Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal.
Infection of primary mouse glial cell cultures with mouse hepatitis virus strain A59 results in a productive, persistent infection, but without any obvious cytopathic effect. Mutant viruses isolated from infected glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type virus but produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion under conditions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late times postinfection, the defect is actually a delay in kinetics rather than an absolute block in activity. Addition of trypsin to mutant-infected fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, suggesting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertants of the mutants supports this hypothesis. Acquisition of fusion competence correlates with the replacement of the negatively charged aspartic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for efficient cell-cell fusion by mouse hepatitis virus but not for virus-cell fusion (infectivity).
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
J L
JL
Gombold
S T
ST
Hingley
S R
SR
Weiss
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001921:Brain / Q000166:cytology; D002455:Cell Division; D002459:Cell Fusion; D002478:Cells, Cultured; D004247:DNA; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D009154:Mutation; D009457:Neuroglia / Q000166:cytology* / Q000382:microbiology; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D004247:DNA