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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   

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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.

  1. Pipette tips (Corning, catalog number: 4154 )
  2. 6-well clear polystyrene flat-bottomed tissue culture plate (Corning, Falcon®, catalog number: 351146 )
  3. Polyallomer centrifuge tubes (Beckman Coulter, catalog number: 344060 )
  4. Polypropylene microcentrifuge tubes and tips that are certified to be Ribonuclease free
  5. iBlotTM PVDF membrane (0.2 μm) (Thermo Fisher Scientific, InvitrogenTM, catalog number: IB401001 )
  6. Razor blade (GEM, catalog number: RB-GEM-080014 )
  7. 0.22 μm filter (EMD Millipore, catalog number: SCGPU05RE )
  8. Zymo RNA Clean & Concentrator (ZYMO RESEARCH, catalog number: R1015 )
  9. 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.
  10. 10x Fragmentation Reagent and Stop Solution: 200 mM EDTA (pH 8.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM8740 )
  11. PierceTM Protein A/G magnetic beads (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88847 )
  12. Anti-BMV CP antibody
    Note: This is a polyclonal antiserum we generated in rabbits immunized with highly purified BMV capsid protein.
  13. NuPAGE® Novex® 4-12% Bis-Tris Gel, NuPAGE® MES-SDS running buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0321BOX )
  14. 1x MES SDS running buffer
  15. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  16. 0.1% Ponceau-S (Sigma-Aldrich, catalog number: P3504 )
  17. Protease K (Fungal) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530015 )
  18. T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201L )
  19. Small RNA kit (Illumina, catalog number: RS-200-0036 )
  20. 1.8x SPRI beads (Beckman Coulter, catalog number: 41105518 )
  21. SPRIselect reagent (Beckman Coulter, catalog number: B23317 )
  22. Sodium hydroxide (NaOH) (Avantor Performance Materials, Macron, catalog number: 7680 )
  23. HT1 buffer (Component of the NexSeq 500 kit) (Illumina, catalog number: FC-404-2005 )
  24. Sodium acetate, Na(OAc) (EMD Millipore, catalog number: 106268 )
  25. Magnesium acetate tetrahydrate, Mg(OAc)2·4H2O (Sigma-Aldrich, catalog number: M0631 )
  26. Glacial acetic acid (EMD Millipore, catalog number: AX0073-9 )
  27. Ethanol, absolute (Decon Labs, catalog number: 2716 )
  28. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  29. Tris base (Sigma-Aldrich, catalog number: T6066 )
  30. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  31. Sodium dodecyl sulfate, sodium salt (SDS) (EMD Millipore, catalog number: 7910-OP )
  32. Glycerol (VWR, BDH®, catalog number: BDH1172 )
  33. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  34. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP120 )
  35. SAMA buffer (see Recipes)
  36. 3 M sodium acetate (pH 5.2) (see Recipes)
  37. IP binding buffer (see Recipes)
  38. IP wash buffer (see Recipes)
  39. 4x Laemmli sample buffer (see Recipes)
  40. PK buffer (see Recipes)

Equipment

  1. Ultraviolet Cross-linker (UVP, model: CL-1000 , catalog number: 95-0174-01)
  2. Beckman Coulter Ultima Max-XP ultracentrifuge (Beckman Coulter, model: OptimaTM Max-XP , catalog number: 393315)
  3. DynaMag-2 (Thermo Fisher Scientific, catalog number: 12321D )
  4. SureLock X Mini-gel electrophoresis system (Thermo Fisher Scientific, InvitrogenTM, catalog number: El0001 )
  5. iBlot® Dry Blotting System (Thermo Fisher Scientific, model: iBlotTM 2, catalog number: IB21001 )
  6. Agilent 2200 Tape station (Agilent Technology, model: Agilent 2200 )
  7. Miseq or NexSeq500 (Illumina, model: NexSeqTM 500 )

Software

  1. Mapping program: bowtie2 ver. 2.2.6
  2. Graphing program: JBrowse ver. 1.12.1

Procedure

  1. UV crosslinking
    1. Add 400 µl of 100 µg/ml each of purified BMV virions into 3 wells of a 6-well cell culture plate. Incubated the plate on ice for at least 5 min. Place the plate without the cover in the crosslinking machine and irradiate three times at 254 nm to 400 mJ/cm2 (Figure 1). Between irradiations, agitate the samples with gently tapping the plate to mix the samples and incubate the plate on ice for 3 min to prevent the samples from overheating. Repeat the irradiation, agitation, and incubation on ice two more times.
      Notes:
      1. BMV virions are purified to homogeneity using cesium chloride density gradients as described in Ni et al., 2014. The concentration of the purified virion was determined in Ni et al., 2014.
      2. The dose of irradiation that provided optimal crosslinking of the BMV virion RNAs was determined empirically by sequencing the RNA fragments processed through the protocol. The same conditions were also successfully used for crosslinking of bacteriophage MS2 RNA (Rolfsson et al., 2016). Nonetheless, it is quite likely that modifications in the crosslinking time should be empirically determined for other virions.


        Figure 1. Location of the plate containing BMV virions in the UV crosslinker

    2. For a negative control, repeat step A1, but do not irradiate the virions. This control reaction can be processed in parallel with the irradiated virion.
    3. Harvest and pool the irradiated virion solutions into a small polyallomer centrifuge tube and centrifuge at 150,000 x g for 30 min using a preparative ultracentrifuge. Resuspend the virion pellet in 150 µl 100 mM Tris (pH 7.0).
    4. Add 15 µl of RNA Fragmentation Reagent, mix and incubate at 70 °C for 15 min.
      Note: We have empirically examined several combinations of ribonucleases and observed more bias in the cleavage of the RNAs. Zinc-induced cleavage of the RNA produced more random cleavage of the RNA, is easier to use, and is easily removed during the processing of the samples. For BMV, the conditions used will fragment the BMV RNA to ~50-nt in length (Figure 2). During the fragmentation step, the pH of the solution and the incubation at 70 °C will cause the BMV virion to dissociate and denature. The virion solution should become cloudy soon after incubation. With other virions, it will be important to try different conditions to identify those that can generate relatively short RNA fragments.
    5. Add 15 µl 0.2 M EDTA (pH 8.0), mix and incubate on ice for 10 min to stop the reaction.


      Figure 2. Time-dependent fragmentation of RNAs from UV-crosslinked BMV virions. The image shown is from ethidium bromide-stained agarose gel containing 100 μg BMV virions that were resuspended in 150 μl Tris buffer (pH: 7.0) containing 10 Mm ZnCl2, and heated at 70 °C to fragment BMV RNA for the time indicated in the gel image.

  2. Immunoprecipitation and RNA extraction
    1. Add 50 µl of the Pierce Protein A/G magnetic beads suspension into a 1.5 ml microcentrifuge tube. Add 1 ml of ice-cold IP binding buffer (see Recipes), mix, and then concentrate the beads by placing the tube against the magnetic tube holder. The beads should be concentrated on the side of the microcentrifuge tube. Use a pipette to remove the supernatant. Repeat the washing of the beads.
    2. Resuspend beads in 100 µl ice-cold IP binding buffer, add the anti-BMV CP antibody and the crosslinked CP-RNA solution from step A5 and place the tube to mix on a rocking platform set at 50 rpm at 4 °C for 2 h.
    3. Collect the beads and co-precipitated protein using a magnetic tube holder. Remove the supernatant. Add 1 ml ice-cold IP binding buffer to wash the beads four times, each time collect the beads using Magnastrip and remove the supernatant.
    4. Remove final wash solution, add 100 µl of Laemmli sample buffer (see Recipes) to the beads, mix and heat at 95 °C for 2 min, load up to 30 µl samples on precast 4-12% Bis-Tris gel, and run the gel for 1 h at 150 V in 1x MES SDS running buffer.
    5. Transfer protein-RNA complexes from the gel to a PVDF membrane using a wet transfer apparatus (Bio-Rad Laboratories). Transfer should be at 100 V for 2.5 h.
      Note: Transferring the protein from the denaturing PAGE to the PVDF membrane will help to remove non-covalently bound RNA fragments, and will decrease the background.
    6. Rinse the membrane with sterile PBS buffer twice, stain with 0.1% Ponceau-S for 10 min. then rinse the membrane with sterile PBS twice to remove excess dye. Cut out the portion of the membrane at ca. 3-4 mm above the BMV CP band using a new razor blade (Figure 3). Transfer the membrane slice that contains the CP and crosslinked RNA fragments into a 1.5 ml sterile microcentrifuge tube, rinse with sterile PBS twice.
      Note: The membrane slices can be stored at -80 °C until use.
    7. Add 200 µl of 1 mg/ml Proteinase K diluted in the PK buffer (see Recipes) to the membrane slice. Incubate at 37 °C for 1 h with occasional mixing to digest the CP.


      Figure 3. Location of the RNA excised from crosslinked BMV coat protein. The image is of the membrane following Western blot transfer. The boxes with dashed lines denote the area of the membrane excise with a razor blade.

  3. cDNA library construction and Illumina DNA sequencing
    1. Apply the solution from step B7 that contains eluted RNA fragments to a Zymo RNA Clean & Concentrator column. Elute the RNA using 35 µl elution buffer from the kit.
    2. Add 5 µl of 10x PNK buffer and 50 U of the PNK enzyme and 5 µl of 10 mM ATP. Incubate the mixture at 37 °C for 30 min.
      Note: The PNK adds a 5’-phosphate to the RNAs to enable the addition of oligonucleotides adaptors needed for library construction.
    3. Transfer the RNA to a fresh Zymo RNA Clean & Concentrator column and elute the RNA in 7 µl elution buffer from the kit. Analyze 2 µl of the eluant on an Agilent High-sensitivity RNA TapeStation tape to determine the RNA concentration.
    4. Use a volume of remaining eluant that contains between 200 ng to 1 µg of the eluted RNA to construct a cDNA library using an Illumina Small RNA kit. Construction of the library as per the manufacturer’s instruction using 11 to 15 cycles of DNA amplification, with the cycle number being dependent on the mass of the RNA present.
    5. Estimate the concentration of the libraries using the Agilent High-sensitivity DNA TapeStation.
    6. Pool the libraries then clean and concentrate the pool using a 1.8x ratio of SPRIselect beads (Beckman Coulter) and eluted in 22 µl.
      Note: In the TapeStation, the libraries of DNA fragments are separated by electrophoresis from the adaptors, the primer dimers and other DNA molecules generated during the amplification process. The DNAs of lengths expected to have adaptors flanking the cDNA are excised and eluted.
    7. The libraries (5 µl with ca. 500 ng of each library) were denatured with 5 µl of 0.2 N NaOH for 5 min, then neutralized with 985 µl of the HT1 buffer provided by Illumina. 5 µl of the 200 mM Tris pH 7.5 was added to supplement the buffering capacity of the HT1 buffer.
      Note: Given the small sizes of viral genomes, multiple viral libraries can be a small percentage of an Illumina sequencing run. Even 1% of the sample on a run can still result in several hundred thousand reads for each of the libraries.
    8. The Illumina NextSeq500 run was performed by members of the Indiana University CGB staff using a 75-High output flow cell designated for an 80-bp read.

Data analysis

  1. Trim and quality-filter the reads using Trimmomatic ver. 0.33, with a minimal cutoff score of 20 (Bolger et al., 2014). Reads shorter than 20 nucleotides post trimming were excluded.
  2. Map the reads to corresponding reference sequences using the computer program Bowtie2 ver 2.2.6 (Langmead and Salzberg, 2012) with default mapping parameter values. For each read, only the best alignments, as defined by least edit distance, were used.
  3. Display the number of reads spanning each base position of the reference sequence on JBrowse ver. 1.12.1 as BigWig tracks (Skinner et al., 2009).

Notes

While this protocol has been optimized for the analysis of the RNAs that bind to the BMV capsid, the protocol can be directly adapted to map the virions of interest. The key will be the availability of highly pure virions and an antibody that can efficiently precipitate the capsid protein of interest. The construction of cDNA libraries, nex-gen DNA sequencing and data analysis can be performed in facilities that specialize in genomic analysis.

Recipes

  1. SAMA buffer
    50 mM Na(OAc)
    8 mM Mg(OAc)2, pH 5.5
  2. 3 M sodium acetate (pH 5.2)
    Dissolve 12.3 g sodium acetate in 30 ml H2O
    Adjust pH to 5.2 with glacial acetic acid
    Add H2O to 50 ml
    Filter with a device containing 0.22 μm filter
    Store at room temperature
  3. IP binding buffer*
    150 mM NaCl
    25 mM Tris (pH 7.2)
    0.05% Tween 20
  4. IP wash buffer*
    500 mM NaCl
    25 mM Tris (pH 7.2)
    0.05% Tween-20

    *Note: For convenience, make 20x stocks of the buffers and dilute with sterile water to 1x for use.

  5. 4x Laemmli sample buffer
    8% SDS
    40% glycerol
    240 mM Tris (pH 6.8)
    0.04% bromophenol blue
  6. PK buffer
    50 mM Tris (pH 7.5)
    75 mM NaCl
    6.3 mM EDTA
    1% SDS

Acknowledgments

This protocol was established in the work supported by grant NIH 1R01AI090280 to CK. This protocol was adapted from the work of Ni et al. (2013) and Hoover et al. (2016) and unpublished results of E. Chuang. We thank A. Kao for photography used in this protocol.

References

  1. Bolger, A. M., Lohse, M. and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114-2120.
  2. Hoover, H. S., Wang, J. C., Middleton, S., Ni, P., Zlotnick, A., Vaughan, R. C. and Kao, C. C. (2016). Phosphorylation of the brome mosaic virus capsid regulates the timing of viral infection. J Virol 90(17): 7748-7760.
  3. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
  4. Ni, P., Vaughan, R. C., Tragesser, B., Hoover, H. and Kao, C. C. (2013). The plant host can affect the encapsidation of brome mosaic virus (BMV) RNA: BMV virions are surprisingly heterogeneous. J Mol Biol 426(5): 1061-1076.
  5. Rolfsson, O., Middleton, S., Manfield, I. W., White, S. J., Fan, B., Vaughan, R., Ranson, N. A., Dykeman, E., Twarock, R., Ford, J., Kao, C. C. and Stockley, P. G. (2016). Direct evidence for packaging signal-mediated assembly of bacteriophage MS2. J Mol Biol 428(2 Pt B): 431-448.
  6. Skinner, M. E., Uzilov, A. V., Stein, L. D., Mungall, C. J. and Holmes, I. H. (2009). JBrowse: a next-generation genome brower. Genome Res 19(9): 1630-1638.
  7. Stockley, P. G, Ranson, N. A. and Twarock, R. (2013). A new paradigm for the roles of the genome in ssRNA viruses. Future Virol 8: 531-543.
  8. Vaughan, R., Tragesser, B., Ni, P., Ma, X., Dragnea, B. and Kao, C. C. (2014). The tripartite virions of the brome mosaic virus have distinct physical properties that affect the timing of the infection process. J Virol 88(11): 6483-6491.

简介

我们已经调整了CLIP-seq(交联 - 免疫沉淀和DNA测序)的方法来绘制与病毒粒子的蛋白质壳接触的壳化RNA片段。 关于接触病毒衣壳的RNA序列的方案报告的结果。
【背景】正义RNA病毒包括所有生命形式的病原体。具有二十面体形状的病毒具有病毒外壳蛋白在RNA基因组周围形成保护壳(Stockley等人,2013)。在噬菌体MS2和植物感染的Brome花叶病毒(BMV)中,外壳蛋白优先接触特异性RNA序列(Ni等人,2013; Hoover等人。 ,2016; Rolfsson等人,2016)。这些接触可以调节感染期间RNA释放的时间,病毒基因表达和病毒RNA复制(Hoover等人,2016)。鉴定衣壳RNA相互作用可以提供对病毒感染的规定的见解,并提供抑制病毒感染的手段。考虑到这一点,我们已经开发了一种方法,使用UV交联,RNA断裂,外壳蛋白的选择性沉淀和由RNA片段制备的cDNA的下一代测序来鉴定纯化的病毒体中的衣壳RNA接触。以下协议是针对BMV病毒粒子开发的。

关键字:蛋白质-RNA相互作用, RNA病毒, 病毒衣壳, 病毒装配, CLIP-Seq

材料和试剂

注意:所有解决方案应使用电阻率高于18.3MΩ的无菌水和分析纯试剂。

  1. 移液器提示(Corning,目录号:4154)
  2. 6孔透明聚苯乙烯平底组织培养板(Corning,Falcon ®,目录号:351146)
  3. Polyallomer离心管(Beckman Coulter,目录号:344060)
  4. 经过认证无核糖酶的聚丙烯微量离心管和提示
  5. iBlot TM PVDF膜(0.2μm)(Thermo Fisher Scientific,Invitrogen TM,目录号:IB401001)
  6. 剃须刀片(GEM,目录号:RB-GEM-080014)
  7. 0.22μm过滤器(EMD Millipore,目录号:SCGPU05RE)
  8. Zymo RNA Clean&集中器(ZYMO RESEARCH,目录号:R1015)
  9. 使用氯化铯密度梯度纯化并悬浮在SAMA缓冲液中的BMV病毒体 注意:可以如Vaughan等人所述获得高纯度BMV病毒粒子。 (2014)。病毒粒子保持在酸性缓冲液中,因为pH影响BMV病毒粒子的肿胀,并且可以改变CP-RNA相互作用。
  10. 10x片段化试剂和终止液:200mM EDTA(pH 8.0)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM8740)
  11. Pierce TM 蛋白A / G磁珠(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88847)
  12. 抗BMV CP抗体
    注意:这是我们在用高度纯化的BMV衣壳蛋白免疫的兔子中产生的多克隆抗血清。
  13. NuPAGE ® Novex ®4-12%Bis-Tris Gel,NuPAGE MES-SDS运行缓冲液(Thermo Fisher Scientific,Invitrogen TM ,目录号:NP0321BOX)
  14. 1x MES SDS运行缓冲区
  15. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  16. 0.1%Ponceau-S(Sigma-Aldrich,目录号:P3504)
  17. 蛋白酶K(真菌)(Thermo Fisher Scientific,Invitrogen TM,目录号:25530015)
  18. T4多核苷酸激酶(New England Biolabs,目录号:M0201L)
  19. 小RNA试剂盒(Illumina,目录号:RS-200-0036)
  20. 1.8倍SPRI珠(Beckman Coulter,目录号:41105518)
  21. SPRI选择试剂(Beckman Coulter,目录号:B23317)
  22. 氢氧化钠(NaOH)(Avantor Performance Materials,Macron,目录号:7680)
  23. HT1缓冲液(NexSeq 500试剂盒的组分)(Illumina,目录号:FC-404-2005)
  24. 乙酸钠,Na(OAc)(EMD Millipore,目录号:106268)
  25. 四水镁酸镁,Mg(OAc)2·4H 2 O(Sigma-Aldrich,目录号:M0631)
  26. 冰醋酸(EMD Millipore,目录号:AX0073-9)
  27. 乙醇,绝对(Decon Labs,目录号:2716)
  28. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  29. Tris碱(Sigma-Aldrich,目录号:T6066)
  30. 吐温20(Sigma-Aldrich,目录号:P1379)
  31. 十二烷基硫酸钠钠盐(SDS)(EMD Millipore,目录号:7910-OP)
  32. 甘油(VWR,BDH ,目录号:BDH1172)
  33. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  34. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:BP120)
  35. SAMA缓冲区(请参阅配方)
  36. 3 M醋酸钠(pH 5.2)(见配方)
  37. IP绑定缓冲区(请参阅配方)
  38. IP洗涤缓冲液(见配方)
  39. 4x Laemmli样品缓冲液(参见食谱)
  40. PK缓冲(见配方)

设备

  1. 紫外线交联剂(UVP,型号:CL-1000,目录号:95-0174-01)
  2. Beckman Coulter Ultima Max-XP超速离心机(Beckman Coulter,型号:Optima TM Max-XP,目录号:393315)
  3. DynaMag-2(Thermo Fisher Scientific,目录号:12321D)
  4. SureLock X迷你凝胶电泳系统(Thermo Fisher Scientific,Invitrogen TM,目录号:El0001)
  5. iBlot ®干式印迹系统(Thermo Fisher Scientific,型号:iBlot TM 2,目录号:IB21001)
  6. Agilent 2200磁带站(Agilent Technologies,型号:Agilent 2200)
  7. Miseq或NexSeq500(Illumina,型号:NexSeq TM 500)

软件

  1. 绘图程序:bowtie2 ver。 2.2.6
  2. 图形程序:JBrowse ver。 1.12.1

程序

  1. 紫外线交联
    1. 将每个纯化的BMV病毒粒子的100μg/ ml的400μl加入6孔细胞培养板的3孔中。将板在冰上孵育至少5分钟。将没有盖子的板放在交联机中,并以254nm至400mJ / cm 2(图1)照射三次。在照射之间,用轻轻敲打板搅拌样品,将样品混合并在冰上孵育3分钟,以防止样品过热。在冰上重复照射,搅动和孵育两次。
      注意:
      1. 使用Ni等人,2014中所述的使用氯化铯浓度梯度将BMV病毒粒子纯化至均匀。在Ni等人,2014中确定纯化的病毒粒子的浓度。
      2. 通过对通过方案处理的RNA片段进行测序,经验地确定提供BMV病毒体RNA最佳交联的照射剂量。同样的条件也成功地用于噬菌体MS2 RNA的交联(Rolfsson等,2016)。尽管如此,交联时间的修改很可能是经验地确定其他病毒粒子。


        图1.含有BMV病毒粒子的板在UV交联剂中的位置

    2. 对于阴性对照,重复步骤A1,但不要照射病毒粒子。该对照反应可以与照射的病毒粒子平行处理
    3. 将照射的病毒粒子溶液收集并收集到小型聚集体离心管中,并使用制备型超速离心机以150,000 x g离心30分钟。将病毒颗粒重悬于150μl100 mM Tris(pH 7.0)中。
    4. 加入15μlRNA片段化试剂,混合并在70℃孵育15分钟 注意:我们经验性地检查了核糖核酸酶的几种组合,并且在RNA切割中观察到更多的偏倚。锌诱导的RNA切割产生更多的RNA的随机切割,更容易使用,并且在样品加工期间容易除去。对于BMV,使用的条件将使BMV RNA片段长度为〜50-nt(图2)。在碎裂步骤期间,溶液的pH值和70℃下的温育将导致BMV病毒粒子解离和变性。孵育后,病毒粒子溶液应该变得阴天。对于其他病毒粒子,重要的是尝试不同的条件来鉴定那些可以产生相对较短的RNA片段的条件。
    5. 加入15μl0.2 M EDTA(pH 8.0),混合并在冰上孵育10分钟以停止反应

      图2.来自UV-交联的BMV病毒粒子的RNA的时间依赖性断裂。所示图像来自含有100μgBMV病毒粒子的溴化乙锭染色的琼脂糖凝胶,其被重悬于150μlTris缓冲液(pH: 7.0),并在70℃下加热至片状BMV RNA,凝胶图像上述时间。

  2. 免疫沉淀和RNA提取
    1. 将50μlPierce Protein A / G磁珠悬浮液加入1.5 ml微量离心管中。加入1ml冰冷的IP结合缓冲液(参见食谱),混合,然后通过将管子放置在磁性管支架上来浓缩珠子。珠子应该集中在微量离心管的一侧。使用移液管去除上清液。重复清洗珠子。
    2. 将珠重悬于100μl冰冷的IP结合缓冲液中,加入抗BMV CP抗体和来自步骤A5的交联的CP-RNA溶液,并将管置于在4℃设定的摇床上,以50rpm设定2小时。
    3. 使用磁性管支架收集珠子并共沉淀蛋白质。去除上清液。加入1毫升冰冷的IP结合缓冲液洗涤珠子四次,每次使用Magnastrip收集珠粒并去除上清液。
    4. 去除最终的洗液,加入100μl的Laemmli样品缓冲液(参见食谱)到珠上,在95℃下混合和加热2分钟,在预制的4-12%Bis-Tris凝胶上加载30μl样品,并运行凝胶在1x MES SDS运行缓冲液中在150V下保持1小时。
    5. 使用湿转移装置(Bio-Rad Laboratories)将蛋白质 - RNA复合物从凝胶转移到PVDF膜。转移应在100 V 2.5 h。
      注意:将蛋白质从变性PAGE转移到PVDF膜将有助于去除非共价结合的RNA片段,并将降低背景。
    6. 用无菌PBS缓冲液冲洗膜两次,用0.1%Ponceau-S染色10分钟。然后用无菌PBS冲洗膜两次以除去多余的染料。切断大部分膜的部分使用新的剃须刀片(图3),BMV CP带以上3-4毫米。将含有CP和交联RNA片段的膜片转移到1.5 ml无菌微量离心管中,用无菌PBS冲洗两次。
      注意:膜片可以储存在-80°C直到使用。
    7. 加入200μl在PK缓冲液中稀释的1mg / ml蛋白酶K(参见食谱)至膜切片。在37℃下孵育1小时,偶尔混合消化CP。


      图3.从交联的BMV外壳蛋白切下的RNA的位置。图像是Western印迹转移后的膜。带有虚线的盒子表示用剃刀刀片消除的膜的面积。

  3. cDNA文库构建和Illumina DNA测序
    1. 将含有洗脱的RNA片段的步骤B7的溶液施用于Zymo RNA Clean&集中器柱。使用试剂盒中的35μl洗脱缓冲液洗脱RNA
    2. 加入5μl10x PNK缓冲液和50 U PNK酶和5μl10 mM ATP。将混合物在37℃下孵育30分钟。
      注意:PNK在RNA中添加了5'-磷酸化物,以增加文库构建所需的寡核苷酸适配器。
    3. 将RNA转移到新鲜的Zymo RNA Clean&浓缩柱,并从试剂盒中将7μl洗脱缓冲液中的RNA洗脱。在Agilent高灵敏度RNA TapeStation磁带上分析2μl洗脱液以确定RNA浓度。
    4. 使用含有200ng至1μg洗脱的RNA的剩余洗脱液体积,使用Illumina Small RNA试剂盒构建cDNA文库。根据制造商的说明使用11至15个循环的DNA扩增构建文库,其循环数取决于存在的RNA的质量。
    5. 使用安捷伦高灵敏度DNA TapeStation估算文库的浓度。
    6. 然后使用1.8倍比例的SPRI选择珠(Beckman Coulter)清洗并浓缩池,并以22μl洗脱。
      注意:在TapeStation中,DNA片段的文库通过电泳从衔接子,引物二聚体和扩增过程中产生的其他DNA分子分离。预期具有cDNA侧翼的衔接长度的DNA被切除并洗脱。
    7. 将文库(5μl,每个文库约500ng)用5μl0.2N NaOH变性5分钟,然后用985μl由Illumina提供的HT1缓冲液中和。加入5μl200mM Tris pH 7.5以补充HT1缓冲液的缓冲能力。
      注意:鉴于病毒基因组的规模很小,多个病毒库可以是Illumina测序运行的一小部分。即使1%的运行样本仍然可以导致每个图书馆数十万次读取。
    8. Illumina NextSeq500运行由印第安那大学CGB工作人员使用75-high输出流动池指定为80 bp读取。

数据分析

  1. 修剪和质量过滤读取使用Trimmomatic ver。 0.33,截止分数最小为20(Bolger等人,2014)。不包括修剪后短于20个核苷酸的读数
  2. 使用具有默认映射参数值的计算机程序Bowtie2 ver 2.2.6(Langmead和Salzberg,2012)将读取映射到相应的引用序列。对于每次读取,仅使用由最小编辑距离定义的最佳对齐。
  3. 显示跨越JBrowse版本的参考序列的每个基本位置的读取次数。 1.12.1作为BigWig曲目(Skinner等人,2009)。

笔记

尽管该协议已经针对结合BMV衣壳的RNA的分析进行了优化,但是该方案可以直接适用于图形感兴趣的病毒粒子。关键在于高纯度病毒粒子和可以有效沉淀感兴趣的衣壳蛋白质的抗体。可以在专门从事基因组分析的设施中进行cDNA文库的构建,nex-gen DNA测序和数据分析。

食谱

  1. SAMA缓冲区
    50 mM Na(OAc)
    8mM Mg(OAc)2,pH5.5
  2. 3 M醋酸钠(pH 5.2)
    将12.3g乙酸钠溶解在30ml H 2 O 3中 用冰醋酸调节pH至5.2
    将H 2 O 2加入到50ml的
    中 用含有0.22μm过滤器的设备进行过滤 在室温下存放
  3. IP绑定缓冲区*
    150 mM NaCl
    25mM Tris(pH7.2)
    0.05%吐温20
  4. IP洗涤缓冲区*
    500 mM NaCl
    25mM Tris(pH7.2)
    0.05%Tween-20

    注意:为了方便起见,请将20x储存的缓冲液用无菌水稀释至1x使用。

  5. 4x Laemmli样品缓冲液
    8%SDS
    40%甘油
    240mM Tris(pH6.8)
    0.04%溴酚蓝
  6. PK缓冲区
    50mM Tris(pH7.5)
    75 mM NaCl
    6.3 mM EDTA
    1%SDS

致谢

该协议是由NIH 1R01AI090280授予CK的工作成立的。该协议由Ni等人(2013)和Hoover等人的工作进行了改编。 (2016)和未发表的E. Chuang的结果。感谢A. Kao在本协议中使用的摄影。

参考

  1. Bolger,AM,Lohse,M.和Usadel,B。(2014)。 Trimmomatic:Illumina序列数据的灵活修剪器。生物信息学 30(15):2114-2120。
  2. Hoover,HS,Wang,JC,Middleton,S.,Ni,P.,Zlotnick,A.,Vaughan,RC和Kao,CC(2016)。  罗马花叶病毒衣壳的磷酸化调节病毒感染的时间。 J Virol 90 (17):7748-7760。
  3. Langmead,B。和Salzberg,SL(2012)。与Bowtie 2快速的间隙读取对齐。 Nat方法 9(4):357-359。
  4. Ni,P.,Vaughan,RC,Tragesser,B.,Hoover,H. and Kao,CC(2013)。  植物宿主可以影响brome花叶病毒(BMV)RNA的壳化:BMV病毒粒子出人意料地是异质性的。 426(5):1061-1076。
  5. Rolfsson,O.,Middleton,S.,Manfield,IW,White,SJ,Fan,B.,Vaughan,R.,Ranson,NA,Dykeman,E.,Twarock,R.,Ford,J.,Kao,CC和Stockley,PG(2016)。  包装的直接证据信号介导的噬菌体MS2的组装.7Mol Biol 428(2 Pt B):431-448。
  6. Skinner,ME,Uzilov,AV,Stein,LD,Mungall,CJ和Holmes,IH(2009)。  JBrowse:下一代基因组浏览器。 Genome Res 19(9):1630-1638。
  7. Stockley,P.G,Ranson,NA和Twarock,R。(2013)。< a class =“ke-insertfile”href =“http://www.futuremedicine.com/doi/abs/10.2217/fvl。 12.84“target =”_ blank“>基因组在ssRNA病毒中的作用的新范例。未来Virol 8:531-543。
  8. Vaughan,R.,Tragesser,B.,Ni,P.,Ma,X.,Dragnea,B. and Kao,CC(2014)。  马铃薯病毒的三分体病毒粒子具有不同的物理性质,影响感染过程的时间。 88(11):6483-6491。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kao, 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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