Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

Abstract

Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt؊ or NAt؊) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M 1 ) protein was also found to be associated with DIGs, but this association was decreased in cells infected withHAt؊ or NAt؊ virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M 2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt؊ and NAt؊ is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt؊ and NAt؊ viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.

Influenza virus morphogenesis and budding

“…The viral envelope consists of a lipid bilayer containing three transmembrane proteins HA (hemagglutinin), NA (neuraminidase), and M2 (ion channel) on the outside and M1 (matrix protein) underneath the membrane. The influenza virus lipid bilayer is a mosaic structure containing both cholesterol-enriched lipid rafts and nonraft lipids derived from the host plasma membrane (Scheiffele et al, 1999;Zhang et al, 2000). HA and NA are anchored in the lipid raft domain of the viral envelope, whereas M2, though a cholesterol-binding protein, is not tightly associated with lipid rafts (Schroeder et al, 2005).…”

“…In the envelope of released virions, both HA and NA remain raft associated, whereas M2 does not associate with lipid rafts. Therefore, the influenza viral envelope is not a homogeneous lipid membrane but a mosaic mixture of both raft-associated and non-raft-associated lipid microdomains even though the majority of lipids present in the viral envelope are in the highly ordered l o phase, and the influenza virus envelope is enriched in cholesterol-dependent detergent-insoluble lipids (Zhang et al, 2000;Scheiffele et al, 1999). Furthermore, protein-protein interactions may facilitate bringing non-raft-associated proteins to lipid raft microdomains.…”

“…However, little is known about the role of lipid rafts in the budding process, specifically in bud release. Cholesterol is a known critical structural component of lipid rafts and depletion of cholesterol leads to disorganization of lipid raft microdomains and dissociation of proteins bound to the lipid rafts (Hanada et al, 1995;Hannan and Edidin, 1996;Zhang et al, 2000;Scheiffele et al, 1999). Recently, it was shown that depletion of cholesterol from virus-infected cells by M␤CD treatment for a short time at the late phase of infection facilitated bud completion and increased virus particle release (Barman and Nayak, 2007).…”

“…M␤CD is a strictly surface-acting agent and can selectively and rapidly remove cholesterol from the plasma membrane in preference to other membrane lipids and has been widely used in studying the effects of cholesterol depletion and lipid raft disassembly (Gosselin-Grenet et al, 2006;Ono and Freed, 2001). Therefore, short M␤CD treatment at the late phase of infection was done to avoid the effect of lipid raft disruption on protein transport (Carrasco et al, 2004;Keller and Simons, 1998;Prydz and Simons, 2001) and virus assembly (Leser and Lamb, 2005;Scheiffele et al, 1999;Takeda et al, 2003;Zhang et al, 2000).…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…When expressed alone, the matrix protein of influenza virus does not bind to detergentresistant membranes (Ali et al, 2000). However, in the presence of viral envelope glycoproteins, the matrix protein of influenza interacts with HA and NA localized in rafts, and the matrix protein associated with membranes becomes detergent resistant (Ali et al, 2000;Zhang et al, 2000b). Thus the envelope glycoproteins, which are targeted to rafts, can drag other viral components so as to promote assembly within rafts, a location where influenza virus has been shown to assemble and bud (ScheiVele et al, 1999).…”