Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA.

The interaction of the ß-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA-N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few µm) of N protein-genomic RNA formed by liquid-liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA-N protein interaction.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

Single domain antibodies derived from ancient animals as broadly neutralizing agents for SARS-CoV-2 and other coronaviruses.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.

Advances in the development of therapeutic strategies against COVID-19 and perspectives in the drug design for emerging SARS-CoV-2 variants.

Since Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has challenged public health around the world. Currently, there is an urgent need to explore antiviral therapeutic targets and effective clinical drugs. In this study, we systematically summarized two main therapeutic strategies against COVID-19, namely drugs targeting the SARS-CoV-2 life cycle and SARS-CoV-2-induced inflammation in host cells. The development of above two strategies is implemented by repurposing drugs and exploring potential targets. A comprehensive summary of promising drugs, especially cytokine inhibitors, and traditional Chinese medicine (TCM), provides recommendations for clinicians as evidence-based medicine in the actual clinical COVID-19 treatment. Considering the emerging SARS-CoV-2 variants greatly impact the effectiveness of drugs and vaccines, we reviewed the appearance and details of SARS-CoV-2 variants for further perspectives in drug design, which brings updating clues to develop therapeutical agents against the variants. Based on this, the development of broadly antiviral drugs, combined with immunomodulatory, or holistic therapy in the host, is prior to being considered for therapeutic interventions on mutant strains of SARS-CoV-2. Therefore, it is highly acclaimed the requirements of the concerted efforts from multi-disciplinary basic studies and clinical trials, which improves the accurate treatment of COVID-19 and optimizes the contingency measures to emerging SARS-CoV-2 variants.

The status and analysis of common mutations found in the SARS-CoV-2 whole genome sequences from Bangladesh.

Rapid emergence of covid-19 variants by continuous mutation made the world experience continuous waves of infections and as a result, a huge number of death-toll recorded so far. It is, therefore, very important to investigate the diversity and nature of the mutations in the SARS-CoV-2 genomes. In this study, the common mutations occurred in the whole genome sequences of SARS-CoV-2 variants of Bangladesh in a certain timeline were analyzed to better understand its status. Hence, a total of 78 complete genome sequences available in the NCBI database were obtained, aligned and further analyzed. Scattered Single Nucleotide Polymorphisms (SNPs) were identified throughout the genome of variants and common SNPs such as: 241:C>T in the 5'UTR of Open Reading Frame 1A (ORF1A), 3037: C>T in Non-structural Protein 3 (NSP3), 14,408: C>T in ORF6 and 23,402: A>G, 23,403: A>G in Spike Protein (S) were observed, but all of them were synonymous mutations. About 97% of the studied genomes showed a block of tri-nucleotide alteration (GGG>AAC), the most common non-synonymous mutation in the 28,881-28,883 location of the genome. This block results in two amino acid changes (203-204: RG>KR) in the SR rich motif of the nucleocapsid (N) protein of SARS-CoV-2, introducing a lysine in between serine and arginine. The N protein structure of the mutant was predicted through protein modeling. However, no observable difference was found between the mutant and the reference (Wuhan) protein. Further, the protein stability changes upon mutations were analyzed using the I-Mutant2.0 tool. The alteration of the arginine to lysine at the amino acid position 203, showed reduction of entropy, suggesting a possible impact on the overall stability of the N protein. The estimation of the non-synonymous to synonymous substitution ratio (dN/dS) were analyzed for the common mutations and the results showed that the overall mean distance among the N-protein variants were statistically significant, supporting the non-synonymous nature of the mutations. The phylogenetic analysis of the selected 78 genomes, compared with the most common genomic variants of this virus across the globe showed a distinct cluster for the analyzed Bangladeshi sequences. Further studies are warranted for conferring any plausible association of these mutations with the clinical manifestation.

Highlight of potential impact of new viral genotypes of SARS-CoV-2 on vaccines and anti-viral therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of the coronavirus disease (COVID-19) pandemic, has infected millions of people globally. Genetic variation and selective pressures lead to the accumulation of single nucleotide polymorphism (SNP) within the viral genome that may affect virulence, transmission rate, viral recognition and the efficacy of prophylactic and interventional measures. To address these concerns at the genomic level, we assessed the phylogeny and SNPs of the SARS-CoV-2 mutant population collected to date in Iran in relation to globally reported variants. Phylogenetic analysis of mutant strains revealed the occurrence of the variants known as B.1.1.7 (Alpha), B.1.525 (Eta), and B.1.617 (Delta) that appear to have delineated independently in Iran. SNP analysis of the Iranian sequences revealed that the mutations were predominantly positioned within the S protein-coding region, with most SNPs localizing to the S1 subunit. Seventeen S1-localizing SNPs occurred in the RNA binding domain that interacts with ACE2 of the host cell. Importantly, many of these SNPs are predicted to influence the binding of antibodies and anti-viral therapeutics, indicating that the adaptive host response appears to be imposing a selective pressure that is driving the evolution of the virus in this closed population through enhancing virulence. The SNPs detected within these mutant cohorts are addressed with respect to current prophylactic measures and therapeutic interventions.

Emergence, evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions.

The emergence of coronavirus disease 2019 (COVID-19) pandemic in Wuhan city, China at the end of 2019 made it urgent to identify the origin of the causal pathogen and its molecular evolution, to appropriately design an effective vaccine. This study analyzes the evolutionary background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS-2) in accordance with its close relative SARS-CoV (SARS-1), which was emerged in 2002. A comparative genomic and proteomic study was conducted on SARS-2, SARS-1, and Middle East respiratory syndrome coronavirus (MERS), which was emerged in 2012. In silico analysis inferred the genetic variability among the tested viruses. The SARS-1 genome harbored 11 genes encoding 12 proteins, while SARS-2 genome contained only 10 genes encoding for 10 proteins. MERS genome contained 11 genes encoding 11 proteins. The analysis also revealed a slight variation in the whole genome size of SARS-2 comparing to its siblings resulting from sequential insertions and deletions (indels) throughout the viral genome particularly ORF1AB, spike, ORF10 and ORF8. The effective indels were observed in the gene encoding the spike protein that is responsible for viral attachment to the angiotensin-converting enzyme 2 (ACE2) cell receptor and initiating infection. These indels are responsible for the newly emerging COVID-19 variants αCoV, ßCoV, γCoV and δCoV. Nowadays, few effective COVID-19 vaccines developed based on spike (S) glycoprotein were approved and become available worldwide. Currently available vaccines can relatively prevent the spread of COVID-19 and suppress the disease. The traditional (killed or attenuated virus vaccine and antibody-based vaccine) and innovated vaccine production technologies (RNA- and DNA-based vaccines and viral vectors) are summarized in this review. We finally highlight the most common questions related to COVID-19 disease and the benefits of getting vaccinated.

The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour.

The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. Simulations of the D614G mutant show differences in behaviour between these clinical variants of the spike: the D614G mutant shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.

Analysis of SARS-COV2 spike protein variants among Iraqi isolates.

The ongoing pandemic of COVID-19 caused by the SARS-COV2 virus has triggered millions of deaths around the globe. Emerging several variants of the virus with increased transmissibility, the severity of disease, and the ability of the virus to escape from the immune system has a cause for concerns. Here, we compared the spike protein sequence of 91 human SARS CoV2 strains of Iraq to the first reported sequence of SARS-CoV2 isolate from Wuhan Hu-1/China. The strains were isolated between June 2020 and March 2021. Twenty-two distinct mutations were identified within the spike protein regions which were: L5F, L18F, T19R, S151T, G181A, A222V, A348S, L452 (Q or M), T478K, N501Y, A520S, A522V, A570D, S605A, D614G, Q675H, N679K, P681H, T716I, S982A, A1020S, D1118H. The most frequently mutations occurred at the D614G (87/91), followed by S982A (50/91), and A570D (48/91), respectively. In addition, a distinct shift was observed in the type of SARS-COV2 variants present in 2020 compared to 2021 isolates. In 2020, B.1.428.1 lineage was appeared to be a dominant variant (85%). However, the diversity of the variants increased in 2021, and the majority (73%) of the isolated were appeared to belong to B.1.1.7 lineage (VOC/alpha variants). To our knowledge, this is the first major genome analysis of SARS-CoV2 in Iraq. The data from this research could provide insights into SARS-CoV2 evolution, and can be potentially used to recognize the effective vaccine against the disease.

Characterizing genomic variants and mutations in SARS-CoV-2 proteins from Indian isolates.

SARS-CoV-2 is mutating and creating divergent variants by altering the composition of essential constituent proteins. Pharmacologically, it is crucial to understand the diverse mechanism of mutations for stable vaccine or anti-viral drug design. Our current study concentrates on all the constituent proteins of 469 SARS-CoV-2 genome samples, derived from Indian patients. However, the study may easily be extended to the samples across the globe. We perform clustering analysis towards identifying unique variants in each of the SARS-CoV-2 proteins. A total of 536 mutated positions within the coding regions of SARS-CoV-2 proteins are detected among the identified variants from Indian isolates. We quantify mutations by focusing on the unique variants of each SARS-CoV-2 protein. We report the average number of mutation per variant, percentage of mutated positions, synonymous and non-synonymous mutations, mutations occurring in three codon positions and so on. Our study reveals the most susceptible six (06) proteins, which are ORF1ab, Spike (S), Nucleocapsid (N), ORF3a, ORF7a, and ORF8. Several non-synonymous substitutions are observed to be unique in different SARS-CoV-2 proteins. A total of 57 possible deleterious amino acid substitutions are predicted, which may impact on the protein functions. Several mutations show a large decrease in protein stability and are observed in putative functional domains of the proteins that might have some role in disease pathogenesis. We observe a good number of physicochemical property change during above deleterious substitutions.

Unraveling the molecular basis of host cell receptor usage in SARS-CoV-2 and other human pathogenic ß-CoVs.

The recent emergence of the novel SARS-CoV-2 in China and its rapid spread in the human population has led to a public health crisis worldwide. Like in SARS-CoV, horseshoe bats currently represent the most likely candidate animal source for SARS-CoV-2. Yet, the specific mechanisms of cross-species transmission and adaptation to the human host remain unknown. Here we show that the unsupervised analysis of conservation patterns across the ß-CoV spike protein family, using sequence information alone, can provide valuable insights on the molecular basis of the specificity of ß-CoVs to different host cell receptors. More precisely, our results indicate that host cell receptor usage is encoded in the amino acid sequences of different CoV spike proteins in the form of a set of specificity determining positions (SDPs). Furthermore, by integrating structural data, in silico mutagenesis and coevolution analysis we could elucidate the role of SDPs in mediating ACE2 binding across the Sarbecovirus lineage, either by engaging the receptor through direct intermolecular interactions or by affecting the local environment of the receptor binding motif. Finally, by the analysis of coevolving mutations across a paired MSA we were able to identify key intermolecular contacts occurring at the spike-ACE2 interface. These results show that effective mining of the evolutionary records held in the sequence of the spike protein family can help tracing the molecular mechanisms behind the evolution and host-receptor adaptation of circulating and future novel ß-CoVs.

Unlocking COVID therapeutic targets: A structure-based rationale against SARS-CoV-2, SARS-CoV and MERS-CoV Spike.

There are no approved target therapeutics against SARS-CoV-2 or other beta-CoVs. The beta-CoV Spike protein is a promising target considering the critical role in viral infection and pathogenesis and its surface exposed features. We performed a structure-based strategy targeting highly conserved druggable regions resulting from a comprehensive large-scale sequence analysis and structural characterization of Spike domains across SARSr- and MERSr-CoVs. We have disclosed 28 main consensus druggable pockets within the Spike. The RBD and SD1 (S1 subunit); and the CR, HR1 and CH (S2 subunit) represent the most promising conserved druggable regions. Additionally, we have identified 181 new potential hot spot residues for the hSARSr-CoVs and 72 new hot spot residues for the SARSr- and MERSr-CoVs, which have not been described before in the literature. These sites/residues exhibit advantageous structural features for targeted molecular and pharmacological modulation. This study establishes the Spike as a promising anti-CoV target using an approach with a potential higher resilience to resistance development and directed to a broad spectrum of Beta-CoVs, including the new SARS-CoV-2 responsible for COVID-19. This research also provides a structure-based rationale for the design and discovery of chemical inhibitors, antibodies or other therapeutic modalities successfully targeting the Beta-CoV Spike protein.

Synergistic antiviral effect of hydroxychloroquine and azithromycin in combination against SARS-CoV-2: What molecular dynamics studies of virus-host interactions reveal.

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.