Can molecular mimicry explain the cytokine storm of SARS‐CoV‐2?: An in silico approach

Abstract PARP14 and PARP9 play a key role in macrophage immune regulation. SARS‐CoV‐2 is an emerging viral disease that triggers hyper‐inflammation known as a cytokine storm. In this study, using in silico tools, we hypothesize about the immunological phenomena of molecular mimicry between SARS‐CoV‐2 Nsp3 and the human PARP14 and PARP9. The results showed an epitope of SARS‐CoV‐2 Nsp3 protein that contains consensus sequences for both human PARP14 and PARP9 that are antigens for MHC Classes 1 and 2, which can potentially induce an immune response against human PARP14 and PARP9; while its depletion causes a hyper‐inflammatory state in SARS‐CoV‐2 patients.


| INTRODUCTION
SARS-COV-2 was described in December 2019 as being the result of zoonotic transmission from wild animals traded in the Wuhan market 1 to humans presenting primarily symptoms such as fever, nonproductive cough, and myalgia or fatigue, normal or decreased leukocyte counts, and radiographic evidence of pneumonia. 2  Syndrome -CoV (SARS-CoV) genome, 3 and researchers changed the initial name to Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). 4 In a year, by March 21, 2021, 5 this new disease produced a pandemic with 122.524 million cases and over 2.7 million deaths worldwide.
The SARS-CoV-2 is an enveloped positive-sense RNA virus belonging to the Coronaviridae, genus Betacoronavirus 6,7 family. The SARS-CoV-2 genome contains 14 open reading frames (ORFs), 4 encoded structural proteins, spikes (S), a membrane (M), an envelope (E), and a nucleocapsid (N) that constitute a protective shell surrounding the genetic material. 8 Other remaining proteins, such as the NsP1-16 and 9 other accessory proteins enhance its virulence. 8 The nonstructural protein 3 (Nsp3) is an important protein for the virus to process viral polyproteins and build a fully functional complex allowing viral propagation. 9 Another function of the Nsp3 is its significant role in regulating the host's inflammatory and immune response. 1 Several studies demonstrate that the Nsp3 and the human poly-adenosine diphosphate-ribose polymerase 9 (parp9) 10 and parp14 1 have identical residues that could produce molecular mimicry, leading to leukopenia and an altered inflammatory response. 1,10 This condition can be explained by a cytokine storm state, related to the macrophage activation syndrome (MAS). 11,12 The PARPs are a family of important enzymes that catalyze posttranslational ribosylation modification of proteins using NAD + as a substrate to carry out mono or poly ADP-ribosylation modification on target proteins to trigger many processes of cellular metabolism, such as DNA repair, 13 regulation of disease pathogenesis, 14 modulation of immune response 1 and is involved in viral infections. 15 Seventeen PARP family members have been described, 16 the most important in immune regulation being PARP14 and PARP9. 13,17 PARP14 increases IL-4 induced cytokine through STAT6, 14 responsible for anti-inflammatory macrophage activation (M2), 17 immune homeostasis, 1,13 tissue injury and inflammatory macrophage regeneration and regulation (M1). 17 PARP9 as PARP14 is responsible for macrophage activation. 17 In the SARS-COV-2 disease, a molecular mimicry phenomenon has been observed, and two recent studies have reported this phenomenon between viral proteins against PARP9 10 and PARP14. 18 The aim of this study is to analyze the molecular mimicry phenomena using in silico tools between SARS-Cov-2 and human proteins.

| Prediction of B cell, cytotoxic T lymphocytes, and helper T lymphocytes epitopes
The B-cell epitopes were predicted using the BepiPred server and the Elliprot server to examine the epitope position in a 3D structure. 21 Cytotoxic T lymphocyte (CTL) epitopes were predicted using the IEDB MHC I algorithm (http://tools.iedb.org/mhci) and helper T lymphocyte (HTL) epitopes were predicted using the MHC II binding prediction tools (http://tools.iedb.org/mhcii). The antigenic properties of the epitopes were studied using the Vaxijen 2.0 server at a threshold of 0.4. Peptide toxicity was predicted from the ToxinPred server (http://crdd.osdd.net/ raghava/toxinpred/), and allergenicity was predicted from the AllegernFP 1.0 server (http://ddg-pharmfac.net/AllergenFP/). All of these analyses were taken into account to select the epitopes.

| Hydrophobic and antigenic protein analysis
The hydrophobic and antigenic analysis was performed using the Kyte-Doolittle and Jameson-Wolf algorithms of human and viral protein.
To determine whether the epitope found was located on the outer surface of the protein where antigen-antibody formation occurs, an overlay of the predicted epitopes and the results of hydrophobic and antigenic analysis was performed. 22 The DNASTAR Protean program was used for this method.

| Protein modeling and molecular docking
The three-dimensional modeling of PARP9 was performed using the I-TASSER online server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), while the Z-score was used to verify the quality of the 3D protein modeling to select the best model. 23 Human PARP14 and Sars-Cov-2 Nsp3 were extracted from PDB (3Q6Z and 6WEY, respectively).
In addition, the 3D structure of the selected epitopes was modeled with the PEPFOLD 3 server. 24 26 The HawKRank server was used for scoring. 27 3 | RESULTS for human PARP14 and PARP9 respectively (Table 1 and Figure 1).

| Prediction of B cell, cytotoxic T lymphocytes, and helper T lymphocytes epitopes
The predicted epitope PTVVVNAANVYLKHGGGVAGAL of Nsp3  Figure 2). BepiPred showed that the epitopes have a large gradient and that the surface of the structure is exposed ( Table 2). As mentioned, the predicted epitope of SARS-CoV-2, for CTL and HTL cells shows predicted epitopes with a high antigenic and allergenic property capable of inducing a large autoimmune response once recognized by the MHC Class I and MHC Class II allele numbers ( Table 2).

| Hydrophobic and antigenicity prediction
Hydrophobic and antigenic analyses of SARS-CoV-2 Nsp3 protein and human Parp14/Parp9 for the consensus amino acid sequence are Parp14 epitope framed in green and Parp9 epitope framed in blue were selected with prediction of B cell, cytotoxic T lymphocytes, and helper T lymphocytes epitopes tools F I G U R E 2 SARS-CoV-2 Nsp3 protein (PDB 6WEY) with consensus sequences with Parp9 in blue (A) and Parp14 in red (B). Human Parp9 (C) and human Parp14 (D) with consensus sequences in red. All epitopes are exposed shown in Table 3. This data showed that the amino acids of Nsp3,

| DISCUSSION
SARS-CoV-2 is a new and serious infectious disease that affects the entire world, threatening the health and life of the human population. 28 The infection produced by the virus results in a strong immune response that releases large amounts of cytokines and chemokines, a phenomenon known as cytokine storm, exhibiting systemic hyper-inflammation, leading to a high incidence of immune disorders, organ failure, and mortality. 11,29,30 This systemic hyperinflammatory condition is known as MAS that can be associated with SARS-CoV-2 pneumonia and its exacerbation. 11,12 MAS can occur in severe infections caused by a wide variety of bacterial, fungal, protozoal, rickettsial, and viral pathogens, [31][32][33] is also found in patients with severe sepsis with a high risk of mortality. 34 In patients with severe SARS-CoV-2, the MAS profile related to inflammatory macrophage M1 35  This in silico study showed that the SARS-CoV-2 Nsp3 protein, is critical for the virus replication in cells, 40  which is responsible for the SARS-CoV-2 related macrophage activation syndrome, however, the presence of this cytokine produces the decrease of NK and CD8 + T cells, 28,47 a condition found in patients with severe SARS-CoV-2 infections. 28,48,49 This scenario produces in the SARS-CoV-2 patient a state of constant hyper-inflammation, without macrophage immune regulation. The combination of the decrease of PARP14/PARP9 responsible for the polarization of anti-inflammatory macrophages (M2) and a weak host's viral response due to the decrease of NK and CD8 + cells will lead to a fatal outcome for the patient.
The results of this study are in agreement with previous studies denoting the presence of molecular mimicry in SARS-CoV-2 between PARP14/PARP9 and the viral proteome 10,18 and its importance in the SARS-CoV-2 infection. 50

| CONCLUSION
Within the limits of this study, it can be assumed that, in patients with severe SARS-CoV-2 infections, a molecular mimicry phenomenon of human PARP14 and PARP9 may be present leading to a hyperinflammatory state due to the macrophage activation syndrome known as cytokine storm-related to SARS-CoV-2.

ACKNOWLEDGMENTS
The author thanks Miguel Chavez Fumagalli for the extensive review of this study. This study is supported by Vicerrectorado de Investigación of Universidad Católica de Santa María, Arequipa, Perú.