ADAPTATION OF EGG-GROWN AND TRANSFECTANT INFLUENZA-VIRUSES FOR GROWTHIN MAMMALIAN-CELLS - SELECTION OF HEMAGGLUTININ MUTANTS WITH ELEVATEDPH OF MEMBRANE-FUSION

A series of eight transfectant influenza viruses was generated by reve rse genetics for studies of the palmitylated cysteine residues in the cytoplasmic tail of the hemagglutinin glycoprotein (HA). Following amp lification of these viruses in MDCK cells we found that all had develo ped an elevated pH of membrane fusion - an unexpected result since pre vious mutant HA expression studies had shown that substitutions of the cysteine residues had no effect on fusion properties. Sequence analys es revealed that each of the viruses had at least one additional mutat ion in the ectodomain of HA which was responsible for the increase in fusion pH. Similarly, when we passaged egg-grown wild-type X-31 virus in three different lines of MDCK cells or in MDBK cells, high pH fusio n mutants were selected within a few passages in every case. The locat ions of the substitutions in the HA structure are in or near the ''fus ion peptide'' or at subunit interfaces throughout the length of the tr imer - reminiscent of the changes selected in earlier studies on amant adine resistance. The observation that passage of certain viruses in m ammalian cells can result in the selection of mutants with elevated fu sion pH has potential implications both for reverse genetic experiment s and, perhaps more importantly, for the choice of substrates for prop agation of vaccine viruses. (C) 1997 Academic Press.