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Influenza virus-mediated membrane fusion: Structural insights from electron microscopy.

ABSTRACT: Influenza virus, the causative agent of flu, enters the host cell by endocytosis. The low pH encountered inside endosomes triggers conformational changes in the viral glycoprotein hemagglutinin (HA), that mediate fusion of the viral and cellular membranes. This releases the viral genome into the cytoplasm of the infected cell, establishing the onset of the replication cycle. To investigate the structural basis of HA-mediated membrane fusion, a number of techniques have been employed. These include X-ray crystallography, which has provided atomic models of the HA ectodomain in its initial (pre-fusion) state and of part of HA in its final (post-fusion) state. However, this left an information deficit concerning many other aspects of the fusion process. Electron microscopy (EM) approaches are helping to fill this void. For example, influenza virions at neutral pH have been imaged by cryo-EM and cryo-electron tomography (cryo-ET); thin section EM has shown that influenza viruses enter the cell by endocytosis; the large-scale structural changes in HA when virions are exposed to low pH (pre-fusion to post-fusion states) have been visualized by negative staining and cryo-EM; acidification also induces structural changes in the M1 matrix layer and its separation from the viral envelope; intermediate HA conformations between its pre- and post-fusion states have been detected by cryo-ET supplemented with subtomogram averaging; and fusion of influenza virions with liposomes has been visualized by cryo-ET. In this review, we survey EM-based contributions towards the characterization of influenza virus-mediated membrane fusion and anticipate the potential for future developments.

SUBMITTER: Fontana J

PROVIDER: S-EPMC4543556 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Influenza virus-mediated membrane fusion: Structural insights from electron microscopy.

Fontana Juan J Steven Alasdair C AC

Archives of biochemistry and biophysics 20150506

Influenza virus, the causative agent of flu, enters the host cell by endocytosis. The low pH encountered inside endosomes triggers conformational changes in the viral glycoprotein hemagglutinin (HA), that mediate fusion of the viral and cellular membranes. This releases the viral genome into the cytoplasm of the infected cell, establishing the onset of the replication cycle. To investigate the structural basis of HA-mediated membrane fusion, a number of techniques have been employed. These inclu ...[more]

PMID: 25958107

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Project description:The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion.IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.

Dataset Information

Influenza virus-mediated membrane fusion: Structural insights from electron microscopy.

Publications

Influenza virus-mediated membrane fusion: Structural insights from electron microscopy.

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