COVID-19: Attacks Immune Cells and Interferences With Antigen Presentation Through MHC-Like Decoy System

The high mortality of coronavirus disease 2019 is related to poor antigen presentation and lymphopenia. Cytomegalovirus and the herpes family encode a series of major histocompatibility complex (MHC)-like molecules required for targeted immune responses to achieve immune escape. In this present study, domain search results showed that many proteins of the severe acute respiratory syndrome coronavirus 2 virus had MHC-like domains, which were similar to decoys for the human immune system. MHC-like structures could bind to MHC receptors of immune cells (such as CD4+ T-cell, CD8+ T-cell, and natural killer-cell), interfering with antigen presentation. Then the oxygen free radicals generated by E protein destroyed immune cells after MHC-like of S protein could bind to them. Mutations in the MHC-like region of the viral proteins such as S promoted weaker immune resistance and more robust transmission. S 127–194 were the primary reason for the robust transmission of delta variants. The S 144–162 regulated the formation of S trimer. The mutations of RdRP: G671S and N: D63G of delta variant caused high viral load. S 62–80 of alpha, beta, lambda variants were the important factor for fast-spreading. S 616–676 and 1014–1114 were causes of high mortality for gamma variants infections. These sites were in the MHC-like structure regions.


BACKGROUND
The lymphopenia of coronavirus disease 2019 (COVID-19) patients includes CD4 + T cells, CD8 + T cells, B cells, and natural killer (NK) cells, with the damage of CD8 + T cells being more significant. 1,2 No obvious virus infection is detected in lymphocytes and mesenchymal cell. 3 Lymphopenia at the initial appearance of COVID-19 is associated with poor prognosis. 4 Lymphopenia and its severity are reliable predictors of the clinical outcome of COVID-19, including mortality, intensive care needs, and oxygen requirements. 4 Besides, the high fatality rate of COVID-19 is related to the poor performance of major histocompatibility complex (MHC) II and the low coverage of MHC II. 5 The quality of MHC II presented by T cells is an essential prerequisite for T-cell-dependent antibody production. The binding capacity of MHC I epitope load and severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) peptide affects the immunity of T cells to infection. 6 Therefore, MHC presentation is closely related to lymphopenia of COVID-19.
Lymphopenia, cell degeneration, necrosis, and atrophy are found in SARS and COVID-19 patients. 7 The sort of lymphopenia and apoptosis between SARs and COVID-19 patients appears different. Namely, lymphopenia in SARs patients precedes apoptosis, while apoptosis in COVID-19 patients precedes lymphopenia. 8 The frequency and activation of SARS-COV-2 specific CD8 + T cells increase during severe illness, highlighting differences in T-cell responses associated with disease progression. 9 The number of regulatory T cells (Treg) has nothing to do with the severity of the disease, suggesting that T-cell exhaustion occurs in a process independent of Treg. 10 SARS-COV-2 generates reactive oxygen species (ROS) through the combination of E protein and heme, 11 in which hydroxyl free radicals can directly destroy the cell membrane and cause damage to immune cells. Immune cells would be directly attacked along the route of antigen recognition and antigen presentation. So, CD4 + T cells, CD8 + T cells, and NK cells are most likely to be destroy and apoptosis in this link of binding to MHC molecules.
Mononuclear macrophages ingest antigens and process them into antigen peptides. Then antigen peptides are combined with surface MHC molecules and are expressed on the cell surface, effectively presenting antigens to helper T lymphocytes. B lymphocytes also have a similar antigen presentation effect. T cells combine with MHC II/antigen to activate B cells. While the BCR of the memory B-cell binds to a specific antigen, the antigen is endocytosed by the B-cell. After these antigens are cut into fragments, they return to the cell membrane in a state combined with MHC molecules. 12 T cells express CD4 or CD8 co-receptors. They recognize non-polymorphic regions of MHC protein on target cells and can bind to partial MHC protein regions. 13 Helper T cells express CD4 and recognize MHC class II proteins, while cytotoxic T cells express CD8 and recognize MHC class I proteins. 13 NK cells express inhibitory receptors (KIR) of MHC class I molecules. These inhibitory receptors include the human KIR [killer cell immunoglobulin (Ig)-like receptor]. 14 Another function of NK cells is recognizing and eliminating cells that cannot express their MHC class I molecules. 15 It is interesting to note that, individuals with specific MHC alleles are less susceptible to severe forms of malaria. 13 It means that the combination of immune cells and MHC is closely related to the susceptibility of certain diseases.
The MHC class II transactivator CIITA induces cell resistance to the Ebola virus and SARS-like coronavirus. 16 However, the apparent CD4 conserved residues at the receptor-binding domain (RBD)-S1 site of SARS-COV-2 interrupt the CD4-MHC-II interaction for adaptive immune activation. 17 The immunity of CD8 + T cells to SARS-COV-2 is related to the severity of COVID-19 and virus control. SARS-COV-2 evades CD8 + T-cell surveillance by mutation of the MHC I restricted epitope of CD8 + T cells. 18 Mutant peptides exhibit reduced or abolished MHC I binding, which is related to the loss of recognition and functional response of CD8 + T cells isolated from human leukocyte antigen (HLA)-matched COVID-19 patients. However, the proportion of interferon-γ-producing cells in SARS-COV-2 specific CD8 + T cells expressing PD-1 is higher than that of PD-1 cells in multimer + cells. The SARS-COV-2 specific CD8 + T cells expressing PD-1 are not exhausted and function normally. 19 It meant SARS-COV-2 had evolved an MHC-like structure that could bind to the MHC receptor of immune cells. Immune cells that could not attach to the MHC-like form of the virus had survived.
Some viruses have acquired inhibitors that target the MHC class I antigen presentation pathway. 20 The cytomegalovirus (CMV) and herpes family encode a series of key molecules required for a targeted immune response. 21 All aspects of acute and chronic CMV disease may be controlled by antibodies, NK, and other cells of the innate immune system, as well as CD8 + T and CD4 + T-cell. 22 About half of the identified genes in CMV 23 and beta herpes virus 24 have HCMV homologs. 22 The m144 25 and m145 family of CMV (m17, m145-m158), 22 m157, 26 UL37 27 are all MHC I-like molecules. The Ly49H NK-cell activation receptor recognizes m157. 28 Ly49 receptor binds m157 glycoprotein encoded by mouse CMV (MCMV). 21 Human CMV(HCMV) UL18 binds inhibitory leukocyte immunoglobulin-like receptor R-1. 29 Human CMV express and distribute a complete library of immune evasion factors for a single MHC class I target. 30 Human CMV encodes glycoproteins homologous to MHC class I. 31 The MHC class I homologs encoded by human CMV binds to endogenous peptides. 32 Many viruses have evolved surprising strategies to interfere with the MHC class I antigen presentation pathway. 33 After the initial NK response, 34 the host will produce adaptive CD8 + T 35 and CD4 + T 36 cellular responses. Viral MHC class I molecules allow evasion of NK-cell effector responses in the body 26 and contribute to immune evasion. 22 Many studies have shown that MHC class I virus proteins interfere with infected cells recognizing, antigen processing, and presentation. 22 The specific recognition of MHC by inhibitory KIR provides excellent protection against a decoy molecule of virus evolution. 37 The diversity of the receptor system may be the result of this specific interaction between MHC and KIR molecules. However, NK cells in severely ill patients with COVID-19 are severely depleted. The protective function of inhibitory KIR shows signs of failure. It shows that some regions of human MHC have an irreplaceable role. The MHC-like structures of the virus were precisely in these areas. If a mutation site was in the MHC-like domain, the mutation enhanced the MHC decoy function. In other words, the human immune system hard to neutralize these MHC-like sites by producing antibodies. Otherwise, the antibodies could also bind to MHC proteins. Then the antibodies would affect normal MHC antigen presentation function, causing autoimmune diseases.
The N-terminal domain (NTD) of the S protein and the S2 membrane fusion region may be MHC-like structural sites for the challenging battle between the immune system and the virus. Most antibodies that recognize the SARS-CoV-2 S protein are directed against the RBD. 38 Analysis of the human monoclonal antibody (mAb) library in the sera of convalescent patients showed that most anti-S antibodies recognize RBD, and a small portion of antibodies recognizes NTD. 39 Some NTD-targeted mAbs can effectively inhibit SARS-CoV-2 infection in vitro; in vivo, the immune system uses neutralization and Fc-mediated effector function activities. 40 Fc receptor cells generally include B cells, killer cells, and macrophages. Compared with neutralizing RBD targeting antibodies that recognize multiple nonoverlapping epitopes, effective NTD targeting neutralizing antibodies appear to target a single supersite: 41 N17, N74, N122, and N149. However, popular variants will partially or completely escape the neutralization mediated by human mAbs targeting the antigen supersite (site i). 39 The variants include B.1.1.7, B. 1.35, and P.1 pedigree. It is difficult for immune system antibodies to neutralize part of the mutation sites in the NTD and the fusion region of the S2 membrane.
In this present study, we used the domain search method to find that many proteins of the SARS-COV-2 virus have MHC-like structures. It indicates that SARS-COV-2 interferes with antigen presentation and attacks immune cells through the MHC-like systems. The SARS-COV-2 virus protein with MHC-like forms could interfere with the antigen presentation response by binding to the MHC receptor of immune cells. The SARS-COV-2 virus employees the MHClike structures of the S protein as bait. After the SARS-COV-2 S protein binds to CD4 + T, CD8 + T, and NK cells, the oxygen free radicals (ROS) generated by the E protein destroys these immune cells, resulting in a decrease in the number of lymphocytes. Through the analysis of the MHClike enhanced regions of existing popular variants, we found that: 127-194 and 144-162 areas of S MHC-like of delta variants were in the NTD; the 62-80 regions of S MHC-like of alpha, beta, lambda variants were also in the NTD; the 616-676 and 1014-1114 regions of S MHC-like of gamma variants were in the S2 membrane fusion region.

MHC-Related Sequence
We downloaded 18,112 protein sequences of MHCrelated from the UniProt data set and searched keyword was "MHC." The MHC-related sequences were compared with the viral proteins to search for the conserved domains.

Localized MEME Tool to Scan for Conserved Domains
The analysis steps are listed as follows: (1) Download MEME from the official website and subsequently install in the virtual machine Ubuntu operating system. The virtual machine was VM 15.2.

RESULTS
We downloaded MHC-related sequences from the UniProt database. Then compared these sequences with the SARS-COV-2 protein sequences to find the domains related to MHC function. We merged the motif sequences according to the domains of the search results. Both MHC 1 and MHC 2 structures include Ig-like and MHC domains. If a viral protein could bind to the antigen peptide like the MHC protein, the viral protein would have both domains.

SARS-COV-2 Virus Proteins Had Ig-Like Domains
Ig-like domains are involved in multiple functions, including cell-to-cell recognition, cell surface receptors, muscle structure, and the immune system. We first listed Ig-like domains of viral proteins in Table 1. Table 1 shows the structural proteins (S, E, N, M) and nonstructural proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF10, ORF1ab) of SARS-COV-2 all have Ig-like domains. Ig-like (IPR032165) is a domain composed of ∼100 residues. Smaller domains (74-90 residues) are observed in several Ig-related molecules (CD2, CD4). The Ig-like motifs of ORF10, E , some subprotein of ORF1ab are the short. ORF7a Ig-like A, ORF8 Ig-like A, ORF3a Ig-like C, N Ig-like B and C, M Ig-like A and C, S Iglike B and H, 3′-to-5′ exonuclease C, 3′-to-5′ exonuclease C, helicase B motifs are longer. The Ig-like structures may help the receptor of CD4 + T, CD8 + T, and NK-cell recognize the MHC-like area of the viral proteins.

SARS-COV-2 Virus Proteins had MHC Domains
We listed MHC-like domains of viral proteins in Table 2. Table 2  We downloaded the functional descriptions of the relevant domains from the InterPro database.
The members of MHC_I_2 (PF14586) are called retinoic acid-inducible proteins. They are ligands that activate the immune receptor NKG2D. NKG2D is widely expressed on NK cells, T cells, and macrophages. MHC_I_C (PF06623) represents the C-terminal region of MHC class I antigen. MHC_I-like_Ag-recog (IPR011161) is an MHC class I antigen recognition sample. Class I MHC glycoproteins are expressed on the surface of all somatic nucleated cells, except neurons. MHC class I receptors present peptide antigens synthesized in the cytoplasm, including self-peptides (offered for self-tolerance) and foreign peptides (such as viral proteins). These antigens are produced by degraded protein fragments transported by the TAP protein (antigenic peptide transporter) to the endoplasmic reticulum, where they can bind to MHC I molecules and then transport them to the cell surface through the Golgi apparatus. MHC class I receptors display antigens recognized by cytotoxic T cells that can destroy virus-infected or malignant (self-peptide excess) cells. CD8 + T toxic cells and NK cells can recognize class I MHC proteins.
MHC_II_alpha (SM00920) is the alpha domain of class II histocompatibility antigen. MHC_II_beta (SM00921) is the beta domain of class II histocompatibility antigen. Class II MHC glycoproteins are expressed on the surface of antigen-presenting cells, including macrophages, dendritic cells, and B cells. MHC II protein presents extracellular peptide antigens derived from foreign substances such as bacteria. Proteins from pathogens are degraded into peptide fragments within the antigen-presenting cells. These fragments are sequestered into endosomes to bind to MHC class II proteins before being transported to the cell surface. MHC class II receptors display antigens for recognition by helper T cells and Inflammatory T cells. CD4 + T helper cells recognize MHC class II proteins.
MHC2-interact (PF09307) is the interaction domain of class II invariant chain-related peptides and MHC2. Members of this family are found in class II invariant chainrelated peptides. They are required for binding to the class II MHC in the MHC class II processing pathway. MHCas-soc_trimer (PF08831) is an invariant chain trimerization domain related to class II MHC. The folding and positioning of MHC class II heterodimers require class II-related consistent chain peptides. This domain participates in the trimerization of the ectoderm and interferes with DM/class II binding. The trimeric protein forms a cylindrical shape, which is considered necessary for the interaction between the invariant and class II molecules.
We noticed that S protein could form a trimer structure and had three MHCassoc_trimer domains: MHCassoc_trimer A, B, and C. MHCassoc_trimer A is in S1 protein, but MHCassoc_trimer B and C in S2 protein. It represents that MHCassoc_trimer plays an important role in the formation of S protein trimer.

MHC-like Structures Had a Decoy Function Against the Immune System
Previous analysis shows that structural proteins and nonstructural proteins can bind to T (CD4 + T and CD8 + T) and NK immune cells through MHC-like structures. The binding prevented the MHC receptors of immune cells from securing to MHC, causing interference in antigen presentation. In addition, E protein generates oxygen free radicals (ROS) after attaching to heme, and the hydroxyl free radicals directly damaged cell membranes. 11 After CD4 + T, CD8 + T, and NK cells were bound to the MHC-like structure of S protein, the hydroxyl free radicals generated by E protein destroyed these immune cell membranes. It caused immune cells to die because of oxidative stress. For these two reasons, the MHC-like structure of the SARS-COV-2 virus protein had a decoy function against immune cells. We noticed that N protein could form a multimer, S protein could form a trimer structure, and E could form a pentameric channel structure. The ORF3a protein and ORF8 protein can form a dimer structure, respectively. The dimer of ORF3a protein (or ORF8 protein) has a groove structure. The Ig-like sites of the ORF3a protein do not fully overlap with the MHC-like areas (Tables 1, 2). However, Table 2 shows that the MHC II and MHC I of ORF3a are located at the "CWKCR" heme-binding area 42 and upstream and downstream. But the sites of MHC structures are not near the groove structure on the ORF3a crystal structure view (PDBID: 6xdc). Therefore, ORF3a is unlikely to have the ability to bind antigen peptides.
The Ig-like structure of the transmembrane protein ORF8 overlaps with the MHC-like system. The dimer of ORF8 has no rod-like structure. Table 2 and the crystal structure view of ORF8 (PDBID: 7jtl) show that the MHC II and MHC I structures of ORF8 include sites near the groove structure. Therefore, ORF8 may trapp antigen peptides through the MHC structure and interfering with antigen presentation. Besides, ORF8 captures MHC-1 and reroutes to autophagosomes for degradation. 43 Table 2 indicates that the MHC I-like domain of ORF8 is MHC_I-like_Ag-recog. ORF8 also has the MHCassoc_trimer domain. The MHC II-like domain of ORF8 overlaps with the MHC_I-like_Ag-recog and MHCassoc_trimer structures. So ORF8 may trap MHC I by MHC_I-like_Ag-recog and MHCassoc_trimer domains. Therefore, the MHC-like system of ORF8 has a decoy function for MHC I or antigen peptides.

MHC-like Enhanced Regions of S Protein Mutation
If the S mutation site was in the MHC-like domain, the mutation enhanced the MHC decoy function. Then the human immune system hard to neutralize these MHC-like sites by producing antibodies. Otherwise, it would affect the normal MHC antigen presentation function by combing MHC and the antibodies. On the basis of this principle, we analyzed several significant variants that were now popular to determine the MHC-like enhanced region of the S protein.

SARS-COV-2 Delta Variant
The B.1.617.2/Delta variant is highly confluent, especially in infected hamsters more pathogenic than the prototype SARS-COV-2. 44 The virus is more infectious and directly reduces the efficacy of antibodies produced by infection and vaccines. It is the most prevalent and difficult mutant virus strain in the world.

SARS-COV-2 Beta Variant
The vaccine is effective against the B.  Table 6 shows that D80A is in the MHC_II_beta A domains. Most of the other mutation sites are not in the MHC-like domains. It shows that most of the mutation sites do not affect the MHC-like domain.
Mutation sites G75V, T76I of the SARS-COV-2 C.37/ lambda 51 variant are both in the MHC_II_beta A domain. So S 62-80 of SARS-COV-2 alpha, beta, lambda variants were the first MHC-like enhanced distribution area. The second MHC-like enhanced distribution area of S protein is 127-194, located in the NTD (14-305 residues) of S1 protein. MHCassoc_trimer (144-162) is a trimer of S Formation, which plays an important role. The third and fourth MHC-like enhanced distribution areas of S protein are MHC_I-like_Ag-recog E (616-676), MHC_I-like_Ag-recog G (1014-1114).    The viral load of Delta variant patients is very high. It shows that the replication activity of the Delta variant of the SARS-COV-2 virus is very active. The viral proteins directly related to viral replication activities are orf1ab and N proteins. We searched the orf1ab and N protein mutation sites of five variants of Alpha, Beta, Gama, Delta, and Lambda from "CORONAVIRUS CORONAVIRUS ANTIVIRAL & RESISTANCE DATABASE" (https: // covdb. stanford. edu/ page/ mutation-viewer). Then compared with Table 2 to find the MHC-like enhanced region sites of orf1ab and N proteins ( Table 7). Table 7 shows that the orf1ab and N protein mutation sites of Alpha, Beta, and Gama variants are not in the MHC-like region. The N protein mutation site of the Lambda variant is also not in the MHC-like area. However, the mutation sites of Delta (orf1ab and N protein) and Lambda (orf1ab protein) are in the MHC-like region. The RdRP: G671S mutation site of Delta variant orf1ab is at MHC_I-like_Ag-recog C. The nsp3:F1569V of Delta variant orf1ab is at MHC_II_beta B and MHC_I-like_Agrecog E. The D63G of Dalta variant N is in MHC_II_beta A, MHC_I-like_Ag-recog A. Both N and RNA-dependent RNA polymerase are directly related to virus replication. In addition, the interaction between N and Nsp3 is essential for connecting the viral genome for processing. So, Table 7 indicates that the mutations of RdRP : G671S and N : D63G enhanced the immune escape ability of the Delta variant virus during the replication process. Therefore, the replication activity for this variant is very active.

Genetic Variation of MHC Protected Immune Cells
The MHC molecule is a cell surface protein complex encoded in the HLA locus. 52 The genetic variation of the three MHC class I genes (HLA-A, -B, and -C genes) affect the susceptibility and severity of COVID-19 disease. 53 The HLA gene complex is closely linked to genes, and there is little exchange between homologous chromosomes. HLA loci located on the same chromosome constitute a closely linked gene group (including HLA-I and II genes), called haplotype or haplotype. A haplotype is inherited as a unit. To the offspring, it is called haplotype genetics. However, HLA haplotypes are not the main risk/protective factor for SARS-COV-2 infection or severity in the Israeli population. 54 It indicates that the genetic variation of MHC structure may protect immune cells that can bind to MHC to a certain extent.
In this present study, we found that many proteins of the SARS-COV-2 virus have MHC-like structures recognized by MHC receptors. CD4 or CD8 co-receptors expressed by T cells can bind to part of MHC proteins. 13 The inhibitory receptors of NK cells can also bind to the MHC-1 receptor recognition structure. Therefore, the S protein of the SARS-COV-2 virus could bind to CD4 + T, CD8 + T, and NK cells through MHC-like structures. Then the ROS generated by the E protein destroyed these immune cells, 11 resulting in a decrease in lymphocytes. The genetic variation of HLA may produce MHC molecules that could not bind to the viral MHC-like structure. It was helpful for immune cells to evade the attachment and positioning of