title: Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.
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.
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. ycdongfmmu@gmail.com.
authors.forename: Yangchao
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. cherry.yj@gmail.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. ahjxyw@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. judy_625@163.com.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. yechuantao2008@163.com.
authors.forename: Chuantao
authors.initials: C
authors.lastname: Ye
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. paradise_009@163.com.
authors.forename: Daihui
authors.initials: D
authors.lastname: Weng
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. gaohuan954227274@163.com.
authors.forename: Huan
authors.initials: H
authors.lastname: Gao
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. flzhang@fmmu.edu.cn.
authors.forename: Fanglin
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. zhikaixu523@163.com.
authors.forename: Zhikai
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Microbiology, Faculty of Preclinical Medicine, the Fourth Military Medical University, Xi'an 710032, China. yflei@fmmu.edu.cn.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Lei
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods; D006224:Cricetinae; D003716:Dengue Virus / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins; D019809:Streptavidin
keywords: 40S ribosomal protein S8; DENV; RNA binding protein; affinity purification; living mammalian cells; streptavidin aptamer S1