Native Morphology of Influenza Virions

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.

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway.