Influenza virus pleiomorphy characterized by cryoelectron tomography

Audray Harris

Giovanni Cardone

Dennis Winkler

J Heymann

Matthew Brecher

Judith White

Alasdair Steven

*Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; and

^{Department of Microbiology, University of Virginia, Charlottesville, VA 22908}

^{To whom correspondence should be addressed. E-mail:}vog.hin.liam@anevets

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved October 13, 2006

Author contributions: A.H. and G.C. contributed equally to this work; A.H., J.M.W., and A.C.S. designed research; A.H., G.C., D.C.W., J.B.H., and M.B. performed research; G.C. and J.B.H. contributed new reagents/analytic tools; A.H., G.C., D.C.W., and J.B.H. analyzed data; and A.H., G.C., and A.C.S. wrote the paper.

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved October 13, 2006

Received 2006 Sep 7

Abstract

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer (“molecular fontanels”), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Keywords: envelope glycoproteins, matrix protein, ribonucleoprotein particles, virus assembly, virus structure

Abstract

Influenza virus belongs to the orthomyxoviridae, a family of enveloped viruses with segmented genomes of single-stranded negative-sense RNA (1). Although high-resolution structures have been determined by x-ray crystallography for several viral components or fragments thereof (2 –4), information about the 3D structure(s) of complete virions has remained scanty. Current models, based primarily on electron microscopy of negatively stained samples (5 –7), envisage an envelope containing the trimeric hemagglutinin (HA), tetrameric neuraminidase (NA) (8), and M2 (9) glycoproteins, lined with a continuous layer of matrix protein, enclosing multiple ribonucleoprotein (RNP) complexes (10). An RNP complex consists of a segment of genomic RNA coated by the nucleocapsid protein, with a loop at one end, whereas the other end has a duplex formed by base pairing of the termini to which the viral polymerase is bound (11 –14).

Influenza virions assemble as they bud from the surface of infected cells. In this process, the glycoproteins accumulate in lipid rafts, where they interact with the underlying matrix protein. Host factors also participate, particularly in the final stage of pinching off (15). During assembly, a dilemma common to all viruses with segmented genomes must be resolved: how to assign the various segments appropriately to nascent virions? In transverse thin-section electron micrographs of budding influenza virions, a commonly observed motif is a ring with seven features, each thought to be an RNP, surrounding a central such feature (16, 17). Their total number, eight, matches the number of distinct RNA segments in the influenza virus genome. However, it has not been determined whether each virion receives one copy of each segment or a random selection, nor how RNPs are recruited to the budding site (15).

To date, influenza virus has been refractory to 3D structural analysis, in large part because its pleiomorphy has precluded visualization by image reconstruction of electron micrographs by procedures that rely on averaging many identical particles, which have been applied successfully to many icosahedrally symmetric viruses (18). The virion structure is of interest not only in the context of virus assembly but also in light of the possibility that pleiomorphic variations may correlate with infectivity and/or pathogenicity. As a step toward addressing these questions, we have used cryoelectron tomography, a technique capable of rendering the 3D structures of individual macromolecular particles in their native states (19 –25), to visualize influenza virions of the type A egg-adapted X-31 strain (26).

Acknowledgments

We thank Charles S. Smith and Brittney Manvilla for help with electron microscopy. This work was supported in part by the Intramural Research Program of National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institutes of Health (NIH) Intramural Targeted Antiviral Program (A.C.S.) and an NIH R01 award, AI22470 (to J.M.W.). M.B. was supported in part by an NIH training grant (5T32AI07047).

Acknowledgments

Abbreviations

NA	neuraminidase
RNP	ribonucleoprotein.

Abbreviations

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Footnotes

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