Role of protease in mouse hepatitis virus-induced cell fusion. Studies with a cold-sensitive mutant isolated from a persistent infection

Abstract

A plaque mutant was isolated from Kirsten mouse sarcoma virus-transformed BALB/c cells persistently infected with a mouse hepatitis virus strain MHV-S. While the wild type produced large plaques consisting of fused cells (fusion type) both at 39 and at 32°, the mutant produced small fusion-type plaques at 39°, and at 32°, only after longer incubation, plaques consisting of round dead cells (non-fusion type) were obtained. The mutant grew equally well at both temperatures. Thus, the mutant was cold sensitive in inducing cell fusion, but not in replication or in ultimate cell killing. The cold sensitivity was overcome by the addition of trypsin (0.04 to 0.05%) to the culture medium, but not by treating the virions with trypsin. SDS-polyacrylamide gel electrophoresis of the virion proteins failed to detect differences between the wild type and the mutant. In intracellular viral proteins immunoprecipitated with anti-MHV-S rabbit serum, a protein of 68,000 daltons (68K protein) which was present in the wild type-infected cells was absent in the mutant-infected cells, but trypsin treatment or incubation at 39° of the mutant-infected cells failed to induce the 68K protein. After six to seven undiluted passages, the mutant segregated varieties of mutants which were partially or totally reverted to the wild type in phenotype, and also those whose cell fusion induction was absent even at 39°. All these mutants failed to induce the 68K protein in the infected cells. Thus, there was no linkage between the presence of 68K protein and the fusion induction. Trypsin treatment of the infected cells enabled MHV-S to form fusion plaques on otherwise resistant cells, and MHV-2, a producer of non-fusion-type plaques, to form fusion-type plaques. Protease appears to play an important role in MHV-induced cell fusion in general.