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 17 Cl 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 El, 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 Saccells, 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. 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 of the cell membranes to the fusing effects of cleaved E2 may also determine the extent...

Coronavirus Spike Protein and Tropism Changes

“…Virus-cell fusion thus not only critically depends on the conformational changes following spike-receptor engagement, and perhaps on acidification of endosomal vesicles (Eifart et al, 2007;Matsuyama and Taguchi, 2009;Zelus et al, 2003), but also on proteolytic activation of the S protein by proteases along the endocytic route (Burkard et al, 2014;Simmons et al, 2005). Indeed, inhibition of intracellular proteases has been shown to block virus entry and virus-cell fusion (Burkard et al, 2014;Frana et al, 1985;Simmons et al, 2005;). The specific proteolytic cleavage requirements of the S protein at the S 1 /S 2 boundary and particularly at the S 2 0 site may furthermore determine the intracellular site of fusion (Burkard et al, 2014).…”

Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses

“…MHV-1 and IAV (A/Puerto Rico/8/1934 (H1N1)) were obtained from the American Type Culture Collection and BEI Resources, respectively. MHV-1 was propagated in 17Cl-1 cells, purified by sucrose gradient centrifugation, and titrated by plaque assay on 17Cl-1 cells, as previously described (Frana et al, 1985;Sturman et al, 1980). PR8 was propagated and titrated by plaque assay in MDCK cells in media containing 1% BSA and TPCK-trypsin (1 g/ml).…”

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

“…The MHV S glycoprotein is 1324 amino acids long; the primary 146 kDa translation product is acylated, N-glycosylated, and trimmed subsequently to yield the mature 180 kDa protein. The mature S protein is post-translationally cleaved in most MHV strains by host proteases into two 90 kDa subunits: an amino terminal subunit (S1) and a carboxy terminal subunit (S2) (Frana et al, 1985). S1-S2 cleavage does not destroy virus infectivity; http://dx.doi.org/10.1016/j.virusres.2014.05.023 0168-1702/© 2014 Elsevier B.V. All rights reserved.…”

“…S1-S2 cleavage does not destroy virus infectivity; http://dx.doi.org/10.1016/j.virusres.2014.05.023 0168-1702/© 2014 Elsevier B.V. All rights reserved. rather it increases the potency of coronaviruses to mediate cell-cell fusion and promotes rapid virus dissemination (Frana et al, 1985;de Haan et al, 2004). S1 makes up the unique globular-head structure of the spike and contains the receptor binding activity; the S2 subunit contains two heptad repeats regions that are part of the stalk structure of the spike and are required for membrane fusion, a trans-membrane domain, and a cytoplasmic domain (in the cell) that is in the interior of the virus particle after budding is completed [reviewed in Heald-Sargent and Gallagher (2012)].…”