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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.

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.

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.

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.

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.

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.

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.

Host-based antivirals could offer broad-spectrum therapeutics and prophylactics against the constantly-mutating viruses including the currently-ravaging coronavirus, yet must target cellular vulnerabilities of viruses without grossly endangering the host. Here we show that the master lipid regulator SREBP1 couples the phospholipid scramblase TMEM41B to constitute a host “metabolism-to-manufacture” cascade that maximizes membrane supplies to support coronaviral genome replication, harboring biosynthetic enzymes including Lipin1 as druggable viral-specific-essential (VSE) host genes. Moreover, pharmacological inhibition of Lipin1, by a moonlight function of the widely-prescribed beta-blocker Propranolol, metabolically uncouples the SREBP1-TMEM41B cascade and consequently exhibits broad-spectrum antiviral effects against coronaviruses, Zika virus, and Dengue virus. The data implicate a metabolism-based antiviral strategy that is well tolerated by the host, and a potential broad-spectrum medication against current and future coronavirus diseases.

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 R. pusillus bats, in the mainland of China. Among the 146 sequences, only 4 are L-Rs. Importantly, none belongs 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.

The spread of Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has progressed into a global pandemic. To date, thousands of genetic variants have been identified across SARS-CoV-2 isolates from patients. Sequence analysis reveals that the codon usage of viral sequences decreased over time but fluctuated from time to time. In this study, through evolution modeling, we found that this phenomenon might result from the virus’ preference for mutations during transmission. Using dual luciferase assays, we further discovered that the deoptimization of codons on viruses might weaken protein expression during the virus evolution, indicating that the choice of codon usage might play important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccine, we designed several omicron BA.2.12.1 and BA.4/5 spike mRNA vaccine candidates based on codon optimization, and experimentally validated their high levels of expression. Our study highlights the importance of codon usage in virus evolution and mRNA vaccine development.

In this paper, the unsteady natural cavitation and ventilated cavitation are investigated with the large eddy simulation method. The predicted cavity morphology and evolution process before and after ventilation agree well with the available experimental data. The results indicate that in natural cavitation, the formation of a U-type cavity is closely related to the separation of a re-entrant jet and the effect of vortex evolution, while the local high pressure region plays an important role in shaping the appearance of the primary and secondary U-type cavity in the process of moving downstream. During ventilated cavitation, the deflation effect of the air drives the pair of vortices to shed periodically, accompanied by the downstream transportation of vorticity, and promotes the formation of arched air cavities. In the anticlockwise vortex shedding region, the vortex dilatation term has a concentrated distribution on the air-liquid interface which is due to the intense mixing of the phases. Further analysis demonstrates that, in the air cavity fluttering area, the magnitude of the velocity pulsation produces a significant downward tendency due to the gradual weakening of vortex intensity. Moreover, the interaction between vortices and turbulence shows a high degree of consistency in the wake flow.

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 to overcome 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 the specificity and simultaneously bound to the two distinct epitopes on 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 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) and 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= 27.3 ng/mL) and Omicron BA.2 (IC50= 121.1 ng/mL) compared with their parental mAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern.ImportanceThe 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, that simultaneously targets SARS-CoV-2 RBD and S2 and improved 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 to treat and prevent COVID-19.

Various plants use antimicrobial proteins/peptides as a means 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 also present 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 to 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.

Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 have been thought to originate from bat, but whether the cross-species transmission occurred directly from bat to human or through an intermediate host remains elusive. In this study, we performed CoV screening of 102 samples collected from animal-selling stalls of Wuhan Huanan Market (WHM) and pharyngeal and anal swabs from13,064 bats collected at 703 locations across China, covering almost all known southern hotspots for sarbecovirus, between 2016 and 2021. This is the first systematic survey of bat CoV in China during the outbreak of Corona Virus Disease 2019. We found four non-sarbeco CoVs in samples of WHM, and 142 SARS-CoV related CoVs (SARSr-CoV) and 4 recombinant CoVs in bats, of which YN2020B-G share the highest sequence identity with SARS-CoV among all known bat CoVs, suggesting endemic SARSr-CoVs in bats in China. However, we did not find any SARS-CoV-2 related CoVs (SC2r-CoV) in any samples, including specimens collected from the only two domestic places where RaTG13 and RmYN02 were previously reported (the Tongguan caves and the karst caves around the Xishuangbanna Tropical Botanical Garden), indicating that SC2r-CoVs might not actively circulate among bats in China. Phylogenetic analysis showed that there are three different lineages of sarbecoviruses, L1 (SARSr-CoV), L2 (SC2r-CoV), and L-R (a novel CoV lineage from L1 and L2 recombination), in China. Of note, L-R CoVs are only found in R. pusillus. Further macroscopical analysis of the genetic diversity, host specificity for colonization and accidental infection, and geographical characteristics of available CoVs in database revealed the presence of a general geographical distribution pattern for bat sarbecoviruses, with the highest genetic diversity and sequence homology to SARS-CoV or SARS-CoV-2 along the southwest border of China, the least in the northwest of China. Considering the receptor binding motifs for spike gene of sarbecoviruses in Indochina Peninsula show the greatest diversity, our data provide the rationale that extensive surveys in further south and southwest to or of China might be needed for finding closer ancestors of SARS-CoV and SARS-CoV-2.

Large eddy simulations were used to investigate unsteady tip-leakage cavitating flow over a National Advisory Committee for Aeronautics 0009 hydrofoil. The proper orthogonal decomposition (POD) was combined with fast Fourier transforms to help better understand the deformation dynamics of the vortex and cavity. The results show three typical vortices, i.e., the tip-leakage vortex (TLV), tip-separation vortex (TSV), and shedding–trailing vortex (STV) with their own helical core lines in the averaged cavitating flows. Upstream of x/ C = 1.8, the mean TLV core carries the breathing mode waves through the vortex dilatation effect, while the TSV core generates the dilatation, shrinkage, and bending distortions near the cavity interface by vortex stretching and deflection effects. Further downstream, the TLV starts to intertwine with the TSV, and the STV gradually diffuses. The TLV wandering encourages large cavity deformations dominated by the breathing and bending modes. However, in the lower-order POD modes, the contribution of bending modes to the cavity fluctuation energy is greatly weakened due to the variation of the relative cavity radius, r*, which indicates that the breathing mode and the double helix mode play major roles in the cavity interfacial waves. The results also show that the cavity interfacial oscillations are highly correlated with the surrounding velocity fluctuations. Moreover, a modified theoretical dispersion equation can well reproduce the quantitative relation between the vibration frequencies and the axial wavenumbers of the TLV cavity, especially for the wave dynamics of the breathing modes and the double helix modes.

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with SARS-CoV-2 among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although RaACE2 binding to the receptor binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but limited for mouse, ACE2s enter cells. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). To date, no prophylactic vaccines or approved therapeutic agents are available for preventing and treating this highly transmittable disease. Here we report two monoclonal antibodies (mAbs) cloned from memory B cells of patients recently recovered from COVID-19, and both mAbs specifically bind to the spike (S) protein of SARS-CoV-2, block the binding of receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin converting enzyme 2 (hACE2), and effectively neutralize S protein-pseudotyped virus infection. These human mAbs hold the promise for the prevention and treatment of the ongoing pandemic of COVID-19.

The SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China, and has since impacted a large portion of China and raised major global concern. Herein, we investigated the extent of molecular divergence between SARS-CoV-2 and other related coronaviruses. Although we found only 4% variability in genomic nucleotides between SARS-CoV-2 and a bat SARS-related coronavirus (SARSr-CoV; RaTG13), the difference at neutral sites was 17%, suggesting the divergence between the two viruses is much larger than previously estimated. Our results suggest that the development of new variations in functional sites in the receptor-binding domain (RBD) of the spike seen in SARS-CoV-2 and viruses from pangolin SARSr-CoVs are likely caused by mutations and natural selection besides recombination. Population genetic analyses of 103 SARS-CoV-2 genomes indicated that these viruses evolved into two major types (designated L and S), that are well defined by two different SNPs that show nearly complete linkage across the viral strains sequenced to date. Although the L type (∼70%) is more prevalent than the S type (∼30%), the S type was found to be the ancestral version. Whereas the L type was more prevalent in the early stages of the outbreak in Wuhan, the frequency of the L type decreased after early January 2020. Human intervention may have placed more severe selective pressure on the L type, which might be more aggressive and spread more quickly. On the other hand, the S type, which is evolutionarily older and less aggressive, might have increased in relative frequency due to relatively weaker selective pressure. These findings strongly support an urgent need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with coronavirus disease 2019 (COVID-19).

Since beginning of this century, there have already been three zoonotic outbreaks caused by beta coronaviruses (CoV), SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly identified 2019-nCoV in late 2019, Wuhan, China. As to Feb 10th, 2020, there are over 40,000 confirmed cases and over 900 deaths. However, little is known about the biology of this newly emerged virus. Here we developed a lentiviral based pseudovirus system for S protein of 2019-nCoV to study virus entry in BSL2 settings. First, we confirmed that human angiotensin converting enzyme 2 (hACE2) is the main entry receptor for 2019-nCoV. Second, we found that 2019-nCoV S protein mediated entry on 293/hACE2 cells was mainly through endocytosis, and PIKfyve, TPC2, and cathepsin L are critical for virus entry. Third, 2019-nCoV S protein is less stable than SARS-CoV, and it could trigger protease-independent and receptor dependent cell-cell fusion, which might help virus rapidly spread from cell to cell. Finally and more importantly, polyclonal anti-SARS S1 antibodies T62 effectively inhibited entry of SARS-CoV S pseudovirions, but almost had no effect on entry of 2019-nCoV S pseudovirions. Further studies using sera from one recovered SARS-CoV patient and five 2019-nCoV patients showed that there was only limited cross-neutralization activities between SARS-CoV and 2019-nCoV sera, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for 2019-nCoV.

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002–2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients’ sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2., SARS-CoV-2 has spread globally. Here, the authors characterize the entry pathway of SARS-CoV-2, show that the SARS-CoV-2 spike protein is less stable than that of SARS-CoV, and show limited cross-neutralization activities between SARS-CoV and SARS-CoV-2 sera.

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696–10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered. IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide ( 955 IAGVGWTAGL 964 ) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution. IMPORTANCE Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.