EXPRESSION OF INFLUENZA-B VIRUS HEMAGGLUTININ CONTAINING MULTIBASIC RESIDUE CLEAVAGE SITES

The hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA(0) precursor is not c leaved to the HA(1) and HA(2) subunits by tissue culture cell-associat ed proteases. To investigate if an HA protein could be obtained that c ould be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. T hree HA mutant proteins were constructed, through substitution or inse rtion of arginine residues, that have 4, 5, or 6 basic residues at the ir cleavage sites. Chemical cross linking studies indicated that a II th ree HA cleavage site mutants could oligomerize to a trimeric specie s, like WT HA, The three HA cleavage site mutant proteins were efficie ntly transported to the cell surface and bound erythrocytes in hemadso rption assays. The mutants were cleaved at a low level to HA(1) and HA (2) by an endogenous host cell protease and cleavage could be increase d 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 usi ng an R18 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 p roteins were able to induce fusion in the absence of trypsin when assa yed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing a ssays, but were unable to induce syncytium formation in either the pre sence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the in fluenza B virus HA(1)/HA(2) boundary does enable some HA(0) molecules to be cleaved intracellularly, it alone is not sufficient for efficien t cleavage. (C) 1997 Academic Press.