An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern.

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.

Adaptation, spread and transmission of SARS-CoV-2 in farmed minks and associated humans in the Netherlands.

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface.

Transformations, Lineage Comparisons, and Analysis of Down-to-Up Protomer States of Variants of the SARS-CoV-2 Prefusion Spike Protein, Including the UK Variant B.1.1.7.

Monitoring and strategic response to variants in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent a considerable challenge in the current pandemic and for future viral outbreaks. Mutations/deletions of the virion's prefusion Spike protein may have significant impact on vaccines and therapeutics that utilize this key structural protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings ("glue points") based on ab inito all-atom force fields coupled with phylogenetic sequence alignment information can identify key residue mutations and deletions associated with variants. We also found several examples of excellent homology of stabilizing residue glue points across the lineages of betacoronavirus Spike proteins that we have called "sequence homologous glue points." SARS-CoV-2 demonstrates the least number of stabilizing glue points associated with interchain interactions among Down-state protomers across lineages. Additionally, we computationally studied variants among the trimeric Spike protein of SARS-CoV-2 using all-atom molecular dynamics to ascertain structural and energetic changes among variants. We examined both a theoretically based triple mutant and the UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through alanine substitutions that three key residues could cause the transition of Down-to-Up protomer states, where the transition is characterized by the "arm" length of the receptor-binding domain (RBD) rather than the hinge angle. For the B.1.1.7 variant, we demonstrated the critical importance of mutations D614G and N501Y on the structure and binding, respectively, of the Spike protein. We note that these same two key mutations are also found in the South African B.1.351 variant. IMPORTANCE Viral variants represent a major challenge to monitoring viral outbreaks and formulating strategic health care responses. Variants represent transmitting viruses that have specific mutations and deletions associated with their genome. In the case of SARS-CoV-2 and other related viruses (betacoronaviruses), many of these mutations and deletions are associated with the Spike protein that the virus uses to infect cells. Here, we have analyzed both SARS-CoV-2 variants and related viruses, such as Middle Eastern respiratory syndrome coronavirus (MERS-CoV), in order to understand not only differences, but also key similarities between them. Understanding similarities can be as important as differences in determining key functional features of a class of viruses, such as the betacoronaviruses. We have used both phylogenetic analysis, which traces genetic similarities and differences, along with independent biophysics analysis, which adds function or behavior, in order to determine possible functional differences and hence possible transmission and infection differences among variants and lineages.

SARS-CoV-2 variants, spike mutations and immune escape.

Although most mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome are expected to be either deleterious and swiftly purged or relatively neutral, a small proportion will affect functional properties and may alter infectivity, disease severity or interactions with host immunity. The emergence of SARS-CoV-2 in late 2019 was followed by a period of relative evolutionary stasis lasting about 11 months. Since late 2020, however, SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations, in the context of 'variants of concern', that impact virus characteristics, including transmissibility and antigenicity, probably in response to the changing immune profile of the human population. There is emerging evidence of reduced neutralization of some SARS-CoV-2 variants by postvaccination serum; however, a greater understanding of correlates of protection is required to evaluate how this may impact vaccine effectiveness. Nonetheless, manufacturers are preparing platforms for a possible update of vaccine sequences, and it is crucial that surveillance of genetic and antigenic changes in the global virus population is done alongside experiments to elucidate the phenotypic impacts of mutations. In this Review, we summarize the literature on mutations of the SARS-CoV-2 spike protein, the primary antigen, focusing on their impacts on antigenicity and contextualizing them in the protein structure, and discuss them in the context of observed mutation frequencies in global sequence datasets.

Identification of Novel Candidate CD8+ T Cell Epitopes of the SARS-CoV2 with Homology to Other Seasonal Coronaviruses.

Cross-reactive T cell immunity to seasonal coronaviruses (HCoVs) may lead to immunopathology or protection during SARS-CoV2 infection. To understand the influence of cross-reactive T cell responses, we used IEDB (Immune epitope database) and NetMHCpan (ver. 4.1) to identify candidate CD8+ T cell epitopes, restricted through HLA-A and B alleles. Conservation analysis was carried out for these epitopes with HCoVs, OC43, HKU1, and NL63. 12/18 the candidate CD8+ T cell epitopes (binding score of ≥0.90), which had a high degree of homology (>75%) with the other three HCoVs were within the NSP12 and NSP13 proteins. They were predicted to be restricted through HLA-A*2402, HLA-A*201, HLA-A*206, and HLA-B alleles B*3501. Thirty-one candidate CD8+ T cell epitopes that were specific to SARS-CoV2 virus (<25% homology with other HCoVs) were predominantly identified within the structural proteins (spike, envelop, membrane, and nucleocapsid) and the NSP1, NSP2, and NSP3. They were predominantly restricted through HLA-B*3501 (6/31), HLA-B*4001 (6/31), HLA-B*4403 (7/31), and HLA-A*2402 (8/31). It would be crucial to understand T cell responses that associate with protection, and the differences in the functionality and phenotype of epitope specific T cell responses, presented through different HLA alleles common in different geographical groups, to understand disease pathogenesis.

Evolutionary analysis of SARS-CoV-2 spike protein for its different clades.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the main target for antiviral and vaccine development. Despite its relevance, e information is scarse about its evolutionary traces. The aim of this study was to investigate the diversification patterns of the spike for each clade of SARS-CoV-2 through different approaches. Two thousand and one hundred sequences representing the seven clades of the SARS-CoV-2 were included. Patterns of genetic diversifications and nucleotide evolutionary rate were estimated for the spike genomic region. The haplotype networks showed a star shape, where multiple haplotypes with few nucleotide differences diverge from a common ancestor. Four hundred seventy-nine different haplotypes were defined in the seven analyzed clades. The main haplotype, named Hap-1, was the most frequent for clades G (54%), GH (54%), and GR (56%) and a different haplotype (named Hap-252) was the most important for clades L (63.3%), O (39.7%), S (51.7%), and V (70%). The evolutionary rate for the spike protein was estimated as 1.08 × 10-3 nucleotide substitutions/site/year. Moreover, the nucleotide evolutionary rate after nine months of the pandemic was similar for each clade. In conclusion, the present evolutionary analysis is relevant as the spike protein of SARS-CoV-2 is the target for most therapeutic candidates; besides, changes in this protein could have consequences on viral transmission, response to antivirals and efficacy of vaccines. Moreover, the evolutionary characterization of clades improves knowledge of SARS-CoV-2 and deserves to be assessed in more detail as re-infection by different phylogenetic clades has been reported.

Comparative phylogenetic analysis of SARS-CoV-2 spike protein-possibility effect on virus spillover.

Coronavirus disease 2019 has developed into a dramatic pandemic with tremendous global impact. The receptor-binding motif (RBM) region of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to host angiotensin-converting enzyme 2 (ACE2) receptors for infection. As ACE2 receptors are highly conserved within vertebrate species, SARS-CoV-2 can infect significant animal species as well as human populations. An analysis of SARS-CoV-2 genotypes isolated from human and significant animal species was conducted to compare and identify mutation and adaptation patterns across different animal species. The phylogenetic data revealed seven distinct phylogenetic clades with no significant relationship between the clades and geographical locations. A high rate of variation within SARS-CoV-2 mink isolates implies that mink populations were infected before human populations. Positions of most single-nucleotide polymorphisms (SNPs) within the spike (S) protein of SARS-CoV-2 genotypes from the different hosts are mostly accumulated in the RBM region and highlight the pronounced accumulation of variants with mutations in the RBM region in comparison with other variants. These SNPs play a crucial role in viral transmission and pathogenicity and are keys in identifying other animal species as potential intermediate hosts of SARS-CoV-2. The possible roles in the emergence of new viral strains and the possible implications of these changes, in compromising vaccine effectiveness, deserve urgent considerations.

Research progress on coronavirus S proteins and their receptors.

Coronaviruses are a large family of important pathogens that cause human and animal diseases. At the end of 2019, a pneumonia epidemic caused by a novel coronavirus brought attention to coronaviruses. Exploring the interaction between the virus and its receptor will be helpful in developing preventive vaccines and therapeutic drugs. The coronavirus spike protein (S) plays an important role in both binding to receptors on host cells and fusion of the viral membrane with the host cell membrane. This review introduces the structure and function of the S protein and its receptor, focusing on the binding mode and binding region of both.

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.

Predicting mammalian species at risk of being infected by SARS-CoV-2 from an ACE2 perspective.

SARS-CoV-2 can transmit efficiently in humans, but it is less clear which other mammals are at risk of being infected. SARS-CoV-2 encodes a Spike (S) protein that binds to human ACE2 receptor to mediate cell entry. A species with a human-like ACE2 receptor could therefore be at risk of being infected by SARS-CoV-2. We compared between 132 mammalian ACE2 genes and between 17 coronavirus S proteins. We showed that while global similarities reflected by whole ACE2 gene alignments are poor predictors of high-risk mammals, local similarities at key S protein-binding sites highlight several high-risk mammals that share good ACE2 homology with human. Bats are likely reservoirs of SARS-CoV-2, but there are other high-risk mammals that share better ACE2 homologies with human. Both SARS-CoV-2 and SARS-CoV are closely related to bat coronavirus. Yet, among host-specific coronaviruses infecting high-risk mammals, key ACE2-binding sites on S proteins share highest similarities between SARS-CoV-2 and Pangolin-CoV and between SARS-CoV and Civet-CoV. These results suggest that direct coronavirus transmission from bat to human is unlikely, and that rapid adaptation of a bat SARS-like coronavirus in different high-risk intermediate hosts could have allowed it to acquire distinct high binding potential between S protein and human-like ACE2 receptors.

In silico studies on the comparative characterization of the interactions of SARS-CoV-2 spike glycoprotein with ACE-2 receptor homologs and human TLRs.

Coronavirus disease-2019 (COVID-19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has come out as a major threat for mankind in recent times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS-CoV-2), its mode of interaction with the angiotensin-converting enzyme-2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll-like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS-CoV-2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS-CoV-2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS-CoV-2 from a bat and also revealed that TLR4 may have a crucial role in the virus-induced inflammatory consequences associated with COVID-19. Therefore, selective targeting of TLR4-spike protein interaction by designing competitive TLR4-antagonists could pave a new way to treat COVID-19. Finally, this study is expected to improve our understanding on the immunobiology of SARS-CoV-2 and could be useful in adopting spike protein, ACE2, or TLR-guided intervention strategy against COVID-19 shortly.

Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2.

To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.

Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS-CoV-2.

From the beginning of 2002 and 2012, severe respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) crossed the species barriers to infect humans, causing thousands of infections and hundreds of deaths, respectively. Currently, a novel coronavirus (SARS-CoV-2), which has become the cause of the outbreak of Coronavirus Disease 2019 (COVID-19), was discovered. Until 18 February 2020, there were 72 533 confirmed COVID-19 cases (including 10 644 severe cases) and 1872 deaths in China. SARS-CoV-2 is spreading among the public and causing substantial burden due to its human-to-human transmission. However, the intermediate host of SARS-CoV-2 is still unclear. Finding the possible intermediate host of SARS-CoV-2 is imperative to prevent further spread of the epidemic. In this study, we used systematic comparison and analysis to predict the interaction between the receptor-binding domain (RBD) of coronavirus spike protein and the host receptor, angiotensin-converting enzyme 2 (ACE2). The interaction between the key amino acids of S protein RBD and ACE2 indicated that, other than pangolins and snakes, as previously suggested, turtles (Chrysemys picta bellii, Chelonia mydas, and Pelodiscus sinensis) may act as the potential intermediate hosts transmitting SARS-CoV-2 to humans.