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

Abstract

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 17CL-1 cell line, in which only small syncytia were observed 18 h postinoculation, and > 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 ines. Since the E2 glycoprotein is responsible for virus-induced cell fusion, we attempted to correlated 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 consiste 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 protease, since extensive fusion was induced by infection of L2 and DBT cells but not 17 Cl 1cells, although all three cell lines cleaved E2 to the same extent. Differences observed between the molecular weights of the E2 cleavage products in several cell lines could result from host cell-dependent differences in glycosylation or cleavage of E2. Such changes in E2 processing could affect the cell-fusing activity of the glycoprotein. Cell lines also differ in susceptibility to the immediate cell-fusing effects of concentrated MHV (Sturman et al., J. Virol. 56:904-911, 1985). Thus, host-dependent differences in the precise location of the cleavage site of E2, the rate of transport of cleaved E2 to the cell membrane, or the response to the cell membranes to the fusing effects of cleaved E2 may also determine the extent of MHV-induced fusion of various cell types.