Structural organization of a filamentous influenza A virus

Abstract

Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.

Fig. 1.

Tomogram sections of frozen-hydrated influenza A virions recorded in a single tomogram field. (A–G) A/Udorn/72 virions from single field shown in Fig. S1A and Movie S1. Virion in A is identical to that in B, but has been manually colored to indicate features corresponding to RNPs (red), multilayered-coil (blue), NA (purple), and HA (green). (K, L, and M) A/Aichi/68 X-31 virions from single field shown in Fig. S1b and Movie S2. Negative stain images of (H) HA rosette (green HA trimer model inset), (I) NA rosette (purple NA head domain tetramer model inset), and (J) RNP purified from X-31. Red lines are used for comparison of RNP length. Purple arcs in D and K indicate typical NA clusters at one end.

Fig. 2.

Low-dose images of Udorn virions. (A) Capsule-shaped virion at pH 7 and (B and C) after bromelain digestion.

Fig. 3.

Filamentous influenza A Udorn shows a helical organization of the matrix protein. (A) Image of a virion. (C) Fourier transform of the area within the membrane (black box) in A indicates a helical organization. A lattice (red) shows the prominent reflections arising from one side of the helix. The 38-Å reflection arises from a 7-start helix. (B) Images obtained after two-sided (Upper) and one-sided (Lower) filtering of the transform in C on three layer lines and the equator (indicated by black ticks at right).

Fig. 4.

Tomogram (also shown in Movies S3 and S4) section showing frozen-hydrated influenza A Udorn virions after acid treatment for 10 min at pH 4.9 and trypsin treatment after neutralization.

Fig. 5.

Gallery of multilayered coils from acid and trypsin-treated virions. (A) Spherical virion with multilayered coil axis perpendicular to image and resolved membrane. (B) Filamentous particle showing both intact matrix layer and matrix layer peeling off membrane to form a multilayered coil. (C) Small coil showing layered structure. (D) Multilayered coil viewed perpendicular to axis showing membrane attachment. (E) Side-on view of multilayered coil attached to membrane. (F) Same coil as E viewed down axis.