Abstract
The viral ribonucleoprotein (vRNP) of influenza A virus is formed by virion RNA (vRNA), viral polymerase complex, and nucleoprotein (NP). The NP plays an important role in facilitating the replication and stabilization of viral RNA. To explore host factors that may be involved in the regulation of viral replication through interactions with NP, we conducted an immunoprecipitation experiment followed by mass spectrometry to identify NP-associated cellular proteins. Here, we demonstrate that NP can interact and colocalize with heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in mammalian cells and that the interaction may occur via direct binding to the glycine-rich domain (GRD) of hnRNP A2/B1. In addition, two residues in the tail loop of NP, F412 and R422, are required for the interaction of hnRNP A2/B1. Because the knockdown of hnRNP A2/B1 expression reduces viral RNP activity, hnRNP A2/B1 may act as a positive regulator in viral RNA synthesis of influenza A virus. More importantly, the findings in this research demonstrate that host proteins can regulate the replication of influenza A virus by interacting with NP.
Highlights
Influenza A virus causes respiratory diseases in humans and leads to annual epidemics and pandemics worldwide
For purification of proteins in vitro, the open reading frames (ORFs) of wild type NP were sub-cloned into the pET30a(+) vector (Novagen/EMD Millipore, Billerica, MA), and the ORFs corresponding to the RRM1, RRM2, and glycine-rich domain (GRD) regions of heterogeneous nuclear ribonucleoprotein (hnRNP) A2 were constructed in the pGEX-5X-1 vector (GE Healthcare Life Sciences, Marlborough, MA, USA), which contains a glutathione S-transferase (GST) sequence
To explore novel functions of the NP protein encoded by influenza A virus in host cells, lysates of 293T cells transfected with plasmids expressing the FLAG-tagged NP of influenza 141/H1N1, WSN/H1N1, or an H6N1 virus were subjected to anti-FLAG immunoprecipitation and mass spectrometry (MS) to search for cellular proteins that could associate with the NP protein
Summary
Influenza A virus causes respiratory diseases in humans and leads to annual epidemics and pandemics worldwide. The virion of influenza A virus consists of eight segments of genomic RNA with negative polarity. Each of the virion RNA segments is associated with a viral polymerase complex and bound with viral nucleoprotein (NP) to form ribonucleoprotein (RNP). The viral polymerase complex comprises three subunits, PB1, PB2 and PA, and functions as a transcriptase and replicase to generate viral mRNA and virion RNA [1, 2]. NP from influenza A virus has been demonstrated to interact directly with the viral polymerase complex and enhance unprimed viral RNA replication [3, 4].
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