key: cord-0969071-rocl6k5o authors: Gómez-Icazbalceta, Guillermo; Hussain, Zubair; Vélez-Alavez, Marcela title: In silico evidence of superantigenic features in ORF8 protein from COVID-19 date: 2021-12-14 journal: bioRxiv DOI: 10.1101/2021.12.14.472240 sha: ab157b1d72fab376175169e7e3811129935bc94e doc_id: 969071 cord_uid: rocl6k5o Very early on COVID-19 pandemic outbreak, it was noted that the some of the virus-induced clinical conditions resembled features of toxaemia caused by the toxic shock syndrome toxin type 1, which is a soluble superantigen produced by Staphylococcus aureus. Among all SARS proteins, the ORF8 protein from SARS-2 virus is significantly different from other known SARS-like coronaviruses, and therefore could exhibit unique pathogenic properties. We assess if ORF8 protein bears super antigenic features using in silico tools. We show that ORF8 has properties of an extracellular soluble protein and shares a significant degree of amino acid sequence identity with toxic shock syndrome toxin. Besides, docking and binding affinity analyses between monomeric and homodimeric ORF-8 with Vβ 2.1 and TRBV11-2 reveal strong interaction and high binding affinity. ORF8-TRBV11-2 strong interaction can contribute to the observed clonal expansion of that chain during COVID-19-associated multisystem inflammatory syndrome. Taken together, the evidence presented here supports the hypothesis that ORF8 protein from SARS-2 bears super antigenic properties. It is well established that SARS-2 virus actively replicates in the lungs causing severe pneumonia [1] , along with other related pathologies, whose manifestations differ among age groups and comorbidities presented during infection [2] . However, extrapulmonary conditions overlapping with some features of superantigen-induced toxic shock syndrome (TSS) are also frequently expressed, such as systemic hyper inflammation and coagulation disorders [3] [4] [5] . TSS is caused by the toxic shock syndrome toxin type 1 (TSST), a soluble superantigen (Sag) produced by Staphylococcus aureus [6] . Systemic inflammatory condition during COVID-19 infection, properly referred to as cytokine release syndrome (CRS) [7] , is characterized by the release of elevated levels of inflammatory cytokines, such as IFN-γ, IL-1β, IL-10, IL-6, IL-8, TNFα, MCP-1, and GM-CSF, that induce excessive systemic thromboinflammation, and eventually may lead to disseminated intravascular hypercoagulation, multi-organic failure and a high risk of death. Besides, CRS induced by COVID-19 infection triggers a plethora of systemic immune disorders. More than 50 immune disorders have been described so far [8] . In this regard, a condition referred to as multisystem inflammatory syndrome (MIS), characterized by an immunopathophysiology resembling bacterial toxic shock syndrome has been observed during advanced COVID-19 infection, mainly in pediatric population [9, 10] . During MIS in children (MIS-C), a clonal expansion of the T cell receptor (TCR) β variable gene 11-2 (TRBV11-2) also known as Vβ21.3, is observed, representing up to 24% of the total T cell clonal space [11, 12] . The massive expansion of a single T cell clone is a key feature of bacterial Sag TSST [13] . Also, a cascade of inflammatory cytokines and thrombotic mediators are released, leading to systemic thrombotic inflammation [12] . Taken together, these lines of research indicate that MIS-C has many characteristics of TSS. An in silico research has elucidated a potential superantigen located on the spike protein of COVID-19 virus, that could be able to interact directly with human TCR [14] . Such interaction was discovered to be driven by a peptide high related to enterotoxin B Sag. However, cumulative evidence suggests that thromboimmune exacerbation can be also driven by soluble viral factors acting independently of viral core [15] [16] [17] . We aimed then to discover nonstructural viral proteins that could bear Sag features. An N-terminal signal peptide for COVID-19 ORF8 accessory protein was predicted with high probability by SignalP5 software. The cleavage site was estimated at position 15 (Table 1 and Figure 1 ). The subcellular location of ORF8 was predicted using DeepLoc software. The software predicted ORF8 as an entirely soluble and extracellular protein and bearing a N-terminal signal peptide ( Figure 2 ). The predicted probability of ORF8 for bearing a signal peptide at N-terminal region was nearly 1, while for the rest of the peptide it was zero. The cleavage site was predicted at position 15. Therefore, the N-terminal region is the only region of ORF8 with a putative signal peptide, which is a feature of extracellular proteins. The software also predicted ORF8 as soluble and extracellular protein and gave to each of these properties a score of 1, which is the maximum score. Therefore, ORF8 is predicted to bear properties of a soluble circulating protein. To further determine the properties of ORF8 protein, the secondary structure of ORF8 was predicted using PredictProtein platform ( Table 2 ). The platform predicted ORF8 is constituted by a loop in near 60% of its length. Loops are associated to ligand-receptor interactions and therefore the predicted loop could serve ORF8 to interact with other proteins. Notably, a disulfide bond of 20 aa length was also predicted, spanning from amino acids 68 to 87. The presence of a disulfide bond is a feature of soluble proteins, as it provides resistance to degradation and hence improves their halflifes during exposure to extracellular milieu [42]. To determine if ORF8 shares a degree of sequence similitude with TSST from Staphylococcus aureus, we ran a BLAST analysis using ORF8 as a query. We found that TSST shared 34.29% of amino acid identity with ORF8 (E = 0.049), with a query coverage of 60%, in 3 alignments (Figures 3 and 4) . The shortest alignment, comprising N-terminal region, may belong to signal peptide. The other two alignments spanned aa 32 to 91 of ORF8 and thus may be more related to a superantigen-like function. To obtain additional support to the BLAST findings, we ran a similitude analysis in Clustal Galaxy TBM and Galaxy refine tool is shown in Table 3 . Then, the model quality was evaluated by Verify 3D, ERRAT, and Pro Check. Verify 3D program was used for verification of the correctness of the model ( Figure 6 ). The verified 3D score for ORF8 was 82.64%. Reliability of monomeric ORF8 model were evaluated by ERRAT (Figure 7 ). ERRAT score for the monomeric ORF8 was 83.3%. Finally, Ramachandran plot analyses were carried out by Pro Check. (Figure 8 ). Regarding ORF8, the results showed that 92.5% residues were in the favored region, 7.5% were in allowed region and 0% in disallowed region. The results for quality of monomeric ORF8 by different model evaluation tools were summarized in Table 4 . Overall, the predicted model quality was considered as good and then was used for further analysis. To carry out the docking, binding sites residues were predicted for monomeric ORF8, Vβ chains, the analysis showed that for each Vβ chain, predicted active residues were distributed across the entire molecule. We took the list of common residues from the predicted residues for each protein by SPPIDER and Meta-PPISP and used them as an input parameter for docking, as summarized in table 5. Docking of monomeric and homodimeric ORF8 with Vβ 2.1 and TRBV11-2 were carried out by ClusPro 2.0 webserver by providing the PDB's of the models along with binding site residues. Different clusters were generated by ClusPro 2.0. The best cluster was selected based on maximum numbers of members and more negative energy score. The score for different docked complexes were summarized in table 6. Analysis of interacting residues between monomeric ORF8 and homodimer ORF8 with Vβ 2.1 and TRBV11-2 were carried out in PyMol version 2.4.1. We found that monomeric ORF8 interacted with Vβ 2. Homodimeric ORF8 interacted with TRBV11-2 through its two chains (Figure 10b) Binding affinity of the complexes was analyzed using Prodigy. The analysis showed that all forms of ORF8 were predicted to strongly bind Vβ chains ( probably rendering a non-functional protein (51). These patients exhibited lower levels of inflammatory factors along with milder disease at overall and significantly lower oxygen requirements that wild type infected patients. This study, albeit limited, clearly indicates that ORF8 have an impact in disease severity. In summary, our research establishes that ORF8 contains superantigenic features. Further research will improve our understanding of its role in pathogenesis in order to conceive novel treatments. Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue Host factors facilitating SARS-CoV-2 virus infection and replication in the lungs COVID-19: Pulmonary and Extra Pulmonary Manifestations. 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