioRxiv DOI: 10.1101/2021.06.21.449284 sha: f3b3eca58bbce0b038bf6f50fcda858c09ff3e36 doc_id: 748892 cord_uid: kfn2q7wt Coronavirus disease-2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus infection is a major global public health concern affecting millions of people worldwide. The scientific community has joint efforts to provide effective and rapid solutions to this disease. Knowing the molecular, transmission and clinical features of this disease is of paramount importance to develop effective therapeutic and diagnostic tools. Here, we provide evidence that SARS-CoV-2 hijacks the glycosylation biosynthetic, ER-stress and UPR machineries for viral replication using a time-resolved (0-48 hours post infection, hpi) total, membrane as well as glycoproteome mapping and orthogonal validation. We found that SARS-CoV-2 induces ER stress and UPR is observed in Vero and Calu-3 cell lines with activation of the PERK-eIF2α-ATF4-CHOP signaling pathway. ER-associated protein upregulation was detected in lung biopsies of COVID-19 patients and associated with survival. At later time points, cell death mechanisms are triggered. The data show that ER stress and UPR pathways are required for SARS-CoV-2 infection, therefore representing a potential target to develop/implement anti-CoVID-19 drugs. COVID-19 4 . The host-pathogen dynamics is the key to infection control and 59 minimize spread, incidence, prevalence and mortality [5] [6] [7] [8] . In the host cell, viral 60 lead to upregulation of genes encoding for ER chaperones and components 92 necessary for degradation of unfolded proteins 17 . 93 Recognizing the biomolecular features that facilitate infection and which 94 host-mediated mechanisms the pathogen uses to favor its replication and 95 transmission are substantial to achieve disease control and prevention 18 . 96 Quantifying and analyzing the temporal changes in host and viral proteins over 19 patients and associated with higher survival. We also show that sustained 104 infection prolonged the effects of ER-stress and UPR, leading to cell death related 105 to necroptosis and caspase induced apoptosis pathways. Cell lines, SARS-CoV-2 and infection assays 108 Vero cell line (ATCC CCL-81) were maintained in DMEM medium supplemented 109 with 10% (v/v) FBS, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 110 100 U/mL penicillin-streptomycin and 1.5 g/L NaHCO3 at 37°C with 5% CO2. Microsomal membrane proteome analysis (cell lysis and trypsin digestion) 148 Microsomal membrane protein fraction was isolated as previously described 23 To identify molecular pathways affected by viral-host interplay on the course of were grouped into clusters associated with early, middle, and late events, respectively. 309 Representation of enriched biological processes (BP) per cluster (q-value <0.05) (F). (Supplementary Data Set 3) . 369 By performing an integrated analysis of all MS-based approaches, we identified 370 the formation of four clusters (Figures 4A and B, Supplementary Data Set 3) , 371 demonstrating again that ER-related processes and cell death are being regulated 372 during infection (Figure 4C and D, Supplementary Data Set 3) . ER-related (Figures 5A-D, Supplementary Figures 3 and 4) . These results 425 further confirmed that this UPR pathway was activated by the virus (Figure 5A -426 D, Supplementary Figures 3 and 4) . In addition, higher levels of ATF6 and 427 phosphorylated IRE1α were seen only after 48h of infection ( Figure 5E , 428 Supplementary Figures 3 and 4) . Interestingly, phosphorylated IRE1α was not (Figure 5G, Supplementary Figures 3 and 4) . The Figures 3 and 4) . Interestingly, the proteomic data evidenced (Figure 6B) . PCA analysis showed diverse transcriptome profile 494 between COVID-19 patients and controls. (Figure 6C) . We observed that 495 differentially regulated transcripts were involved in several processes linked to 496 ER stress, such as cell death, chaperone-mediated folding, 'de novo' protein 497 folding, protein localization to ER, programmed cell death, and protein folding, 498 confirming the proteomic data (Figures 6G and H) . Mapping ER-stress 499 transcripts and proteins in clinical specimens from patients infected with SARS-500 CoV-2, it was possible to identify that RCN3, UCHL1, and ERO1A are upregulated 501 in the lung at the level of transcript and proteome 37 . Moreover, we found 51 up-502 regulated and 45 down-regulated confirming the alteration of the host 503 glycosylation biosynthetic machinery upon SARS-CoV-2 infection (Figure 6F) . 504 Interestingly, hierarchal clustering analysis showed that the infected samples 1, hpi. Such pattern was also recently reported in the total proteome analysis 557 performed by Stukalov and collaborators (2021) . Although they did not analyze 558 the proteomic profile at 48 hpi, they observed that proteins were mostly down-559 regulated at 12 and 24 hpi but being up-regulated at 6 hpi 48 . Since it has already 560 been shown that viral replication starts at 6 hpi 57,58 , the observed global protein 561 up-regulation at 6 hpi could imply an initial response from the cell, followed by . In addition, other SARS-Cov-2 proteins were found to interact with proteins 654 involved in ER protein quality control, ER morphology and protein glycosylation 6 . Cell surface GRP78 was identified to interact with the Middle East respiratory 656 syndrome coronavirus spike glycoprotein and increase the viral entry 88 . Furthermore, SARS-CoV S glycoprotein was found to bind calnexin and increase 658 its infectivity by modulating the maturation of the glycans 89 . Another host-virus 659 protein-protein interaction analysis also pointed ER stress as being one of the It has been shown prolonged ER stress can activate apoptosis pathway, which 679 will conclude with the assembly of the apoptosome and caspase-3 activation 96 . 680 We observed that apoptotic-related processes were mainly modulated in the late 681 time events, indicating that cell death may be more frequent at 48 hpi. Additionally, the enriched pathway analysis showed that these processes started 683 at 12 hpi and remained active at 24 hpi. Although ER stress is predominantly the 684 main cause of stress observed in this study, viruses have been shown to induce 685 oxidative stress by ROS production in to facilitate their replication in the host cell 686 39, 40 . Viral infections can induce the release of pro-oxidant cytokines such as the 687 tumor necrosis factor (TNF), which lately will produce the hydroxyl radical OH 97 . 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