A conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions.

The coronavirus spike glycoprotein (S) mediates both the attachment of virus to the host cell receptor and membrane fusion. We describe here the characterization of a temperature-sensitive mutant of the coronavirus mouse hepatitis virus A59 (MHV-A59) having multiple S protein-related defects. The most remarkable of these was that the mutant, designated Albany 18 (Alb18), assembled virions devoid of the S glycoprotein at the nonpermissive temperature. Alb18 also failed to bring about syncytia formation in cells infected at the nonpermissive temperature. Virions of the mutant assembled at the permissive temperature were much more thermolabile than wild type. Moreover, mutant S protein that was incorporated into virions at the permissive temperature showed enhanced pH-dependent thermolability in its ability to bind to the MHV receptor. Alb18 was found to have a single point mutation in S resulting in a change of serine 287 to isoleucine, and it was shown by revertant analysis that this was the lesion responsible for the phenotype of the mutant.

Site-directed mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination.

We have genetically characterized a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV). This mutant, designated Alb4, is both temperature-sensitive and thermolabile, and its N protein is smaller than wild-type N. Sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. PCR analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronaviral genomic RNA and a tailored, synthetic RNA species. To date, we have constructed three additional mutants by this procedure. For two of these, a second-site point mutation that reverts the Alb4 phenotype has been transduced into a wild type background, which does not contain the Alb4 deletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination.

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a tailored, synthetic RNA species.

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.

Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site.

The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L.S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904-911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and JHM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.

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)

Isolation of the subunits of the coronavirus envelope glycoprotein E2 by hydroxyapatite high-performance liquid chromatography.

The coronavirus glycoprotein E2, which is responsible for virus attachment to cell receptors and virus-induced cell fusion, was purified by solubilization of virions with Triton X-114 and phase fractionation. Native E2 and tryptic subunits of the glycoprotein were separated by size-exclusion high-performance liquid chromatography (HPLC). Two distinct 90 kD E2 subunits, which had identical electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were separated by hydroxyapatite HPLC in the presence of sodium dodecyl sulfate.

Virus adsorption to mineral surfaces is reduced by microbial overgrowth and organic coatings.

In experiments with strains of poliovirus, reovirus, echovirus, and Coxsackievirus, overgrowth with exopolymer-forming bacteria reduced virus adsorption to mineral surfaces. Adsorption was improved when organic materials adsorbed to minerals were removed by low-temperature ashing. In a soil series, virus adsorption increased with soil depth. This paralleled a decrease in organic content, but differences in charge development of the soil particles may also be involved. Prolonged (40-year) irrigation with treated sewage effluents, leading to a buildup of organic coatings, also decreased adsorption. However, saturation of the virus-binding capacity of the soil as a result of continuous exposure to virus-containing effluents was not apparent.

Adsorption of reovirus by minerals and soils.

Adsorption of [35S]methionine-labeled reovirus by 30 dry soils, minerals, and finely ground rocks suspended in synthetic freshwater at pH 7 was investigated to determine the conditions necessary for optimum virus removal during land application of wastewaters. All of the minerals and soils studied were excellent adsorbents of reovirus, with greater than 99% of the virus adsorbed after 1 h at 4 degrees C. Thereafter, virus remaining in suspension was significantly inactivated, and within 24 h a three to five log10 reduction in titer occurred. The presence of divalent cations, i.e., Ca2+ and Mg2+, in synthetic freshwater enhanced removal, whereas soluble organic matter decreased the amount of virus adsorbed in secondary effluent. The amount of virus adsorbed by these substrates was inversely correlated with the amount of organic matter, capacity to adsorb cationic polyelectrolyte, and electrophoretic mobility. Adsorption increased with increasing available surface area, as suspended infectivity was reduced further by the more finely divided substrates. However, the organic content of the soils reduced the level of infectious virus adsorbed below that expected from surface area measurements alone. The inverse correlation between virus adsorption and substrate capacity for cationic polyelectrolyte indicates that the adsorption of infectious reovirus particles is predominately a charged colloidal particle-charged surface interaction. Thus, adsorption of polyelectrolyte may be useful in predicting the fate of viruses during land application of sewage effluents and sludges.