Roles and functions of SARS-CoV-2 proteins in host immune evasion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the host immune system through a variety of regulatory mechanisms. The genome of SARS-CoV-2 encodes 16 non-structural proteins (NSPs), four structural proteins, and nine accessory proteins that play indispensable roles to suppress the production and signaling of type I and III interferons (IFNs). In this review, we discussed the functions and the underlying mechanisms of different proteins of SARS-CoV-2 that evade the host immune system by suppressing the IFN-ß production and TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3)/signal transducer and activator of transcription (STAT)1 and STAT2 phosphorylation. We also described different viral proteins inhibiting the nuclear translocation of IRF3, nuclear factor-κB (NF-κB), and STATs. To date, the following proteins of SARS-CoV-2 including NSP1, NSP6, NSP8, NSP12, NSP13, NSP14, NSP15, open reading frame (ORF)3a, ORF6, ORF8, ORF9b, ORF10, and Membrane (M) protein have been well studied. However, the detailed mechanisms of immune evasion by NSP5, ORF3b, ORF9c, and Nucleocapsid (N) proteins are not well elucidated. Additionally, we also elaborated the perspectives of SARS-CoV-2 proteins.

Roles and functions of SARS-CoV-2 proteins in host immune evasion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the host immune system through a variety of regulatory mechanisms. The genome of SARS-CoV-2 encodes 16 non-structural proteins (NSPs), four structural proteins, and nine accessory proteins that play indispensable roles to suppress the production and signaling of type I and III interferons (IFNs). In this review, we discussed the functions and the underlying mechanisms of different proteins of SARS-CoV-2 that evade the host immune system by suppressing the IFN-β production and TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3)/signal transducer and activator of transcription (STAT)1 and STAT2 phosphorylation. We also described different viral proteins inhibiting the nuclear translocation of IRF3, nuclear factor-κB (NF-κB), and STATs. To date, the following proteins of SARS-CoV-2 including NSP1, NSP6, NSP8, NSP12, NSP13, NSP14, NSP15, open reading frame (ORF)3a, ORF6, ORF8, ORF9b, ORF10, and Membrane (M) protein have been well studied. However, the detailed mechanisms of immune evasion by NSP5, ORF3b, ORF9c, and Nucleocapsid (N) proteins are not well elucidated. Additionally, we also elaborated the perspectives of SARS-CoV-2 proteins.

Structural Analysis on the Severe Acute Respiratory Syndrome Coronavirus 2 Non-structural Protein 13 Mutants Revealed Altered Bonding Network With TANK Binding Kinase 1 to Evade Host Immune System.

Mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made this virus more infectious. Previous studies have confirmed that non-structural protein 13 (NSP13) plays an important role in immune evasion by physically interacting with TANK binding kinase 1 (TBK1) to inhibit IFNß production. Mutations have been reported in NSP13; hence, in the current study, biophysical and structural modeling methodologies were adapted to dissect the influence of major mutations in NSP13, i.e., P77L, Q88H, D260Y, E341D, and M429I, on its binding to the TBK1 and to escape the human immune system. The results revealed that these mutations significantly affected the binding of NSP13 and TBK1 by altering the hydrogen bonding network and dynamic structural features. The stability, flexibility, and compactness of these mutants displayed different dynamic features, which are the basis for immune evasion. Moreover, the binding was further validated using the MM/GBSA approach, revealing that these mutations have higher binding energies than the wild-type (WT) NSP13 protein. These findings thus justify the basis of stronger interactions and evasion for these NSP13 mutants. In conclusion, the current findings explored the key features of the NSP13 WT and its mutant complexes, which can be used to design structure-based inhibitors against the SARS-CoV-2 new variants to rescue the host immune system.

Computational modelling of potentially emerging SARS-CoV-2 spike protein RBDs mutations with higher binding affinity towards ACE2: A structural modelling study.

The spike protein of SARS-CoV-2 and the host ACE2 receptor plays a vital role in the entry to the cell. Among which the hotspot residue 501 is continuously subjected to positive selection pressure and induces unusual virulence. Keeping in view the importance of the hot spot residue 501, we predicted the potentially emerging structural variants of 501 residue. We analyzed the binding pattern of wild type and mutants (Spike RBD) to the ACE2 receptor by deciphering variations in the amino acids' interaction networks by graph kernels along with evolutionary, network metrics, and energetic information. Our analysis revealed that N501I, N501T, and N501V increase the binding affinity and alter the intra and inter-residue bonding networks. The N501T has shown strong positive selection and fitness in other animals. Docking results and repeated simulations (three times) confirmed the structural stability and tighter binding of these three variants, correlated with the previous results following the global stability trend. Consequently, we reported three variants N501I, N501T, and N501V could worsen the situation further if they emerged. The relations between the viral fitness and binding affinity is a complicated game thus the emergence of high affinity mutations in the SARS-CoV-2 RBD brings up the question of whether or not positive selection favours these mutations or not?

Comparative mutational analysis of SARS-CoV-2 isolates from Pakistan and structural-functional implications using computational modelling and simulation approaches.

SARS-CoV-2, an RNA virus, has been prone to high mutations since its first emergence in Wuhan, China, and throughout its spread. Its genome has been sequenced continuously by many countries, including Pakistan, but the results vary. Understanding its genomic patterns and connecting them with phenotypic features will help in devising therapeutic strategies. Thus, in this study, we explored the mutation landscape of 250 Pakistani isolates of SARS-CoV-2 genomes to check the genome diversity and examine the impact of these mutations on protein stability and viral pathogenesis in comparison with a reference sequence (Wuhan NC 045512.2). Our results revealed that structural proteins mainly exhibit more mutations than others in the Pakistani isolates; in particular, the nucleocapsid protein is highly mutated. In comparison, the spike protein is the most mutated protein globally. Furthermore, nsp12 was found to be the most mutated NSP in the Pakistani isolates and worldwide. Regarding accessory proteins, ORF3A is the most mutated in the Pakistani isolates, whereas ORF8 is highly mutated in world isolates. These mutations decrease the structural stability of their proteins and alter different biological pathways. Molecular docking, the dissociation constant (KD), and MM/GBSA analysis showed that mutations in the S protein alter its binding with ACE2. The spike protein mutations D614G-S943T-V622F (-75.17 kcal/mol), D614G-Q677H (-75.78 kcal/mol), and N74K-D614G (-73.84 kcal/mol) exhibit stronger binding energy than the wild type (-66.34 kcal/mol), thus increasing infectivity. Furthermore, the simulation results strongly corroborated the predicted protein servers. Our analysis findings also showed that E, M, ORF6, ORF7A, ORF7B, and ORF10 are the most stable coding genes; they may be suitable targets for vaccine and drug development.

Mutations in SARS-CoV-2 ORF8 Altered the Bonding Network With Interferon Regulatory Factor 3 to Evade Host Immune System.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously mutating since its first emergence in early 2020. These alterations have led this virus to gain significant difference in infectivity, pathogenicity, and host immune evasion. We previously found that the open-reading frame 8 (ORF8) of SARS-CoV-2 can inhibit interferon production by decreasing the nuclear translocation of interferon regulatory factor 3 (IRF3). Since several mutations in ORF8 have been observed, therefore, in the present study, we adapted structural and biophysical analysis approaches to explore the impact of various mutations of ORF8, such as S24L, L84S, V62L, and W45L, the recently circulating mutant in Pakistan, on its ability to bind IRF3 and to evade the host immune system. We found that mutations in ORF8 could affect the binding efficiency with IRF3 based on molecular docking analysis, which was further supported by molecular dynamics simulations. Among all the reported mutations, W45L was found to bind most stringently to IRF3. Our analysis revealed that mutations in ORF8 may help the virus evade the immune system by changing its binding affinity with IRF3.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta.

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.

Genetic characterization of structural and open reading Fram-8 proteins of SARS-CoV-2 isolates from different countries.

Since December 2019, a severe pandemic of pneumonia, COVID-19 associated with a novel coronavirus (SARS-CoV-2), have emerged in Wuhan, China and spreading throughout the world. As RNA viruses have a high mutation rate therefore we wanted to identify whether this virus is also prone to mutations. For this reason we selected four major structural (Spike protein (S), Envelope protein (E), Membrane glycoprotein (M), Nucleocapsid phosphoprotein (N)) and ORF8 protein of 100 different SARS-CoV-2 isolates of fifteen countries from NCBI database and compared these to the reference sequence, Wuhan NC_045512.2, which was the first isolate of SARS-CoV-2 that was sequenced. By multiple sequence alignment of amino acids, we observed substitutions and deletion in S protein at 13 different sites in the isolates of five countries (China, USA, Finland, India and Australia) as compared to the reference sequence. Similarly, alignment of N protein revealed substitutions at three different sites in isolates of China, Spain and Japan. M protein exhibits substitution only in one isolates from USA, however, no mutation was observed in E protein of any isolate. Interestingly, in ORF8 substitution of Leucine, a nonpolar to Serine a polar amino acid at same position (aa84 L to S) in 23 isolates of five countries i.e. China, USA, Spain, Taiwan and India were observed, which may affect the conformation of peptides. Thus, we observed several mutations in the isolates thereafter the first sequencing of SARS-CoV-2 isolate, NC_045512.2, which suggested that this virus might be a threat to the whole world and therefore further studies are needed to characterize how these mutations in different proteins affect the functionality and pathogenesis of SARS-CoV-2.

Evidence and speculations: vaccines and therapeutic options for COVID-19 pandemic.

A novel coronavirus (2019-nCov) emerged in China, at the end of December 2019 which posed an International Public Health Emergency, and later declared as a global pandemic by the World Health Organization (WHO). The International Committee on Taxonomy of Viruses (ICTV) named it SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), while the disease was named COVID-19 (Coronavirus Disease- 2019). Many questions related to the exact mode of transmission, animal origins, and antiviral therapeutics are not clear yet. Nevertheless, it is required to urgently launch a new protocol to evaluate the side effects of unapproved vaccines and antiviral therapeutics to accelerate the clinical application of new drugs. In this review, we highlight the most salient characteristics and recent findings of COVID-19 disease, molecular virology, interspecies mechanisms, and health consequences related to this disease.