Abstract
Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.
Highlights
It was well known that host RNA-binding proteins (RBPs) are involved in the viral replication for plus-strand (+) RNA viruses
After endogenous viral RNP was assembled, the RNA-protein complexes were cross-linked by 365 nm UV light irradiation and the RNPs were isolated from whole cell extract through binding of
Were identified by mass spectrometry, and several newly identified protein partners potentially interact with dengue virus (DENV) 5′–3′ untranslated regions (UTRs) RNA
Summary
It was well known that host RNA-binding proteins (RBPs) are involved in the viral replication for plus-strand (+) RNA viruses. After the biotinylated Csy H29A/S50C was immobilized on Avidin agarose, the protein partners binding to Csy hairpin-tagged RNA were selectively purified This RNA affinity purification strategy offers an easy elution procedure and is simple and effective for transcriptome analysis. The S1 RNA aptamer sequence was cloned into the 3′ end of DENV RNA UTR in a plasmid, and after transfection the hybrid RNA was transcribed in living cells This allowed endogenous viral RNP assembly and isolation of RNPs from whole cell extract. By using this method, we identified several novel host proteins binding to DENV RNA. We identified several novel host proteins binding to DENV RNA This method, coupled with LC-MS/MS, will allow quick and easy purification of high- and low-affinity RBPs and RNPs under endogenous conditions
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