MUTATIONS AT PALMITYLATION SITES OF THE INFLUENZA-VIRUS HEMAGGLUTININAFFECT VIRUS FORMATION

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among EW s ubtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attach ment of palmitic acid. In order to study the role of the acylated cyst eines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reve rse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. T he cysteine at position 560 can be changed to alanine or tyrosine to y ield virus strains that ore attenuated in cell cultures. The change fr om cysteine at position 553 to serine or alanine does not significantl y alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytop lasmic tail. This interpretation is further supported by experiments i n which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutatio ns resulted in infectious virus. Selection of revertants of the attenu ated cysteine-to-tyrosine mutant (mutation at position 560) always res ulted in reversion to cysteine rather than to other amino acids. Altho ugh our data indicate a biological role for the conserved cysteine res idues in the cytoplasmic tail of the HA of influenza viruses, we canno t exclude the possibility that structural constraints in the cytoplasm ic tail of the HA-rather than altered palmitylation-are the determinin g factors for infectious-particle formation.