A Way to the Cure for Corona

A study/ research by:

Mukul Sharma

Corona Virus (COVID-19)

Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is caused also by certain other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19.

Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. This is wrapped in a icosahedral protein shell. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, one of the largest among RNA viruses. They have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives.

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Replication cycle�

• This morphology is created by the viral spike peplomers. Infection begins when the viral spike (S) glycoprotein attaches to its complementary host cell receptor. After attachment, a protease of the host cell cleaves and activates the receptor-attached spike protein. Depending on the host cell protease available, cleavage and activation allows the virus to enter the host cell by endocytosis or direct fusion of the viral envelop with the host membrane.
• On entry into the host cell, the virus particle is uncoated, and its genome enters the cell cytoplasm. The coronavirus RNA genome has a 5′ methylated cap and a 3′ polyadenylated tail, which allows the RNA to attach to the host cell's ribosome for translation. The host ribosome translates the initial overlapping open reading frame of the virus genome and forms a long polyprotein. The polyprotein has its own proteases which cleave the polyprotein into multiple nonstructural proteins.

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Replication�

• A number of the nonstructural proteins coalesce to form a multi-protein replicase-transcriptase complex (RTC). The main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). It is directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease nonstructural protein, for instance, provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks.
• One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA.^] The other important function of the complex is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. This is followed by the transcription of these negative-sense subgenomic RNA molecules to their corresponding positive-sense mRNAs.

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Release�

The replicated positive-sense genomic RNA becomes the genome of the progeny viruses. The mRNAs are gene transcripts of the last third of the virus genome after the initial overlapping reading frame. These mRNAs are translated by the host's ribosomes into the structural proteins and a number of accessory proteins. RNA translation occurs inside the endoplasmic reticulum. The viral structural proteins S, E, and M move along the secretory pathway into the Golgi intermediate compartment. There, the M proteins direct most protein-protein interactions required for assembly of viruses following its binding to the nucleocapsid. Progeny viruses are then released from the host cell by exocytosis through secretory vesicles.

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Transmission�

Infected carriers are able to shed viruses into the environment. The interaction of the coronavirus spike protein with its complementary cell receptor is central in determining the tissue tropism, infectivity, and species range of the released virus. Coronaviruses mainly target epithelial cells. They are transmitted from one host to another host, depending on the coronavirus species, by either an aerosol, fomite, or fecal-oral route. Human coronaviruses infect the epithelial cells of the respiratory tract, while animal coronaviruses generally infect the epithelial cells of the digestive tract. SARS coronavirus, for example, infects via an aerosol route, the human epithelial cells of the lungs by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Transmissible gastroenteritis coronavirus (TGEV) infects, via a fecal-oral route, the pig epithelial cells of the digestive tract by binding to the alanine aminopeptidase (APN) receptor.

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Coronavirus proteins

• Spike glycoprotein

Spike protein S1 :- Attaches the virion to the cell membrane by interacting with host receptor, initiating the infection (By similarity). Binding to human ACE2 and CLEC4M/DC-SIGNR receptors and internalization of the virus into the endosomes of the host cell induces conformational changes in the S glycoprotein. Proteolysis by cathepsin CTSL may unmask the fusion peptide of S2 and activate membranes fusion within endosomes.

Spike protein S2 :- Mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and target cell membrane fusion, the coiled coil regions (heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes.

Spike protein S2’ :- Acts as a viral fusion peptide which is unmasked following S2 cleavage occurring upon virus endocytosis.

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• M protein

The coronavirus M protein (formerly called E1) is a multiple membrane-spanning protein containing 221–262 amino acids and it is the most abundant protein in the virus envelope. M protein consists of a short (∼ 25 amino acids for MHV) hydrophilic glycosylated N-terminal domain that is exposed on the external surface of the virion, followed by three transmembrane domains followed by a long C-terminal tail that is positioned in the interior of the virus.

A role of M protein in the induction of an interferon response to coronavirus infection was first demonstrated for TGEV, where the induction of interferon-α in PBMCs by glutaraldehyde-fixed purified virus or virus-infected cells could be blocked by monoclonal antibodies that recognized the N-terminal exposed M protein ectodomain. 

• N protein

The nucleocapsid protein (N), a basic RNA-binding protein encoded in the most 3′ portion of the MHV genome, plays both structural and nonstructural roles in infection. N complexes with genome RNA to form the viral capsid and interacts with the viral membrane protein (M) during assembly as described above. In addition, N associates with genomic and subgenomic messenger RNA, binding specifically to the TRS and significantly enhances recovery of infectious virus from transfected genome length synthetic RNA. It was recently demonstrated that N protein is associated with replication-transcription complexes in infected cells and that recruitment of N to these complexes requires the C-terminal N2b domain, which interacts with other N proteins. Interestingly, N is unique among MHV structural proteins in that it is partially localized to the nucleus of infected cells.

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• E protein

Coronaviruses (CoVs) are enveloped viruses with positive-sense, single-stranded RNA genomes that infect avian and mammalian species. These viruses cause about 20% of common colds in humans.

Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection.

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• Homo sapiens (Human)NKG2D protein

NKG2D is a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. NKG2D is encoded by KLRK1 gene which is located in the NK-gene complex (NKC) situated on chromosome 6 in mice and chromosome 12 in humans. In mice, it is expressed by NK cells, NK1.1⁺ T cells, γδ T cells, activated CD8⁺ αβ T cells and activated macrophages. In humans, it is expressed by NK cells, γδ T cells and CD8⁺ αβ T cells. NKG2D recognizes induced-self proteins from MIC and RAET1/ULBP families which appear on the surface of stressed, malignant transformed, and infected cells.

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NKG2D ligands�

• Human NKG2D receptor complex assembles into a hexameric structure. NKG2D itself forms a homodimer whose ectodomains serve for ligand binding. Each NKG2D monomer is associated with DAP10 dimer. This association is maintained by ionic interaction of a positively charged arginine present in a transmembrane segment of NKG2D and negatively charged aspartic acids within both transmembrane regions of DAP10 dimer. DAP10 functions as an adaptor protein and transduces the signal after the ligand binding by recruiting the p85 subunit of PI3K and Grb2-Vav1 complex which are responsible for subsequent downstream events.
• NKG2D ligands are induced-self proteins which are completely absent or present only at low levels on surface of normal cells, but they are overexpressed by infected, transformed, senescent and stressed cells. Their expression is regulated at different stages (transcription, mRNA and protein stabilization, cleavage from the cell surface) by various stress pathways. Among them, one of the most prominent stress pathways is DNA damage response. Genotoxic stress, stalled DNA replication, poorly regulated cell proliferation in tumorigenesis, viral replication or some viral products activate the ATM and ATR kinases. These kinases initiate the DNA damage response pathway which participates in NKG2D ligand upregulation. DNA damage response thus participate in alerting the immune system to the presence of potentially dangerous cells.

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Function & Role

NKG2D is a major recognition receptor for the detection and elimination of transformed and infected cells as its ligands are induced during cellular stress, either as a result of infection or genomic stress such as in cancer. In NK cells, NKG2D serves as an activating receptor, which itself is able to trigger cytotoxicity. The function of NKG2D on CD8⁺ T cells is to send co-stimulatory signals to activate them. Some autoreactive or immunosupprossive CD4+ T Cell.

The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells.

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NKG2D: A Master Regulator of Immune Cell Responsiveness

NKG2D is an activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system. Ligands of NKG2D are normally of low abundance, but can be induced in virtually any cell in response to stressors, such as infection and oncogenic transformation. Engagement of NKG2D stimulates the production of cytokines and cytotoxic molecules and traditionally this receptor is, therefore, viewed as a molecule that mediates direct responses against cellular threats.

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Process for Polymerization of NKG2D Protein

• Tubulin polymerization-promoting protein
• Tubulin polymerization-promoting protein family member 2
• Tubulin polymerization-promoting protein family member 3
• Chondroitin polymerizing factor [ Homo sapiens (human) ]
• Cancer isolated cell
•  Sickle-cell Haemoglobin

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These are six options to build a vaccine. Choose the best process among them that will be more efficient and workable.

Interferon Type I�

Human type I interferons (IFNs) are a large subgroup of interferon proteins that help regulate the activity of the immune system.Interferons bind to interferon receptors. All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.

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Mammalian types

The mammalian types are designated IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

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• IFN-α

The IFN-α proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection. The genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.

IFN-α is also made synthetically as medication in hairy cell leukemia. The International Nonproprietary Name (INN) for the product is interferon alfa. The recombinant type is interferon alfacon-1. The pegylated types are pegylated interferon alfa-2a and pegylated interferon alfa-2b.

• IFN-β

The IFN-β proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN-β have been described, IFN-β1 (IFNB1) and IFN-β3 (IFNB3)^[5] (a gene designated IFN-β2 is actually IL-6). IFN-β1 is used as a treatment for multiple sclerosis as it reduces the relapse rate.

IFN-β1 is not an appropriate treatment for patients with progressive, non-relapsing forms of multiple sclerosis.

• IFN-ε, -κ, -τ, -δ and -ζ

IFN-ε, -κ, -τ, and -ζ appear, at this time, to come in a single isoform in humans, IFNK. Only ruminants encode IFN-τ, a variant of IFN-ω. So far, IFN-ζ is only found in mice, while a structural homolog, IFN-δ is found in a diverse array of non-primate and non-rodent placental mammals. Most but not all placental mammals encode functional IFN-ε and IFN-κ genes.

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• IFN-ω

IFN-ω, although having only one functional form described to date (IFNW1), has several pseudogenes: IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and IFNWP19 in humans. Many non-primate placental mammals express multiple IFN-ω subtypes.

• IFN-Y

This subtype of type I IFN was recently described as a pseudogene in human, but potentially functional in the domestic cat genome. In all other genomes of non-feline placental mammals, IFN-Y is a pseudogene; in some species, the pseudogene is well preserved, while in others, it is badly mutilated or is undetectable. Moreover, in the cat genome, the IFN-Y promoter is deleteriously mutated. It is likely that the IFN-Y gene family was rendered useless prior to mammalian diversification. Its presence on the edge of the type I IFN locus in mammals may have shielded it from obliteration, allowing its detection.

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Sources and functions�

IFN-α and IFN-β are secreted by many cell types including lymphocytes (NK cells, B-cells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others. They stimulate both macrophages and NK cells to elicit an anti-viral response, involving IRF3/IRF7 antiviral pathways, and are also active against tumors. Plasmacytoid dendritic cells have been identified as being the most potent producers of type I IFNs in response to antigen, and have thus been coined natural IFN producing cells.

IFN-ω is released by leukocytes at the site of viral infection or tumors.

IFN-α acts as a pyrogenic factor by altering the activity of thermosensitive neurons in the hypothalamus thus causing fever. It does this by binding to opioid receptors and eliciting the release of prostaglandin-E₂ (PGE₂).

A similar mechanism is used by IFN-α to reduce pain; IFN-α interacts with the μ-opioid receptor to act as an analgesic.

In mice, IFN-β inhibits immune cell production of growth factors, thereby slowing tumor growth, and inhibits other cells from producing vessel producing growth factors, thereby blocking tumor angiogenesis and hindering the tumour from connecting into the blood vessel system.

In both mice and human, negative regulation of type I interferon is known to be important. Few endogenous regulators have been found to elicit this important regulatory function, such as SOCS1 and Aryl Hydrocarbon Receptor Interacting Protein (AIP).

OBJECTIVE

The polymerization of the NKG2D protein can play a critical role in developing an effective vaccine against COVID-19. This approach leverages the immunological potential of NKG2D to promote a robust immune response, complemented by the immunoregulatory support of interferons (IFNs).

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Main Role Of These

• This process focuses on the polymerization of the NKG2D protein to enhance its stability and antigenicity, enabling the production of a sufficient amount of antibodies against the coronavirus.
• NKG2D acts by targeting and destroying virus-infected cells directly or mimicking antibody-like activity. It boosts immune responses by activating natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), promoting the elimination of infected cells and enhancing overall immunity.
• Interferons serve as adjuvant support, regulating the immune system's activity and aiding in the recovery process by inhibiting viral replication and promoting antiviral responses.

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