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.     

Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions.

Monoclonal antibodies (MAbs) directed against the E2 glycoprotein of mouse hepatitis virus (MHV) have been classified according to their ability to bind to either of the two purified 90,000-molecular-weight subunits (90K subunits) of the 180K peplomeric glycoprotein E2. Correlation with previously reported information about these MAbs suggest that both of the subunits of E2 are important for viral infectivity and cell fusion. Incubation of trypsin-treated virions at pH 8.0 and 37 degrees C released only the E2N subunit from virions. The pattern of MAb reactions suggested that a conformational change occurred in the E2N subunit in association with its release from virions under mildly alkaline conditions at 37 degrees C, the same conditions which are optimal for coronavirus-induced cell fusion.     

Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.     

The N-terminal domain of the murine coronavirus spike glycoprotein determines the CEACAM1 receptor specificity of the virus strain

Using isogenic recombinant murine coronaviruses expressing wild-type murine hepatitis virus strain 4 (MHV-4) or MHV-A59 spike glycoproteins or chimeric MHV-4/MHV-A59 spike glycoproteins, we have demonstrated the biological functionality of the N-terminus of the spike, encompassing the receptor binding domain (RBD). We have used two assays, one an in vitro liposome binding assay and the other a tissue culture replication assay. The liposome binding assay shows that interaction of the receptor with spikes on virions at 37 degrees C causes a conformational change that makes the virions hydrophobic so that they bind to liposomes (B. D. Zelus, J. H. Schickli, D. M. Blau, S. R. Weiss, and K. V. Holmes, J. Virol. 77: 830-840, 2003). Recombinant viruses with spikes containing the RBD of either MHV-A59 or MHV-4 readily associated with liposomes at 37 degrees C in the presence of soluble mCEACAM1(a), except for S(4)R, which expresses the entire wild-type MHV-4 spike and associated only inefficiently with liposomes following incubation with soluble mCEACAM1(a). In contrast, soluble mCEACAM1(b) allowed viruses with the MHV-A59 RBD to associate with liposomes more efficiently than did viruses with the MHV-4 RBD. In the second assay, which requires virus entry and replication, all recombinant viruses replicated efficiently in BHK cells expressing mCEACAM1(a). In BHK cells expressing mCEACAM1(b), only viruses expressing chimeric spikes with the MHV-A59 RBD could replicate, while replication of viruses expressing chimeric spikes with the MHV-4 RBD was undetectable. Despite having the MHV-4 RBD, S(4)R replicated in BHK cells expressing mCEACAM1(b); this is most probably due to spread via CEACAM1 receptor-independent cell-to-cell fusion, an activity displayed only by S(4)R among the recombinant viruses studied here. These data suggest that the RBD domain and the rest of the spike must coevolve to optimize function in viral entry and spread.     

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.     

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.     

Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism

Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein.

In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.     

Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly.

The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.     

Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate

The mechanism of membrane fusion by “class II” viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein (E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The ∼55-aa-residue, membrane proximal “stem” can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4°C rather than 37°C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.     

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.     

Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.     

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.     

Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.     

Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation

Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH₄Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.     

Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity

Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.     

Two distinct low-pH steps promote entry of vaccinia virus

Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37 degrees C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.     

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion.

Cell fusion induced by infection with mouse hepatitis virus strain A59 (MHV-A59) varied markedly in extent and time course in four different murine cell lines. When inoculated at a multiplicity of 3 to 5 PFU per cell, the Sac-, L2, and DBT cell lines began to fuse by 7 h, were fused into confluent syncytia by 9 to 12 h, and peeled from the substrate by 10 to 14 h. These virulent virus-cell interactions were in striking contrast to the moderate interaction of MHV-A59 with the 17 Cl 1 cell line, in which only small syncytia were observed 18 h postinoculation, and greater than 50% of the cells remained unfused by 24 h. The yield of infectious virus produced by 17 Cl 1 cells was 10-fold higher than the yields from the other three cell lines. The processing of the nucleocapsid protein, the membrane glycoprotein E1, and the peplomeric glycoprotein E2 were found to differ significantly in the four cell lines. Since the E2 glycoprotein is responsible for virus-induced cell fusion, we attempted to correlate differences in cellular processing of E2 with differences in fusion of infected cells. The predominant intracellular form of E2 in all cell lines was the 180K species. Pulse-chase experiments showed that a small portion of the 17 Cl 1 cell-associated 180K E2 was cleaved by 1 h after synthesis to yield 90K E2, shown in the preceding paper to consist of two different glycoproteins called 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes, J. Virol. 56:904-911, 1985). This cleavage occurred shortly before the release of virions from cells, as shown by pulse-chase experiments. After budding at intracellular membranes, virions released into the medium by the four cell lines contained different ratios of 180K to 90K E2. Virions from Sac- cells, which contained 100% 90K E2, fused L2 cells rapidly without requiring virus replication, whereas virions from 17 Cl 1 cells, which had 50% 90K E2, required trypsin activation to induce rapid fusion (Sturman et al., J. Virol. 56:904-911, 1985). The addition of protease inhibitors to the medium markedly delayed L2 cell fusion induced by MHV infection. The extent of coronavirus-induced cell fusion does not depend solely upon the percent cleavage of the E2 glycoprotein by cellular proteases, since extensive fusion was induced by infection of L2 and DBT cells but not 17 Cl 1 cells, although all three cell lines cleaved E2 to the same extent.(ABSTRACT TRUNCATED AT 400 WORDS)