Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells
© Volmer et al. 2010
Received: 19 November 2009
Accepted: 17 March 2010
Published: 17 March 2010
In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.
The nucleolus is a highly dynamic multifunctional subnuclear compartment . It is the site of ribosomal RNA synthesis and ribosomal subunits assembly. In addition, the nucleolus is increasingly recognized as a critical regulator of many other cellular functions, including the regulation of mitosis, cell growth and response to stress [1–3]. The nucleolus is also emerging as an important target of various viral proteins . Viral proteins targeting the nucleolus are for example implicated in the regulation of apoptosis, as shown with West Nile virus capsid protein, and in the regulation of viral mRNA export, as shown with human immunodeficiency virus Rev protein and with herpesvirus saimiri ORF57 protein [5–7]. However, for most viruses, consequences of viral protein localization in the nucleolus remain largely unknown [3, 4].
The non-structural 1 (NS1) protein of influenza A viruses NS1 is a multifunctional protein, known to interact with and modify the function of many cellular proteins, thereby creating a cellular environment favouring virus replication . Recently, a nucleolar localization signal (NoLS) has been identified in NS1 . This NoLS targets NS1 to the nucleolus of mammalian cells. Presently, the role of the nucleolar localization of NS1 in the viral cycle is unknown. One can speculate that NS1 proteins targeting the nucleolus of mammalian cells could modify the functions of nucleolar proteins. The mammalian NoLS of NS1 consists of a stretch of C-terminal basic amino acids that are present only in certain strains of influenza A viruses . Thus, only certain NS1 proteins accumulate in the nucleolus of mammalian cells. Whether NS1 proteins accumulate in the nucleolus of avian cells is currently unknown.
In this study, we compared the nucleolar localization of NS1 of different influenza virus strains in mammalian and avian cells using immunocytochemistry and confocal microscopy. Experiments were done in human A549 alveolar epithelial cells and in primary embryonic fibroblasts used between passages 2 and 6, cultured from 11 days old Balb/c mouse (Mus musculus) embryos, from 14 days old Pekin duck (Anas platyrhynchos) embryos or from 12 days old chicken (Gallus gallus) embryos. Cells were infected at a multiplicity of infection (MOI) of 3 plaque forming units (pfu) per cell (MOI = 3) with the human influenza A/Udorn/72(H3N2) strain (designated Udorn), the human laboratory adapted influenza A/PR/8/34(H1N1) strain (designated PR8), the avian influenza A/Turkey/Italy/977/V99(H7N1) strain (designated 977) or the avian influenza A/Turkey/Italy/4426/00(H7N1) strain (designated 4426). At 3, 4, 6, 8 and 12 hours post-infection (hpi), cells were fixed with 4% Paraformaldehyde, permeabilized with Phosphate Buffered Saline (PBS) 0.5% Triton X-100 and incubated for one hour in PBS 0.1% Triton X-100 and 2% Bovine Serum Albumin. Antibody incubation was performed overnight at 4°C.
Presently the role of the nucleolar localization of NS1 in influenza virus cycle is unknown. In mammalian cells, nucleolar accumulation of NS1 occurs only with certain strains of influenza A viruses. As the NS1 of all viruses studied here targeted the nucleolus of avian cell, we speculate that the nucleolar localization of NS1 could be an important step during the viral cycle in avian cells. Whether, nucleolar localization of NS1 contributes to virulence is currently unknown. Valuable information would certainly be obtained by studying the phenotype of a reverse genetics engineered virus lacking a functional NoLS. In order to perform such studies in avian cells, the avian NoLS needs to be identified. Our results show that the avian NoLS relies on an amino acid sequence that is present in all the influenza virus strains tested in this study, and thus could be conserved among most influenza virus strains.
We thank I. Capua and W. Dundon (Istituto Zooprofilattico Sperimentale Delle Venezie, Legnaro, Italy), N. Naffakh (Institut Pasteur, Paris) and R. Fouchier (Erasmus University, Rotterdam, Netherlands) for the kind gift of viruses, D. Marc (INRA, Tours, France), J. Ortin (Centro Nacional de Biotecnología, Madrid, Spain) for the kind gift of antibodies.
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