The crystal structure of the tetrameric DNAbinding domain of the single-stranded DNA binding protein from Escherichia coli was determined at a resolution of 2.9 Å using multiwavelength anomalous dispersion. Each monomer in the tetramer is topologically similar to an oligomer-binding fold. Two monomers each contribute three ␤-strands to a single six-stranded ␤-sheet to form a dimer. Two dimer-dimer interfaces are observed within the crystal. One of these stabilizes the tetramer in solution. The other interface promotes a superhelical structure within the crystal that may ref lect tetramer-tetramer interactions involved in the positive cooperative binding of the single-stranded DNA-binding protein to single-stranded DNA.

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHIV) 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.

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 I 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.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37°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°C (half-life,-24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37°C (half-life,-30 min). Virions treated at pH 8.0 and 37°C formed clumps and large aggregates. With virions treated at pH 8.0 and 37°C, the amino-terminal peptide E2N (or Sl) 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°C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37°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°C or following treatment with guanidine and urea at pH 6.0 and 37°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°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. * Corresponding author. binding to receptors on murine cells (16; S. R. Compton and K. V. Holmes, unpublished data), induction of neutralizing antibodies (7) and cell-mediated cytotoxicity (17, 42), and virus-induced cell fusion (11, 39). The induction of cell fusion by MHV-A59 requires cleavage of the 180,000-molecular weight E2 glycoprotein (180K E2 [or S] glycoprotein)