No abstract

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.

We propose a rational approach to the generation of live viral vaccines: alteration of virally encoded type I IFN antagonists to attenuate virulence while retaining immunogenicity. We have explored this concept by using the influenza virus. Previously we have shown that the NS1 protein of influenza A virus possesses anti-IFN activity. We now present evidence that influenza A and B viruses encoding altered viral NS1 proteins are highly attenuated in the mouse host, yet provide protection from challenge with wild-type viruses.

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.Influenza A, B, and C viruses are human pathogens of the Orthomyxoviridae family. These negative-sense RNA viruses replicate and transcribe their genomes in the nuclei of infected cells. The genomes of influenza A and B viruses are composed of eight segments, while influenza C virus genomes have seven segments (46,48). These RNA segments are encapsidated by the nucleoprotein (NP) and are associated with the viral polymerase (the three P proteins), which together are termed the viral ribonucleoprotein (vRNP) complex (4, 23). After the initial binding, penetration, and uncoating of the viral particle, the vRNPs are released into the cytoplasm of the infected cell. Influenza A vRNP transport into the nucleus is mediated by soluble cellular nuclear import factors karyopherin ␣, karyopherin ␤, Ran, and p10 by a direct interaction between the viral NP protein and karyopherin ␣ (42, 43, 58). Genomic vRNPs are amplified within the nucleus and then must exit the nucleus to accumulate with other viral proteins at the plasma membrane, where packaging and assembly of viral particles occur.The majority of cellular and viral RNA export from the nucleus is thought to be protein mediated. The export of human immunodeficiency virus type 1 (HIV-1) unspliced RNA, for example, is mediated by the virally encoded export protein, Rev. The Rev protein interacts with both a cis-acting sequence present on the viral RNA (the Rev-responsive element, or RRE) and with the karyopherin ␤ family member, Crm1 (reviewed in reference 12).It was origin...