Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic, represents a serious threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using the α-glycosidase inhibitors castanospermine and NB-DNJ, we confirmed that disruption of N-linked glycosylation blocked the maturation of the S protein, leading to the impairment of S protein-mediated membrane fusion. Single-amino-acid substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation. Thus, substitution at these sites resulted in reduced S protein-mediated cell-cell fusion and viral entry. Notably, the N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T/hACE2-KO cells. Additionally, the removal of the furin cleavage site partially compensated for the instability induced by the N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the N669Q and N1080Q mutants exhibited increased fusogenic activity and did induce syncytium formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are highly important for maintaining the pre-fusion state of the S protein. This study revealed the critical roles of N-glycans in S protein maturation and stability, information that has implications for the design of vaccines and antiviral strategies.

A comprehensive survey of bat sarbecoviruses across China in relation to the origins of SARS-CoV and SARS-CoV-2.

SARS-CoV and SARS-CoV-2 have been thought to originate from bats. In this study, we screened pharyngeal and anal swabs from 13 064 bats collected between 2016 and 2021 at 703 locations across China for sarbecoviruses, covering almost all known southern hotspots, and found 146 new bat sarbecoviruses. Phylogenetic analyses of all available sarbecoviruses show that there are three different lineages-L1 as SARS-CoV-related CoVs (SARSr-CoVs), L2 as SARS-CoV-2-related CoVs (SC2r-CoVs) and novel L-R (recombinants of L1 and L2)-present in Rhinolophus pusillus bats, in the mainland of China. Among the 146 sequences, only four are L-Rs. Importantly, none belong in the L2 lineage, indicating that circulation of SC2r-CoVs in China might be very limited. All remaining 142 sequences belong in the L1 lineage, of which YN2020B-G shares the highest overall sequence identity with SARS-CoV (95.8%). The observation suggests endemic circulations of SARSr-CoVs, but not SC2r-CoVs, in bats in China. Geographic analysis of the collection sites in this study, together with all published reports, indicates that SC2r-CoVs may be mainly present in bats of Southeast Asia, including the southern border of Yunnan province, but absent in all other regions within China. In contrast, SARSr-CoVs appear to have broader geographic distribution, with the highest genetic diversity and sequence identity to human sarbecoviruses along the southwest border of China. Our data provide the rationale for further extensive surveys in broader geographical regions within, and beyond, Southeast Asia in order to find the most recent ancestors of human sarbecoviruses.

An RBD bispecific antibody effectively neutralizes a SARS-CoV-2 Omicron variant.

Potent neutralizing antibodies (nAbs) against SARS-CoV-2 are a promising therapeutic against the ongoing COVID-19 pandemic. However, the continuous emergence of neutralizing antibody escape variants makes it challenging for antibody therapeutics based on monospecific nAbs. Here, we generated an IgG-like bispecific antibody (bsAb), Bi-Nab, based on a pair of human neutralizing antibodies targeting multiple and invariant sites of the spike receptor binding domain (RBD): 35B5 and 32C7. We demonstrated that Bi-Nab exhibited higher binding affinity to the Delta spike protein than its parental antibodies and presented an extended inhibition breadth of preventing RBD binding to angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-CoV-2. In addition, pseudovirus neutralization results showed that Bi-Nab improved the neutralization potency and breadth with a lower half maximum inhibitory concentration (IC50) against wild-type SARS-CoV-2, variants being monitored (VBMs) and variants of concern (VOCs). Notably, the IgG-like Bi-Nab enhanced the neutralizing activity against Omicron variants with potent capabilities for transmission and immune evasion in comparison with its parental monoclonal antibody (mAb) 32C7 and a cocktail (with the lowest IC50 values of 31.6 ng/mL against the Omicron BA.1 and 399.2 ng/mL against the Omicron BA.2), showing evidence of synergistic neutralization potency of Bi-Nab against the Omicron variants. Thus, Bi-Nab represents a feasible and effective strategy against SARS-CoV-2 variants of concern.

Host susceptibility and structural and immunological insight of S proteins of two SARS-CoV-2 closely related bat coronaviruses.

The bat coronaviruses (CoV) BANAL-20-52 and BANAL-20-236 are two newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) closely related coronaviruses (SC2r-CoV) and the genome of BANAL-20-52 shares the highest homology with SARS-CoV-2. However, the risk of their potential zoonotic transmission has not been fully evaluated. Here, we determined their potential host susceptibility among 13 different bat species and 26 different animal species, and found that both might have extensive host ranges, indicating high zoonotic transmission potential. We also determined the cryo-EM structures of BANAL-20-52 and BANAL-20-236 S proteins at pH 5.5 and the complex of BANAL-20-236 S1 and Rhinolophus affinis ACE2, and found that both trimeric S proteins adopt all three receptor binding domains (RBDs) in "closed" conformation and are more compact than SARS-CoV-2. Strikingly, the unique sugar moiety at N370 of bat SC2r-CoVs acts like a "bolt" and crosses over two neighboring subunits, facilitating the S proteins in the locked conformation and underpinning the architecture stability. Removal of the glycosylation at N370 by a T372A substitution substantially enhances virus infectivity but becomes highly sensitive to trypsin digestion at pH 5.5, a condition roughly mimicking the insectivorous bat's stomach digestion. In contrast, WT S proteins of SC2r-CoVs showed considerable resistance to trypsin digestion at pH 5.5, indicating that the highly conserved T372 in bat CoVs might result from the selective advantages in stability during the fecal-oral transmission over A372. Moreover, the results of cross-immunogenicity among S proteins of SARS-CoV-2, BANAL-20-52, and BANAL-20-236 showed that A372 pseudoviruses are more sensitive to anti-S sera than T372, indicating that immune evasion might also play a role in the natural selection of T372 over A372 during evolution. Finally, residues 493 and 498 of the S protein affect host susceptibility, and residue 498 also influences the immunogenicity of the S protein. Together, our findings aid a better understanding of the molecular basis of CoV entry, selective evolution, and immunogenicity and highlight the importance of surveillance of susceptible hosts of these viruses to prevent potential outbreaks.

Optimization and Deoptimization of Codons in SARS-CoV-2 and Related Implications for Vaccine Development.

The spread of coronavirus disease 2019 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), has progressed into a global pandemic. To date, thousands of genetic variants have been identified among SARS-CoV-2 isolates collected from patients. Sequence analysis reveals that the codon adaptation index (CAI) values of viral sequences have decreased over time but with occasional fluctuations. Through evolution modeling, it is found that this phenomenon may result from the virus's mutation preference during transmission. Using dual-luciferase assays, it is further discovered that the deoptimization of codons in the viral sequence may weaken protein expression during virus evolution, indicating that codon usage may play an important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccines, it is designed several codon-optimized Omicron BA.2.12.1, BA.4/5, and XBB.1.5 spike mRNA vaccine candidates and experimentally validated their high levels of expression. This study highlights the importance of codon usage in virus evolution and provides guidelines for codon optimization in mRNA and DNA vaccine development.

Adaptive Evolution of the Spike Protein in Coronaviruses.

Coronaviruses are single-stranded, positive-sense RNA viruses that can infect many mammal and avian species. The Spike (S) protein of coronaviruses binds to a receptor on the host cell surface to promote viral entry. The interactions between the S proteins of coronaviruses and receptors of host cells are extraordinarily complex, with coronaviruses from different genera being able to recognize the same receptor and coronaviruses from the same genus able to bind distinct receptors. As the coronavirus disease 2019 pandemic has developed, many changes in the S protein have been under positive selection by altering the receptor-binding affinity, reducing antibody neutralization activities, or affecting T-cell responses. It is intriguing to determine whether the selection pressure on the S gene differs between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses due to the host shift from nonhuman animals to humans. Here, we show that the S gene, particularly the S1 region, has experienced positive selection in both SARS-CoV-2 and other coronaviruses. Although the S1 N-terminal domain exhibits signals of positive selection in the pairwise comparisons in all four coronavirus genera, positive selection is primarily detected in the S1 C-terminal domain (the receptor-binding domain) in the ongoing evolution of SARS-CoV-2, possibly owing to the change in host settings and the widespread natural infection and SARS-CoV-2 vaccination in humans.

A Competitive Panning Method Reveals an Anti-SARS-CoV-2 Nanobody Specific for an RBD-ACE2 Binding Site.

Most neutralizing antibodies neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by directly blocking the interactions between the spike glycoprotein receptor-binding domain (RBD) and its receptor, human angiotensin-converting enzyme 2 (ACE2). Here, we report a novel nanobody (Nb) identified by an RBD-ACE2 competitive panning method that could specifically bind to the RBD of SARS-CoV-2 with a high affinity (EC50 = 0.03 nM) and successfully block the binding between the RBD and ACE2 recombinant protein. A structural simulation of the RBD-VHH complex also supports a mechanism of the Nb to block the interaction between the RBD and ACE2. A pseudovirus assay of the Nb showed it could neutralize the WT pseudovirus with high potency (IC50 = 0.026 µg/mL). Furthermore, we measured its binding to phages displaying RBDs of different SARS-CoV-2 variants and found that it could bind to recombinant phages displaying the RBD of beta and delta variants. This study also provides a method of phage library competitive panning, which could be useful for directly screening high-affinity antibodies targeting important functional regions.

Effect of polymorphism in Rhinolophus affinis ACE2 on entry of SARS-CoV-2 related bat coronaviruses.

Bat coronavirus RaTG13 shares about 96.2% nucleotide sequence identity with that of SARS-CoV-2 and uses human and Rhinolophus affinis (Ra) angiotensin-converting enzyme 2 (ACE2) as entry receptors. Whether there are bat species other than R. affinis susceptible to RaTG13 infection remains elusive. Here, we show that, among 18 different bat ACE2s tested, only RaACE2 is highly susceptible to transduction by RaTG13 S pseudovirions, indicating that the bat species harboring RaTG13 might be very limited. RaACE2 has seven polymorphic variants, RA-01 to RA-07, and they show different susceptibilities to RaTG13 S pseudovirions transduction. Sequence and mutagenesis analyses reveal that residues 34, 38, and 83 in RaACE2 might play critical roles in interaction with the RaTG13 S protein. Of note, RaACE2 polymorphisms have minimal effect on S proteins of SARS-CoV-2 and several SARS-CoV-2 related CoVs (SC2r-CoVs) including BANAL-20-52 and BANAL-20-236 in terms of binding, membrane fusion, and pseudovirus entry. Further mutagenesis analyses identify residues 501 and 505 in S proteins critical for the recognition of different RaACE2 variants and pangolin ACE2 (pACE2), indicating that RaTG13 might have not been well adapted to R. affinis bats. While single D501N and H505Y changes in RaTG13 S protein significantly enhance the infectivity and minimize the difference in susceptibility among different RaACE2 variants, an N501D substitution in SARS-CoV-2 S protein displays marked disparity in transduction efficiencies among RaACE2 variants with a significant reduction in infectivity on several RaACE2 variants. Finally, a T372A substitution in RaTG13 S protein not only significantly increases infectivity on all RaACE2 variants, but also markedly enhances entry on several bat ACE2s including R. sinicus YN, R. pearsonii, and R. ferrumeiqunum. However, the T372A mutant is about 4-fold more sensitive to neutralizing sera from mice immunized with BANAL-20-52 S, suggesting that the better immune evasion ability of T372 over A372 might contribute to the natural selective advantage of T372 over A372 among bat CoVs. Together, our study aids a better understanding of coronavirus entry, vaccine design, and evolution.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

A Lentiviral Pseudotype System to Characterize SARS-CoV-2 Glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 causes worldwide COVID-19 pandemic and poses a great threat to global public health. Due to its high pathogenicity and infectivity, live SARS-CoV-2 is classified as a BSL-3 agent and has to be handled in BSL-3 condition. Nevertheless, entry of SARS-CoV-2 is mediated by viral spike (S) glycoprotein, and pseudovirus with SARS-CoV-2 S protein can mimic every entry step of SARS-CoV-2 virus and be studied in BSL-2 settings. In this chapter, we describe a detailed protocol of production of lentivirus-based SARS-CoV-2 S pseudovirus and its application in study of virus entry and determination of neutralizing antibody titer of human sera against SARS-CoV-2.

Characterization of SARS-CoV-2 Glycoprotein Using a Quantitative Cell-Cell Fusion System.

Coronaviruses (CoVs) infect host cells through the fusion of viral and cellular membrane and may also spread to the neighboring uninfected cells from infected cells through cell-cell fusion. The viral spike (S) glycoproteins play an essential role in mediating membrane fusion. Here, we present a luciferase-based quantitative assay to measure the efficiency of cell-cell fusion mediated by the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This method applies to S proteins of the other coronaviruses and can be adapted to fusion proteins of other enveloped viruses.

Relative vaccine effectiveness against Delta and Omicron COVID-19 after homologous inactivated vaccine boosting: a retrospective cohort study.

ObjectiveTwo COVID-19 outbreaks occurred in Henan province in early 2022-one was a Delta variant outbreak and the other was an Omicron variant outbreak. COVID-19 vaccines used at the time of the outbreak were inactivated, 91.8%; protein subunit, 7.5%; and adenovirus5-vectored, 0.7% vaccines. The outbreaks provided an opportunity to evaluate variant-specific breakthrough infection rates and relative protective effectiveness of homologous inactivated COVID-19 vaccine booster doses against symptomatic infection and pneumonia. DESIGN: Retrospective cohort study METHODS: We evaluated relative vaccine effectiveness (rVE) with a retrospective cohort study of close contacts of infected individuals using a time-dependent Cox regression model. Demographic and epidemiologic data were obtained from the local Centers for Disease Control and Prevention; clinical and laboratory data were obtained from COVID-19-designated hospitals. Vaccination histories were obtained from the national COVID-19 vaccination dataset. All data were linked by national identification number. RESULTS: Among 784 SARS-CoV-2 infections, 379 (48.3%) were caused by Delta and 405 (51.7%) were caused by Omicron, with breakthrough rates of 9.9% and 17.8%, respectively. Breakthrough rates among boosted individuals were 8.1% and 4.9%. Compared with subjects who received primary vaccination series ≥180 days before infection, Cox regression modelling showed that homologous inactivated booster vaccination was statistically significantly associated with protection from symptomatic infection caused by Omicron (rVE 59%; 95% CI 13% to 80%) and pneumonia caused by Delta (rVE 62%; 95% CI 34% to 77%) and Omicron (rVE 87%; 95% CI 3% to 98%). CONCLUSIONS: COVID-19 vaccination in China provided good protection against symptomatic COVID-19 and COVID-19 pneumonia caused by Delta and Omicron variants. Protection declined 6 months after primary series vaccination but was restored by homologous inactivated booster doses given 6 months after the primary series.

Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay.

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen-antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern.

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable in overcoming the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained parental specificity and simultaneously bound to the two distinct epitopes on receptor-binding domain (RBD) and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs), and variants being monitored (VBMs) compared with its parental monoclonal antibodies (MAbs). Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs, including Alpha (B.1.1.7), Beta (B.1.351), and Kappa (B.1.617.1), as well as VOCs, including Delta (B.1.617.2), Omicron BA.1, and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50 = 0.15 nM) and Omicron BA.2 (IC50 = 0.67 nM) compared with its parental MAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. IMPORTANCE The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, which simultaneously targets SARS-CoV-2 RBD and S2 and improves the neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy by which to treat and prevent COVID-19.

A case-case study on the effect of primary and booster immunization with China-produced COVID-19 vaccines on prevention of pneumonia and viral load among vaccinated persons infected by Delta and Omicron variants.

Using a three-prefecture, two-variant COVID-19 outbreak in Henan province in January 2022, we evaluated the associations of primary and booster immunization with China-produced COVID-19 vaccines and COVID-19 pneumonia and SARS-CoV-2 viral load among persons infected by Delta or Omicron variant. We obtained demographic, clinical, vaccination, and multiple Ct values of infections ≥3 years of age. Vaccination status was either primary series ≥180 days prior to infection; primary series <180 days prior to infection, or booster dose recipient. We used logistic regression to determine odds ratios (OR) of Delta and Omicron COVID-19 pneumonia by vaccination status. We analysed minimum Ct values by vaccination status, age, and variant. Of 826 eligible cases, 405 were Delta and 421 were Omicron cases; 48.9% of Delta and 19.0% of Omicron cases had COVID-19 pneumonia. Compared with full primary vaccination ≥180 days before infection, the aOR of pneumonia was 0.48 among those completing primary vaccination <180 days and 0.18 among booster recipients among these Delta infections. Among Omicron infections, the corresponding aOR was 0.34 among those completing primary vaccination <180 days. There were too few (ten) Omicron cases among booster dose recipients to calculate a reliable OR. There were no differences in minimum Ct values by vaccination status among the 356 Delta cases or 70 Omicron cases. COVID-19 pneumonia was less common among Omicron cases than Delta cases. Full primary vaccination reduced pneumonia effectively for 6 months; boosting six months after primary vaccination resulted in further reduction. We recommend accelerating the pace of booster dose administration.

Kathryn V. Holmes: A Career of Contributions to the Coronavirus Field.

Over the past two years, scientific research has moved at an unprecedented rate in response to the COVID-19 pandemic. The rapid development of effective vaccines and therapeutics would not have been possible without extensive background knowledge on coronaviruses developed over decades by researchers, including Kathryn (Kay) Holmes. Kay's research team discovered the first coronavirus receptors for mouse hepatitis virus and human coronavirus 229E and contributed a wealth of information on coronaviral spike glycoproteins and receptor interactions that are critical determinants of host and tissue specificity. She collaborated with several research laboratories to contribute knowledge in additional areas, including coronaviral pathogenesis, epidemiology, and evolution. Throughout her career, Kay was an extremely dedicated and thoughtful mentor to numerous graduate students and post-doctoral fellows. This article provides a review of her contributions to the coronavirus field and her exemplary mentoring.

Evolutionary dynamics of the severe acute respiratory syndrome coronavirus 2 genomes.

The coronavirus disease 2019 (COVID-19) pandemic has caused immense losses in human lives and the global economy and posed significant challenges for global public health. As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has evolved, thousands of single nucleotide variants (SNVs) have been identified across the viral genome. The roles of individual SNVs in the zoonotic origin, evolution, and transmission of SARS-CoV-2 have become the focus of many studies. This review summarizes recent comparative genomic analyses of SARS-CoV-2 and related coronaviruses (SC2r-CoVs) found in non-human animals, including delineation of SARS-CoV-2 lineages based on characteristic SNVs. We also discuss the current understanding of receptor-binding domain (RBD) evolution and characteristic mutations in variants of concern (VOCs) of SARS-CoV-2, as well as possible co-evolution between RBD and its receptor, angiotensin-converting enzyme 2 (ACE2). We propose that the interplay between SARS-CoV-2 and host RNA editing mechanisms might have partially resulted in the bias in nucleotide changes during SARS-CoV-2 evolution. Finally, we outline some current challenges, including difficulty in deciphering the complicated relationship between viral pathogenicity and infectivity of different variants, and monitoring transmission of SARS-CoV-2 between humans and animals as the pandemic progresses.

The stalk domain of SARS-CoV-2 NSP13 is essential for its helicase activity.

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.