Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions
病毒粒子中接触病毒衣壳蛋白RNA的序列图谱绘制
发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2398 浏览次数: 9571
评审: Longping Victor TseWeiyan JiaAnonymous reviewer(s)
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Sep 2016
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Abstract
We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.
Keywords: Protein-RNA interaction (蛋白质-RNA相互作用)
RNA virus (RNA病毒)
Viral capsid (病毒衣壳)
Virion assembly (病毒装配)
CLIP-Seq (CLIP-Seq)
Background
Positive-sense RNA viruses include pathogens of all life forms. Viruses with icosahedral shapes have the viral coat proteins form a protective shell around the RNA genome (Stockley et al., 2013). In the phage MS2 and the plant-infecting Brome mosaic virus (BMV), the coat protein preferentially contacts specific RNA sequences (Ni et al., 2013; Hoover et al., 2016; Rolfsson et al., 2016). These contacts could regulate the timing of RNA release during infection, viral gene expression, and viral RNA replication (Hoover et al., 2016). Identification of the capsid-RNA interactions could thus provide insights into the regulations of viral infection and provide means to inhibit viral infection. With this in mind, we have developed a method to identify the capsid-RNA contacts in purified virions using a combination of UV crosslinking, RNA fragmentation, selective precipitation of the coat protein, and next-generation sequencing of the cDNAs made from RNA fragments. The protocol below was developed for BMV virions.
Materials and Reagents
Note: All solutions should be made using sterile water with resistivity of better than 18.3 MΩ and analytical grade reagents.
- Pipette tips (Corning, catalog number: 4154 )
- 6-well clear polystyrene flat-bottomed tissue culture plate (Corning, Falcon®, catalog number: 351146 )
- Polyallomer centrifuge tubes (Beckman Coulter, catalog number: 344060 )
- Polypropylene microcentrifuge tubes and tips that are certified to be Ribonuclease free
- iBlotTM PVDF membrane (0.2 μm) (Thermo Fisher Scientific, InvitrogenTM, catalog number: IB401001 )
- Razor blade (GEM, catalog number: RB-GEM-080014 )
- 0.22 μm filter (EMD Millipore, catalog number: SCGPU05RE )
- Zymo RNA Clean & Concentrator (ZYMO RESEARCH, catalog number: R1015 )
- BMV virions purified using cesium chloride density gradients and suspended in SAMA buffer
Note: High purity BMV virions can be obtained as described in Vaughan et al. (2014). The virions are kept in an acidic buffer because pH affects the swelling of the BMV virions and can change the CP-RNA interaction. - 10x Fragmentation Reagent and Stop Solution: 200 mM EDTA (pH 8.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM8740 )
- PierceTM Protein A/G magnetic beads (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88847 )
- Anti-BMV CP antibody
Note: This is a polyclonal antiserum we generated in rabbits immunized with highly purified BMV capsid protein. - NuPAGE® Novex® 4-12% Bis-Tris Gel, NuPAGE® MES-SDS running buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0321BOX )
- 1x MES SDS running buffer
- Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
- 0.1% Ponceau-S (Sigma-Aldrich, catalog number: P3504 )
- Protease K (Fungal) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530015 )
- T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201L )
- Small RNA kit (Illumina, catalog number: RS-200-0036 )
- 1.8x SPRI beads (Beckman Coulter, catalog number: 41105518 )
- SPRIselect reagent (Beckman Coulter, catalog number: B23317 )
- Sodium hydroxide (NaOH) (Avantor Performance Materials, Macron, catalog number: 7680 )
- HT1 buffer (Component of the NexSeq 500 kit) (Illumina, catalog number: FC-404-2005 )
- Sodium acetate, Na(OAc) (EMD Millipore, catalog number: 106268 )
- Magnesium acetate tetrahydrate, Mg(OAc)2·4H2O (Sigma-Aldrich, catalog number: M0631 )
- Glacial acetic acid (EMD Millipore, catalog number: AX0073-9 )
- Ethanol, absolute (Decon Labs, catalog number: 2716 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Tris base (Sigma-Aldrich, catalog number: T6066 )
- Tween 20 (Sigma-Aldrich, catalog number: P1379 )
- Sodium dodecyl sulfate, sodium salt (SDS) (EMD Millipore, catalog number: 7910-OP )
- Glycerol (VWR, BDH®, catalog number: BDH1172 )
- Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
- Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP120 )
- SAMA buffer (see Recipes)
- 3 M sodium acetate (pH 5.2) (see Recipes)
- IP binding buffer (see Recipes)
- IP wash buffer (see Recipes)
- 4x Laemmli sample buffer (see Recipes)
- PK buffer (see Recipes)
Equipment
- Ultraviolet Cross-linker (UVP, model: CL-1000 , catalog number: 95-0174-01)
- Beckman Coulter Ultima Max-XP ultracentrifuge (Beckman Coulter, model: OptimaTM Max-XP , catalog number: 393315)
- DynaMag-2 (Thermo Fisher Scientific, catalog number: 12321D )
- SureLock X Mini-gel electrophoresis system (Thermo Fisher Scientific, InvitrogenTM, catalog number: El0001 )
- iBlot® Dry Blotting System (Thermo Fisher Scientific, model: iBlotTM 2, catalog number: IB21001 )
- Agilent 2200 Tape station (Agilent Technology, model: Agilent 2200 )
- Miseq or NexSeq500 (Illumina, model: NexSeqTM 500 )
Software
- Mapping program: bowtie2 ver. 2.2.6
- Graphing program: JBrowse ver. 1.12.1
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kao, C. C., Chuang, E., Ford, J., Huang, J., Podicheti, R. and Rusch, D. B. (2017). Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions. Bio-protocol 7(14): e2398. DOI: 10.21769/BioProtoc.2398.
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分类
微生物学 > 微生物生物化学 > RNA
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > RNA > RNA-蛋白质相互作用
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