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Development of protease activation mutants of HVJ (Sendai virus) in persistently infected cell cultures
Abstract:
HVJ wild-type virus, in which the F protein is activated by trypsin but not by elastase, was spontaneously converted to a mutant with an F protein characterized by being activated by elastase alone. This spontaneous mutation generally occurred during serial passages of cells persistently infected with HVJ, even though the cells were first established by infection with plaque-purified wild-type virus. Multiple-cycle replication, plaque formation, haemolysis and SDS-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed that all the elastase-activated mutants isolated from HVJ carrier cells no longer required trypsin for F protein activation. At early passages, these protease activation mutants did not show temperature-sensitive (ts) growth, while at a later stage the mutants, together with the ts mutation, appeared dominant. The frequency of such a protease activation mutation during passage in the HVJ carrier cells seemed to depend on the cell species, but was increased when compared to lytic infections.
Insights
Hendra virus (HVJ) spontaneously mutated during cell passage, altering its F protein activation from trypsin to elastase alone. This mutation, linked to persistent infections, eventually co-occurred with temperature-sensitive growth properties.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Hendra virus (HVJ) is a paramyxovirus with a fusion (F) protein typically activated by host proteases like trypsin.
- Persistent viral infections in cell cultures can lead to viral adaptation and genetic alterations.
- Understanding viral F protein activation is crucial for studying viral entry and pathogenesis.
Purpose of the Study:
- To investigate the spontaneous mutations in HVJ during persistent cell culture infections.
- To characterize the altered protease activation of the HVJ F protein in isolated mutants.
- To examine the relationship between F protein activation mutations and temperature-sensitive (ts) growth properties.
Main Methods:
- Serial passage of HVJ-infected cells to induce and isolate mutants.
- Analysis of viral replication, plaque formation, and haemolysis assays.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein analysis.
- Assessment of temperature-sensitive (ts) growth characteristics at different passage stages.
Main Results:
- Spontaneous mutation of HVJ resulted in F protein activation by elastase alone, independent of trypsin.
- Elastase-activated mutants were isolated from persistently infected HVJ carrier cells.
- These mutants showed altered protease activation profiles and, at later stages, acquired temperature-sensitive (ts) growth.
- Mutation frequency varied with cell species and was higher in carrier cells than in lytic infections.
Conclusions:
- Persistent HVJ infection in cell cultures can drive spontaneous mutations in viral protease activation mechanisms.
- The acquisition of elastase-dependent F protein activation is a significant adaptation.
- The emergence of temperature-sensitive growth alongside protease activation mutations suggests complex viral evolution under specific culture conditions.