Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we describe detailed methods for successive procedures for identification and purification of IREF-2, including large-scale suspension culture of HeLa S3 cells, preparation of NEs and separation of IREF-2 by sequential column chromatography steps. This protocol can be modified and used for purification and identification of the other unknown nuclear protein(s) of your interest.
[Background] The influenza A virus genome is composed of 8 segmented- and single stranded-RNA (vRNA). Its transcription and replication are catalyzed by virus-encoded RNA-dependent RNA polymerase (RdRP). Several lines of evidence suggest that certain host-derived factors regulate the viral RNA syntheses (Nagata et al., 2008). Recently, a variety of host-derived proteins have been reported as regulator candidates related to the viral RNA syntheses by interactome analyses and genome wide RNAi screening studies. Among them, however, some false-positive interactors and factors involved indirectly in the viral RNA synthesis seem to be included. Instead, to identify reliable and important host factor(s) which play a direct role in the viral RNA synthesis process, we utilized a biochemical complementation assay system. In this system, the viral vRNA replication reaction occurring efficiently in infected cell nuclei is dissected and reconstituted in vitro using viral factors essential for the viral RNA replication such as viral RdRP derived from detergent-solubilized virion particle and model viral genomic RNA templates and uninfected nuclear extracts (NEs).
Recently, we have reported that an efficient vRNA replication is reproduced in vitro with viral factors and a crude fraction of NEs (Sugiyama et al., 2015). This stimulatory activity presented in NEs, designated IREF-2 that allows robust vRNA replication, was further fractionated and purified by sequential column chromatography. Finally, two host-derived paralogous proteins, pp32 (accession number NP_006296) and APRIL (accession number NP_006392), were identified by MS spectrometry analyses. Following experiments using recombinant pp32 and APRIL, and also in vivo analyses using siRNA confirmed that these host proteins are authentic proteins responsible for the IREF-2 activity (Sugiyama et al., 2015).
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