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Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.

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Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA
Flag-TLR3 或 Myc-MSR1 与 HCV RNA的免疫共沉淀

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
作者: Daisuke Yamane
Daisuke YamaneAffiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, USA
For correspondence: yamane@email.unc.edu
Bio-protocol author page: a1194
Hiromichi Dansako
Hiromichi DansakoAffiliation: Tumor Virology Department, Okayama University Graduate School of Medicine, Okayama, Japan
Bio-protocol author page: a1195
 and Stanley M. Lemon
Stanley M. LemonAffiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, USA
Bio-protocol author page: a1196
Vol 4, Iss 5, 3/5/2014, 2966 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1061

[Abstract] Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.
Keywords: PAMP receptor(PAMP受体), Toll-like receptor(Toll样受体), Viral RNA(病毒RNA), Interferon signaling adaptor molecule(干扰素信号适配器分子), Scavenger receptor(清道夫受体)

[Abstract]

Materials and Reagents

  1. Huh-7.5 cells (obtained from Apath, LLC) expressing TLR3-Flag (or other cells stably/transiently expressing Flag/Myc-tagged protein)
  2. DMEM (Life Technologies, catalog number: 11995065 )
  3. 10% heat-inactivated FBS (Life Technologies, catalog number: 26140079 )
  4. Penicillin-streptomycin (Life Technologies, catalog number: 15140-148 )
  5. L-Glutamine (Life Technologies, catalog number: 25030-081 )
  6. Non-essential amino acids (Life Technologies, catalog number: 11140050 )
  7. HCV strain HJ3-5 stock prepared in cell culture medium (see Reference 2)
  8. DPBS (Life Technologies, catalog number: 14190-144 )
  9. RNase inhibitor (RNaseOUT) (Life Technologies, catalog number: 10777-019 )
  10. Protease inhibitor (Complete Protease Inhibitor Cocktail Tablets) (Roche Diagnostics, catalog number: 1697498 )
  11. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  12. Anti-Flag (M2) and anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: F1804 and M4439 ) and mouse IgG control (Life Technologies, catalog number: 02-6502 )
  13. Protein G sepharose (General Electric Company , catalog number: 17-0618-01 )
  14. TRIzol (Life Technologies, catalog number: 15596-026 )
  15. Molecular biology grade water (Corning, catalog number: 46-000-CM )
  16. Agarose (GenePure LE Agarose) (BioExpress, GeneMate, catalog number: E-3120-500 )
  17. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  18. Platinum® Taq DNA Polymerase (Life Technologies)
  19. Culture medium (see Recipes)
  20. Lysis buffer (see Recipes)

Equipment

  1. 100 mm plates (FalconTM, catalog number: 353003 )
  2. Cell scraper (FalconTM, catalog number: 353085 )
  3. Electrophoresis Gel Box
  4. Tube Rotator (Fisher Scientific, catalog number: 05-450-200 )
  5. Centrifuge (Eppendorf, catalog number: 5415R )
  6. Superscript III One-step RT-PCR system (Life Technologies, catalog number: 12574-018 )

Procedure

  1. Preparation of HCV infected cells
    1. Seed the Huh-7.5 cells (1.5 x 106 per dish) stably expressing TLR3-Flag onto a 10 cm dish and incubated overnight.
    2. Inoculate HCV (strain HJ3-5) at an MOI of 1 (5 ml of HJ3-5 stock at 3 x 105 FFU/ml) for 6 h, remove the inoculum, and then replace with 10 ml fresh culture medium.
    3. Incubate cells at 37 °C in 5% CO2 for 72 h.

  2. Preparation of the lysates
    1. Aspirate culture medium, wash cells twice with DPBS, and scrape the cells into 50 ml tube.
    2. Centrifuge at 800 x g for 3 min at 4 °C, and remove the supernatant.
    3. Resuspend the cell pellet in 1 ml lysis buffer, and rotate the lysate for 5-6 h at 4 °C.
    4. Centrifuge the lysate at 15,700 x g for 20 min at 4 °C.
    5. Transfer the supernatant to 1.5 ml tube and place on ice, and determine the protein content using Protein Assay Kit.

  3. Immunoprecipitation
    1. Transfer the cell lysate (20 μg of total protein) to a new 1.5 ml tube.
    2. Add 1 μg of anti-Flag antibody or mouse IgG control to the lysate and rotate overnight at 4 °C.
    3. Add 20 μl Protein G sepharose to the lysate and rotate for 2-3 h at 4 °C.
    4. Centrifuge at 800 x g for 3 min at 4 °C.
    5. Aspirate the supernatant, wash beads with 1 ml lysis buffer and rotate for 10 min at 4 °C.
    6. Centrifuge at 800 x g for 3 min at 4 °C.
    7. Repeat steps C4-5 twice.
    8. Suspend the sepharose in 100 μl DPBS and use half for RNA extraction and the remainder for Western blotting to detect the immunoprecipitated protein.

  4. RNA extraction and RT-PCR
    1. Extract RNA from the sepharose beads by vortexing 15 sec in TRIzol reagent, followed by the standard protocol as indicated in the manufacturer’s instruction, and suspend the RNA pellet in 50 μl of nuclease-free water (optional: Add 1 μl of Glycogen before the precipitation of RNA with Isopropanol).
    2. Detect HCV RNA bound to TLR3-Flag with SuperScriptIII One-Step RT-PCR System with Platinum® Taq DNA Polymerase using an HCV-specific primer pair HCV84FP, 5’-GCCATGGCGTTAGTATGAGTGT-3’; HCV 303RP, 5’-CACCCTATCAGGCAGTACCACAA-3’, at an annealing temperature of 55 °C, followed by gel electrophoresis in 1.5% agarose gel. Specific bands (220 bp) can be detected typically with 35-40 PCR cycles.

Recipes

  1. Culture medium
    DMEM
    10% heat-inactivated FBS
    Penicillin-streptomycin
    L-Glutamine
    Non-essential amino acids
  2. Lysis buffer
    1x DPBS
    0.1% Triton X-100
    1x Protease inhibitor cocktail
    100 U/ml RNaseOUT

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (RO1-AI095690) and the University Cancer Research Fund. This protocol is adapted from previous work by Dansako et al. (2013).

References

  1. Dansako, H., Yamane, D., Welsch, C., McGivern, D. R., Hu, F., Kato, N. and Lemon, S. M. (2013). Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9(5): e1003345.
  2. Yi, M., Ma, Y., Yates, J. and Lemon, S. M. (2007). Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81(2): 629-638.

材料和试剂

  1. 表达TLR3-Flag(或稳定/瞬时表达Flag/Myc标记的蛋白的其他细胞)的Huh-7.5细胞(获自Apath,LLC)
  2. DMEM(Life Technologies,目录号:11995065)
  3. 10%热灭活的FBS(Life Technologies,目录号:26140079)
  4. 青霉素 - 链霉素(Life Technologies,目录号:15140-148)
  5. L-谷氨酰胺(Life Technologies,目录号:25030-081)
  6. 非必需氨基酸(Life Technologies,目录号:11140050)
  7. 在细胞培养基中制备的HCV菌株HJ3-5原液(参见参考文献2 )
  8. DPBS(Life Technologies,目录号:14190-144)
  9. RNase抑制剂(RNaseOUT)(Life Technologies,目录号:10777-019)
  10. 蛋白酶抑制剂(完全蛋白酶抑制剂混合物片)(Roche Diagnostics,目录号:1697498)
  11. Triton X-100(Sigma-Aldrich,目录号:T9284)
  12. 抗Flag(M2)和抗Myc(9E10)单克隆抗体(Sigma-Aldrich,目录号:F1804和M4439)和小鼠IgG对照(Life Technologies,目录号:02-6502)
  13. 蛋白G琼脂糖(General Electric Company,目录号:17-0618-01)
  14. TRIzol(Life Technologies,目录号:15596-026)
  15. 分子生物学级水(Corning,目录号:46-000-CM)
  16. 琼脂糖(GenePure LE agarose)(BioExpress,GeneMate,目录号:E-3120-500)
  17. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  18. Platinum ® Taq DNA Polymerase(Life Technologies)
  19. 培养基(见配方)
  20. 裂解缓冲液(见配方)

设备

  1. 100mm板(Falcon TM ,目录号:353003)
  2. 细胞刮刀(Falcon TM ,目录号:353085)
  3. 电泳凝胶盒
  4. Tube Rotator(Fisher Scientific,目录号:05-450-200)
  5. 离心机(Eppendorf,目录号:5415R)
  6. Superscript III一步RT-PCR系统(Life Technologies,目录号:12574-018)

程序

  1. HCV感染细胞的制备
    1. 将稳定表达TLR3-Flag的Huh-7.5细胞(每皿1.5×10 6个)接种到10cm培养皿上并孵育过夜。
    2. 以MOI为1(5ml HJ3-5原液,3×10 5 FFU/ml)接种6小时的HCV(菌株HJ3-5)6小时,除去接种物,然后用10 ml新鲜培养基
    3. 在37℃下在5%CO 2中孵育细胞72小时
  2. 裂解物的制备
    1. 吸出培养基,用DPBS洗涤细胞两次,并将细胞刮到50ml管中
    2. 在4℃下以800×g离心3分钟,并除去上清液。
    3. 重悬细胞沉淀在1毫升裂解缓冲液,并在4℃下旋转裂解物5-6小时。
    4. 在4℃下以15,700xg离心裂解物20分钟。
    5. 将上清液转移至1.5ml管中并置于冰上,并使用蛋白质测定试剂盒确定蛋白质含量
  3. 免疫沉淀
    1. 将细胞裂解液(20μg总蛋白)转移到新的1.5 ml试管中
    2. 向裂解物中加入1μg抗Flag抗体或小鼠IgG对照,并在4℃下旋转过夜
    3. 加入20微升蛋白G琼脂糖凝胶裂解液,并在4℃下旋转2-3小时
    4. 在4℃下以800xg离心3分钟。
    5. 吸出上清液,用1ml裂解缓冲液洗涤珠子,并在4℃下旋转10分钟
    6. 在4℃下以800xg离心3分钟。
    7. 重复步骤C4-5两次。
    8. 将琼脂糖悬浮在100μlDPBS中,使用一半用于RNA提取,剩余部分用于Western印迹以检测免疫沉淀的蛋白质。

  4. RNA提取和RT-PCR
    1. 通过在TRIzol试剂中涡旋15秒,然后按照制造商的说明书中所述的标准方案从琼脂糖珠提取RNA,并将RNA沉淀悬浮于50μl无核酸酶的水中(任选:在沉淀前加入1μl糖原的RNA与异丙醇)
    2. 使用具有Platinum Taq DNA聚合酶的SuperScriptIII一步RT-PCR系统,使用HCV特异性引物对HCV84FP,5'-GCCATGGCGTTAGTATGAGTGT检测结合至TLR3- -3'; HCV 303RP,5'-CACCCTATCAGGCAGTACCACAA-3',在55℃的退火温度下,随后在1.5%琼脂糖凝胶中进行凝胶电泳。通常可以用35-40个PCR循环检测特异性条带(220bp)

食谱

  1. 培养基
    DMEM
    10%热灭活的FBS 青霉素 - 链霉素 L-谷氨酰胺 非必需氨基酸
  2. 裂解缓冲液
    1x DPBS
    0.1%Triton X-100 1x蛋白酶抑制剂混合物
    100 U/ml RNaseOUT

致谢

这项工作部分得到了国立卫生研究院(RO1-AI095690)和大学癌症研究基金的资助。该协议改编自Dansako等人之前的工作。(2013)。

参考文献

  1. Dansako,H.,Yamane,D.,Welsch,C.,McGivern,D.R.,Hu,F.,Kato,N.and Lemon,S.M。 A类清道夫受体1(MSR1)通过介导toll样受体3识别来限制丙型肝炎病毒复制在邻近细胞中产生的病毒RNA。

    9(5):e1003345。
  2. Yi,M.,Ma,Y.,Yates,J。和Lemon,S.M。(2007)。 E1,p7,NS2和NS3中的补偿性突变增强了细胞培养 - 感染性基因型间嵌合肝炎的产量C.病毒。 J Virol 81(2):629-638。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yamane, D., Dansako, H. and Lemon, S. M. (2014). Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA. Bio-protocol 4(5): e1061. DOI: 10.21769/BioProtoc.1061; Full Text
  2. Dansako, H., Yamane, D., Welsch, C., McGivern, D. R., Hu, F., Kato, N. and Lemon, S. M. (2013). Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9(5): e1003345.




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