Background SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. Results Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. Conclusion Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

SARS-CoV-2, the causative agent of COVID-2019 pandemic is an RNA virus prone to mutations.Information on mutations within the circulating strains of the virus is pivotal to understand disease spread and dynamics. Here, we analyse the mutations associated with 2,954 globally reported high quality genomes of SARS-CoV-2 with special emphasis on genomes of viral strains from India.Molecular phylogenetic analysis suggests that SARS-CoV-2 strains circulating in India form five distinct phyletic clades designated R1-R5. These clades categorize into the previously reported S, G as well as a new unclassified subtype. A detailed analysis of gene encoding the Spike (S) protein in the strains across the globe shows non-synonymous mutations on 54 amino acid residues. Among these, we pinpoint 4 novel mutations in the region that interacts with human ACE2 receptor (RBD). Further in silico molecular docking analyses suggest that these RBD mutations could alter the binding affinity of S-protein with ACE2 that may lead to changes in SARS-CoV-2 infectivity. Strikingly, one of these RBD mutations (S438F) is unique to a subset within the R4 clade suggesting intrinsic S-protein variations in strains currently circulating in India. Together, our findings reveal a unique pattern of SARS-CoV-2 evolution that may alert vaccine and therapeutic development.

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Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to host cell is mediated through the binding of the SARS-CoV-2 Spike protein via receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds inhibiting Spike-ACE2 binding would be a promising, safe antiviral approach against COVID-19. Methods: In the present study, we have used BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) replaced glycoprotein with spike protein of SARS-CoV-2 expressing eGFP reporter system (VSV-eGFP-SARS-CoV2) in a permissive cells harboring cytotoxicity marker. The high-throughput compatible SARS-CoV-2 permissive reporter system that encompasses cells stably expressing hACE2 tagged cerulean and nuclear H2B tagged with mCherry, as a marker of nuclear condensation that also enabled imaging of fused cells among infected EGFP positive cells and could give real-time information of syncytia formation. Results: A limited high-throughput screening identified six natural products with marked VSV-eGFP-SARS-CoV2 inhibition at non cytotoxic dose. Molecular simulation studies with positive hits in complex with wild-type spike reaffirm their potential to impede viral entry. Real-time syncytia formation assay of the molecules revealed inhibition of syncytia with Didemnin B, and delayed inhibition with other natural products such as Scillaren A, Proscillaridin, Acetoxycycloheximide indicating that the assay is a reliable platform for any image based drug screening. Conclusion: BSL-2 compatible assay system equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effect on syncytia. Studies using clinical isolates of SARS-CoV-2 is warranted to confirm the antiviral potency of the leads and the utility of the screening system.