Influenza viruses are responsible for recurrent annual epidemics causing acute febrile respiratory illness. Moreover, they constitute a major threat to public health since they could lead to the emergence of potentially pandemic new variants. These enveloped viruses contain eight single strand negative RNA segments and most of their infectious cycle occurs within the host cellular nucleus. Both viral transcription and replication are associated with host nuclear machinery and many interactions between viral proteins and nuclear components occur during the time course of infection. However, due to the fact that influenza has never constituted tools for studying fundamental molecular mechanisms of the cell, not much data are available concerning ultrastructural and molecular relationships between influenza viruses and human host nuclear domains. Recent publications have mentioned a dynamic nucleolar localization of viral nucleoprotein (NP), early in infection. Moreover, mutations suppressing the nucleolar addressing of NP, result in an abortive viral cycle (Ozawa M. et al., J. of Virology. 2007. Vol. 80, p. 30-41).Although NP is considered as a key adapter molecule between viral genome and host cell machinery, the biological significance of these events needs to be elucidated. Furthermore, the Non Structural protein NS1 was characterized as an interacting partner of nucleolin, a major nucleolar component (Murayama R. et al., Biochem. Biophys. Res. Communications. 2007. Vol. 362 p. 880- 5). Altogether, these results suggest a likely interplay between influenza and the host nucleolus. Nucleoli are known to be the site of ribosomal RNA (rRNA) transcription, processing, and assembly into the ribosomal subunits. In addition, nucleoli are dynamic structures involved in additional non-conventional roles including cell cycle regulation and cellular stress responses (Boisvert et al. Nat. Rev. Mol. Cell Biol. 2007. Vol. 8 p. 574-85). Furthermore, it is now well established that many other viruses, with nuclear replication, induce important remodeling of the ultrastructure, composition and dynamic of nucleoli, and that these modifications are required for optimal infection (Hiscox J.A.,Nature Rev. Microbiol. 2007. Vol. 5, p. 119-127). In this context, we hypothesized that host nucleolus could play a crucial role in determining the outcome of influenza infections. We then explored the potential impact of influenza viruses on the ultrastructure and molecular composition of nucleolus. For this purpose, human epithelium A549 cells were infected with relevant human (A/New Caledonia/20/99, H1N1 and A/Moscow/10/99, H3N2) and avian (A/Turkey/582/2006, H5N1; A/Finch/England/2051/94, H5N2 and A/chicken/Belgium/2003, H7N7) viruses and analysed at several times post-infection by electron microscopy and confocal laser scanning. Our results show that all these influenza type A viruses induce a strong remodelling of nucleolar morphology and a dynamic delocalization of several major constitutive nucleolar markers. These events probably imply interaction between viral nucleoprotein and host endogenous nucleolin which we characterized by a GST pull down assay. Finally, a complementary approach using reverse genetics confirmed that influenza type A viruses have a ?nucleolar experience? for performing effective and optimal viral replication.

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