Abstract
The hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA0 precursor is not cleaved to the HA1 and HA2 subunits by tissue culture cell-associated proteases. To investigate if an HA protein could be obtained that could be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. Three HA mutant proteins were constructed, through substitution or insertion of arginine residues, that have 4, 5, or 6 basic residues at their cleavage sites. Chemical cross-linking studies indicated that all three HA cleavage site mutants could oligomerize to a trimeric species, like WT HA. The three HA cleavage site mutant proteins were efficiently transported to the cell surface and bound erythrocytes in hemadsorption assays. The mutants were cleaved at a low level to HA1 and HA2 by an endogenous host cell protease and cleavage could be increased somewhat by addition of exogenous trypsin. The fusogenic activities of the HA cleavage site mutants were assessed in comparison to the WT HA protein by determining their syncytium formation ability and by using an 818 lipid-mixing assay and a NBD-taurine aqueous-content mixing assay. While the fusion activity of the WT HA protein was dependent on exogenous trypsin to activate HA, the three HA cleavage site mutant proteins were able to induce fusion in the absence of trypsin when assayed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing assays, but were unable to induce syncytium formation in either the presence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the influenza B virus HA1/HA2 boundary does enable some HA0 molecules to be cleaved intracellularly, it alone is not sufficient for efficient cleavage.
Original language | English (US) |
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Pages (from-to) | 234-248 |
Number of pages | 15 |
Journal | Virology |
Volume | 236 |
Issue number | 2 |
DOIs | |
State | Published - Sep 29 1997 |
Funding
We thank Peter Palese and Wendy Barclay for generously providing the cDNA to the influenza B/Maryland/59 HA, Sangeeta Bagai for help in establishing the fusion assays, Becky Dutch for her thoughtful comments on the manuscript, and Helen Nestoras for excellent technical assistance. Some of the site-specific mutants were made by Maris Zivarts as part of a C98 project for undergraduates at Northwestern University. This work was supported by Grant R37 AI-20201 from the National Institute of Allergy and Infectious Diseases. R.A.L is an Investigator of the Howard Hughes Medical Institute.
ASJC Scopus subject areas
- Virology