Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin

Abstract

Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

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).…”

Apical Trafficking Pathways of Influenza A Virus HA and NA via Rab17- and Rab23-Positive Compartments

“…It is well known that influenza A virus HA and NA are associated with lipid raft microdomains (Barman and Nayak, 2000; Zhang et al, 2000; Scolari et al, 2016), which play pivotal roles in apical PM trafficking in polarized cells (Simons and Ikonen, 1997; Rodriguez-Boulan et al, 2005; Rog and Vattulainen, 2014). Previous studies on HA have shown that the disruption of HA association with lipid rafts by the use of non-raft mutants and treatment of cholesterol-lowering drugs cause a delay or block in TGN-to-apical PM trafficking of HA (Keller and Simons, 1998; Ohkura et al, 2014).…”

Identification of a Novel Universal Potential Epitope on the Cytoplasmic Tail of H7N9 Virus Hemagglutinin

“…HA-CT (Siche et al 2015) plays an important role in virus replication (Imai et al 2012), viral infectivity (Siche et al 2015), and virus entry into host cells (Scolari et al 2016). Although its functions have been well studied, little is known about the immunogenicity and sub-localization of HA-CT.…”