Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin

Abstract

The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

“…For enveloped viruses, both cytoplasmic and transmembrane structural proteins use palmitoylation as a mechanism to achieve or enhance membrane association during budding. Palmitoylation of influenza virus hemagglutinin, a transmembrane protein, increases its association with membrane microdomains, where it interacts with the M2 protein during budding (50)(51)(52). In herpes simplex virus, the tegument protein UL51 relies on palmitoylation for association with the Golgi membrane, where secondary envelopment occurs (53,54).…”

Acid-Induced Membrane Fusion by the Hemagglutinin Protein and Its Role in Influenza Virus Biology

“…N-linked glycosylation contributes to protein folding in the ER (Hebert et al 1995). As the HA protein trafficks intracellularly to the cell surface, N-linked glycosylation sites undergo maturation and free cysteine residues in the TM domain and CT are acylated with fatty acids (Naeve and Williams 1990;Veit et al 1991;Steinhauer et al 1991b).…”

Apical Budding of a Recombinant Influenza A Virus Expressing a Hemagglutinin Protein with a Basolateral Localization Signal

“…The cysteine residue 560 in the cytoplasmic tail of the HA of influenza viruses has previously been shown to be palmitoylated (40,41,57,58,64). As HA was apically transported in cells infected with transfectant viruses C560A and C560F, the altered transport of the HA of C560Y virus was due to the presence of a new tyrosine residue in the cytoplasmic tail rather than to the loss of a palmitoylation site.…”