Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology

Abstract

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membraneenveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCEInfluenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two-and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

“…2A and B). HANAM1M2 filamentous particles had NA clusters localized at the tip of the VLP as reported before (8), whereas MAYNAM1M2 filamentous particles contained, overall, more NA glycoproteins, and NA clusters were also present on the cylindrical surface of the filamentous particle. Importantly, 52% (n ϭ 29) of filamentous MAYNAM1M2 particles contained only NA, an observation consistent with the increased release of NA detected by Western blotting.…”

“…A previous study showed that HA expression induces ruffling of the plasma membrane and shedding of HA containing spheroidal particles, suggesting that HA is able to alter membrane curvature (8). To address whether HA acylation has a role in modulating membrane curvature, we compared the diameters of released spheroidal VLPs containing either HANA or MAYNA but with no M1.…”

“…To prevent reentry into the producing cell and promote virion release, NA concentrates at the rear tip of the budding virion where cleavage of sialic acid moieties on the surface of the plasma membrane occurs (7,13). The resulting virus particles have pleomorphic geometry, with a predominance of spheroidal and filamentous shapes (8). Effects of glycoproteins on the viral envelope curvature have been reviewed recently (2).…”

“…Both HA and NA interact with M1 protein via their cytoplasmic tails (CTs) (4,5). M1, which electrostatically binds to negatively charged lipids (6) and assembles in a helical array beneath the inner monolayer of the plasma membrane (7), is an important determinant of virion morphology (8,9) and provides a platform for viral genome incorporation (10). The negative-sense RNA genome is segmented into eight physically independent viral ribonucleoprotein (vRNP) complexes which have an average diameter of ϳ14 nm and a length ranging from ϳ24 to ϳ110 nm (11).…”

Late stages of the influenza A virus replication cycle—a tight interplay between virus and host

“…It has been demonstrated that not only M1 has a major influence on the shape of the particle but also HA, NA, M2 and NP have been suggested to impact virus morphology (Bourmakina & Garcia-Sastre, 2003;Elleman & Barclay, 2004;Rossman et al, 2010a;Bialas et al, 2014;Chlanda et al, 2015). Given that, most up-to-date studies on assembly and egress of influenza virus have been performed with laboratory-adapted spherical virions, this review will focus on budding of spherical virions.…”