人冠状病毒NL63核壳蛋白和棘突蛋白的表达及其在血清学检测中的初步应用

目的:重组表达人冠状病毒NL63(HCoV-NL63)的核壳蛋白(N蛋白)及棘突蛋白(S蛋白),用于检测血清中的相应抗体。方法:用原核表达系统表达HCoV-NL63的N蛋白,建立检测N抗体的Werstern印迹法;用真核表达系统表达HCoV-NL63的S蛋白,建立检测S抗体的间接免疫荧光(IFA)法。结果:经Werstern印迹检测,重组S蛋白和N蛋白表达正确;初步建立了N蛋白纯化方法。利用建立的检测方法,检测了100份正常成人血清,总阳性率为81%。其中S抗体阳性率为66%,N抗体阳性率为38%,S抗体和N抗体均为阳性的占总数的22%,双抗体均为阴性的占总数的19%;S抗体的检出率明显高于N抗体。结论:重组HCoV-NL63N蛋白及S蛋白表达成功;S抗体和N抗体共同检测可获得较好的检测结果,减少漏检。     

Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core

Severe acute respiratory syndrome coronavirus is a newly emergent virus responsible for a recent outbreak of an atypical pneumonia. The coronavirus spike protein, an enveloped glycoprotein essential for viral entry, belongs to the class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions are understood to form a fusion-active conformation similar to those of other typical viral fusion proteins. This hairpin structure likely juxtaposes the viral and cellular membranes, thus facilitating membrane fusion and subsequent viral entry. The fusion core protein of severe acute respiratory syndrome coronavirus spike protein was crystallized, and the structure was determined at 2.8 A of resolution. The fusion core is a six-helix bundle with three HR2 helices packed against the hydrophobic grooves on the surface of central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. This structure shares significant similarity with the fusion core structure of mouse hepatitis virus spike protein and other viral fusion proteins, suggesting a conserved mechanism of membrane fusion. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation, which have been successfully used in human immunodeficiency virus 1 inhibitor development, may be applicable to the inhibition of severe acute respiratory syndrome coronavirus on the basis of structural information provided here. The relatively deep grooves on the surface of the central coiled coil will be a good target site for the design of viral fusion inhibitors.     

The Nucleocapsid Protein of Human Coronavirus NL63

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.     

Influence of hydrophobic and electrostatic residues on SARS‐coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS‐coronavirus (SARS‐CoV). SARS‐CoV entry is facilitated by the spike protein (S), which consists of an N‐terminal domain (S1) responsible for cellular attachment and a C‐terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS‐CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site‐directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH‐independent retroviral fusion proteins, SARS‐CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS‐CoV S2 analysis showed that specific hydrophobic positions at the C‐terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C‐terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C‐terminal hydrophobic residues led us to identify a 42‐residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.     

Mosaic structure of human coronavirus NL63, one thousand years of evolution

Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.     

SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.     

Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.     

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.     

北京地区人冠状病毒NL63 N和E蛋白基因序列及生物信息学分析

为了解北京地区新近发现的新型冠状病毒-人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)的N和E蛋白编码基因的特征,从经RT-PCR检测阳性的临床标本中扩增得到的HCoV-NL63 N蛋白和E蛋白编码基因序列,分别克隆至pCF-T和pUCm-T载体中并进行测序,同时运用生物信息学的方法,对北京HCoV-NL63阳性标本BJ8081 N和E蛋白编码基因的核苷酸和氨基酸序列与HCoV-NL63原型株及其他几种冠状病毒的N和E蛋白编码基因的核苷酸和氨基酸序列进行比较分析和种系进化分析;用SOPMA方法对BJ8081 N和E蛋白的二级结构进行了预测分析,并对N和E蛋白的其他生物学特性进行了预测分析.经序列比对分析发现,BJ8081 N蛋白氨基酸序列在78～85肽段(FYYLGTGP)内与所比较的其他冠状病毒N蛋白相应位置的氨基酸序列完全相同,提示此区段可能为包括HCoV-NL63在内的所有冠状病毒N蛋白的保守区域.在BJ8081 N蛋白氨基酸序列的100～121肽段可能是和基因组RNA相结合的位置;在BJ8081 E蛋白的15～37位氨基酸可能是E蛋白的跨膜区域.研究对BJ8081 N蛋白和E蛋白的编码基因序列进行了测定和生物信息学分析,为今后对HCoV-NL63的进一步深入研究奠定了基础.     

Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.     

Identification of a new human coronavirus

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.     

Trimeric, coiled-coil extension on peptide fusion inhibitor of HIV-1 influences selection of resistance pathways

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.     

Elastase-mediated Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein at Discrete Sites within the S2 Domain

Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr⁷⁹⁵ in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.     

Characterization and Complete Genome Sequence of Human Coronavirus NL63 Isolated in China

Human coronavirus NL63 (HCoV-NL63) was first discovered in Amsterdam in 2004 and was identified as a new human respiratory coronavirus. We here report the first complete genome sequence of HCoV-NL63 strain CBJ 037 isolated in 2008 from a patient with bronchitis in Beijing, China.     

Fusion core structure of the severe acute respiratory syndrome coronavirus (SARS-CoV): in search of potent SARS-CoV entry inhibitors

Severe acute respiratory coronavirus (SARS-CoV) spike (S) glycoprotein fusion core consists of a six-helix bundle with the three C-terminal heptad repeat (HR2) helices packed against a central coiled-coil of the other three N-terminal heptad repeat (HR1) helices. Each of the three peripheral HR2 helices shows prominent contacts with the hydrophobic surface of the central HR1 coiled-coil. The concerted protein-protein interactions among the HR helices are responsible for the fusion event that leads to the release of the SARS-CoV nucleocapsid into the target host-cell. In this investigation, we applied recombinant protein and synthetic peptide-based biophysical assays to characterize the biological activities of the HR helices. In a parallel experiment, we employed a HIV-luc/SARS pseudotyped virus entry inhibition assay to screen for potent inhibitory activities on HR peptides derived from the SARS-CoV S protein HR regions and a series of other small-molecule drugs. Three HR peptides and five small-molecule drugs were identified as potential inhibitors. ADS-J1, which has been used to interfere with the fusogenesis of HIV-1 onto CD4+ cells, demonstrated the highest HIV-luc/SARS pseudotyped virus-entry inhibition activity among the other small-molecule drugs. Molecular modeling analysis suggested that ADS-J1 may bind to the deep pocket of the hydrophobic groove on the surface of the central coiled-coil of SARS-CoV S HR protein and prevent the entrance of the SARS-CoV into the host cells.     

Construct design, biophysical, and biochemical characterization of the fusion core from mouse hepatitis virus (a coronavirus) spike protein

Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses.     

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.