Abstract:
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.
Author(s): Rosa Calatrava Manuel, Moules V., Josset L., Bouscambert M.
Ferraris, O.1; Frobert, E.1; De Chassey, B.3; Hay, A.4; Morfin, F.1; Diaz, J.J.5;
Lina, B.1