Due to the limited coding capacity of the viral genome, the fate of the virus is determined by its interaction with host factors that could either be facilitating or restricting the infection. Influenza A viral nucleoprotein (NP), encoded by segment 5 of negative sense RNA genome is a multifunctional protein and serves as a key connector between virus and host. It shuttles between nucleus and cytoplasm interacting with host proteins to direct various functions during viral replication. Here, we sought to identify the novel host cellular interacting partners to influenza A viral NP by co-immunoprecipitation assay using subcellular fractions of A549 cells transfected with a recombinant viral NP. Host proteins co-precipitated with recombinant viral NP were identified by MALDI-TOF/mass spectrometry analysis. Nucleolin, a major RNA-binding protein of the nucleolus was identified to be a novel interacting partner to viral NP in the cytoplasmic fraction with a significant protein score. This observation was confirmed in cells infected with the corresponding virus, 2009 H1N1 pandemic strain by Co-IP and confocal microscopy. Strongest co-localization of host nucleolin and viral NP was observed in the cytoplasm of infected cells at 6hrs post infection. Surprisingly, quantitative real-time PCR demonstrated increased mRNA levels of late viral genes encoding matrix (M1) and hemagglutinin (HA) proteins upon depletion of endogenous nucleolin by siRNA targeting nucleolin. However, viral infection following transient transfection of A549cells with pEGFP-Nucleolin reduced the late viral gene transcripts, consequently reduced the viral-induced cytopathic effect. Host nucleolin and viral NP association in cells infected with seasonal strains H3N2 and H1N1 independent of each other further confirmed this interaction. Altered expression of late viral gene transcripts following the manipulation of host nucleolin levels proposes the functional importance of host nucleolin and viral NP interaction in influenza A viral infection. Studies are ongoing to investigate this interaction in greater detail.
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