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°C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas El, 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-40disrupted virus at 37°C resulted in formation of a complex between one of the viral glycoproteins, El, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in El, resulting in aggregation of El and interaction with the viral RNA in the nucleocapsid. El also bound rRNA. The El-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein El 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.

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...

No abstract

No abstract

The fast, efficient, and accurate release of proteins from cells and tissues is a critically important initial step in most analytical processes and is essential to reliable proteomic analyses. Two-dimensional gel electrophoresis (2DGE) 1 can be an accurate representation of a proteome only if the entire protein constituency of cells is recovered during the sample preparation process. Pressure cycling technology (PCT) uses alternating cycles of high and low hydrostatic pressure to effectively induce the lysis of cells and tissues in preparation for 2DGE and other analytical or preparative methods. Rapid cycling between high and low pressure is more disruptive than high pressure alone, as evidenced by the increased protein yields from Saccharomyces cerevisae correlating to the number of pressure cycles rather than the total elapsed time at high pressure [1]. Similarly, Herrero and coworkers [2] reported a 20% increase in phycobiliproteins yielded from Spirulina platensis when multiple iterations of a pressurized liquid extraction method were performed. Previously, Geiser and coworkers [3] reported the release of 37% more protein from the nematode Caenorhabditis elegans by PCT than by sonication. From gram-negative bacteria, PCT reportedly yielded 14.2% more protein from Escherichia coli than did bead beating [4,5] and yielded 17.1% more protein from Rhodopseudomonas palustris than did enzymatic lysis with lysozyme [6]. For mammalian tissues, PCT also isolated more protein from liver, including several unique proteins that were not isolated by conventional homogenization techniques [7]. From adipose tissue, PCT extracted more protein than did pulverization under liquid nitrogen and detergent extraction of the triturate. The Barocycler NEP-3229 instrument, disposable polypropylene PULSE Tubes FT-500, and ProteoSOLVE IEF Reagent were obtained from Pressure BioSciences (West Bridgewater, MA, USA). Linear immobilized pH gradients (IPGs, pH 3-10) were obtained from Proteome Systems (Woburn, MA, USA). Nonlinear IPGs and PDQuest version 7.1 image analysis software were obtained from Bio-Rad (Hercules, CA, USA). Ultrafree-CL

2-DE is a powerful protein analytical tool whose major strengths include semiglobal quantitation and charge separation of complex protein mixtures, enabling the analysis of differential protein expression, and variable post-translational modification. One of 2-DE's limitations relates to its limited dynamic range and consequently the number of proteins expressed that can be analyzed on a single gel. In an attempt to improve the yield of detectable proteins during sample preparation, we applied a novel extraction technique called pressure cycling technology.

No abstract

No abstract