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Purification of HCV-remodeled and Control ER Membranes
改型丙肝病毒的纯化和内质网莫的控制   

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Abstract

As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane fractions are first enriched by density gradient centrifugation, followed by HA-specific affinity tag purification. Upon elution under native conditions, purified samples can be subject to a variety of biochemical and functional assays.

Keywords: Hepatitis c virus(丙型肝炎病毒), Double membrane vesicles(双膜囊), Affinity purification(亲和纯化), Plus-strand RNA virus(加链RNA病毒), Replication factory(复制厂)

Materials and Reagents

  1. Huh7 human hepatoma cells, stably replicating a wt JFH1 subgenomic replicon (NS4Bwt) and a recombinant replicon cell line harboring the HA epitope and the adaptive mutation Q31R in NS4B (NS4BHA) (Paul et al., 2013) and Huh7 hepatoma cells stably overexpressing C-terminally HA-tagged calnexin (CANXHA) (Paul et al., 2013)
  2. 100 U/ml Pencillin and Streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
  3. 10% (v/v) Fetal Calf Serum (Life Technologies, Gibco®, catalog number: 10270-106 )
  4. 1x nonessential amino acids (Life Technologies, catalog number: 11140-035 )
  5. Geneticin (Life Technologies, Gibco®, catalog number: 11811-098 )
  6. Blasticidin (Life Technologies, Gibco®, catalog number: R210-01 )
  7. Bradford reagent
  8. NaN3 (Sigma-Aldrich, catalog number: S2002 )
  9. Hemagglutinin peptide (Sigma-Aldrich, catalog number: I2149 )
  10. Anti-HA agarose beads (Sigma-Aldrich, catalog number: A2095 )
  11. 1x protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
  12. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 ) (see Recipes)
  13. Bovine serum albumin (BSA) standard (see Recipes)
  14. PBS (sterile) (see Recipes)
  15. Hypotonic buffer (see Recipes)
  16. Sucrose (see Recipes)
  17. 5 M NaCl solution (see Recipes)
  18. IP washing buffer I (see Recipes)
  19. IP washing buffer II (see Recipes)
  20. IP elution buffer (see Recipes)

Equipment

  1. 15 cm cell culture dishes
  2. 37 °C, 5% CO2 cell culture incubator
  3. Cell scraper (e.g. SARSTEDT AG, catalog number: 83.1830 )
  4. Dounce homogenizer (1 ml) (e.g. WHEATON, catalog number: 357538 )
  5. Table top cooling centrifuge with swinging buckets for 15 ml and 50 ml falcon tubes (e.g. Heraeus Multifuge 3-SR)
  6. Open top polyclear ultracentrifuge tubes for SW40 rotor (Seton Identification Products, catalog number: 7031 )
  7. Ultracentrifuge and SW40 rotor and buckets (e.g. Beckman Optima LE80K Ultracentrifuge and Beckman SW40Ti rotor and buckets)
  8. Lab stand and clamp
  9. 19 G needle (e.g. BD, catalog number: 301500 )
  10. Spectrophotometer (e.g. Amersham Ultrospec 2100 pro) and cuvettes (e.g. SARSTEDT AG, catalog number: 67.742 )
  11. Rotator wheel for Eppendorf tubes (e.g. Labinco, model: L28, catalog number: 2800 )
  12. Table top cooling centrifuge for Eppendorf reaction tubes (e.g. Eppendorf, model: 5417R )
  13. Eppendorf Thermomixer compact

Procedure

  1. Cells are grown in DMEMcplt in the incubator in a 5% CO2 environment at 37 °C, in presence of 0.5 mg/ml geneticin in case of replicon cell lines and 10 µg/ml blasticidin for CANXHA overexpressing cells. Around 5-7.5 x 107 cells are needed for the experiment, which equals six 15 cm cell culture dishes.
    Note: Cell confluence should not exceed 90% at the day of harvest.
  2. Cells are washed twice in PBS and subsequently scraped in 5 ml PBS per dish and transferred into a 50 ml falcon tube.
  3. The cells are pelleted at 700 x g for 10 min at 4 °C.
  4. The supernatant is removed and cells are resuspended in 2 ml hypotonic buffer containing 1x protease inhibitor cocktail.
  5. Cells are incubated on ice for 30 min.
  6. Cells are broken by 40 strokes with the dounce homogenizer.
    Note: Turn pestle on the bottom of mortar chamber by 90° during each stroke.
  7. Cell debris and nuclei are pelleted by centrifugation at 700 x g for 10 min at 4 °C.
  8. Preparation of discontinuous sucrose gradient in ultra-centrifugation SW40 tubes:
    Pipette 4 ml 70% sucrose on the bottom and consecutively overlay carefully with 3 ml 60% sucrose, 2 ml 40% sucrose and 2 ml 30% sucrose.
  9. 2 ml post-nuclear supernatants (see step 7) are carefully layered on top of the sucrose gradient.
  10. Membrane fractions are separated in the sucrose gradient by ultra-centrifugation for 4 h at 130,000 x g using a SW40 rotor.
  11. Place tubes in the clamp of a lab stand, and carefully make a hole at the bottom using a 19 G needle. Collect 13 1 ml fractions, with fraction number 1 being the lowest one and number 13 representing the top of the gradient.
    Note: The elution speed of the gradient should be around 1 drop per second, which can be adjusted by the hole size on the bottom.
  12. Gradient fractions can be analyzed for their protein and HCV RNA content. Typically the majority of HCV RNA and proteins are found in fractions 7-11 (Paul et al., 2013), which thus are subsequently pooled.
  13. Equilibrate pooled fractions 7-11 to 150 mM NaCl by adding an appropriate volume of 5 M NaCl solution (e.g. 150 µl 5 M NaCl for 5 ml fractions 7-11), and store a small aliquot (~50 µl as input samples).
  14. Determine protein concentration in pooled fractions by using the Bradford assay and a BSA standard curve.
  15. Wash 25 µl anti-HA agarose beads with 500 µl IP washing buffer I in 2 ml Eppendorf tubes (for each NS4Bwt, NS4BHA and CANXHA). Pellet beads by centrifugation at 3,800 x g for 3 min at 4 °C and remove supernatant.
  16. Incubate beads with membrane fraction containing a total of around 1 mg protein (approximately 1-1.5 ml) for 4 h on a rotator wheel at 4 °C.
    Note: The total protein input should be the same for the three IP samples.
  17. Pellet beads and bound material by centrifugation at 3,800 x g for 3 min at 4 °C and remove supernatant.
  18. Wash beads three times with 1 ml IP washing buffer I and in between each washing step pellet beads as described in step 17.
  19. Add 1ml of IP washing buffer I and transfer beads and bound material into a new eppendorf tube and pellet beads as described in step 17.
  20. Wash beads with 1 ml IP washing buffer II and pellet beads as described in step 17.
  21. Elution of bound material:
    1. Add 100 µl IP elution buffer to the beads and shake the samples for 15 min at 1,100 rpm at 25 °C.
    2. Pellet beads by centrifugation at 3,800 x g for 3 min at 25 °C.
    3. Collect and keep supernatant.
    4. Add 100 µl IP elution buffer to the beads and shake the samples for 15 min at 1,100 rpm at 25 °C.
    5. Pellet beads by centrifugation at 3,800 x g for 3 min at 25 °C.
    6. Collect and pool with supernatant from step 21c.
  22. IP elution fractions can be analyzed in a variety of subsequent assays, including western blot, RNA quantification as well as electron microscopy analyses (Paul et al., 2013).

Recipes

  1. Complete Dulbecco’s Modified Eagle’s Medium (DMEMcplt)
    2 mM L-glutamine
    1x nonessential amino acids
    Supplemented with 100 U/ml Pencillin and Streptomycin
    10% (v/v) Fetal Calf Serum (heat inactivated at 56 °C for 30 min)
  2. BSA standard
    Dissolve 0.1 g of BSA in 10 ml IP washing buffer I (final concentration 10 µg/µl)
    Use 1, 2, 3 and 4 µl for the BSA standard in the Bradford assay
  3. PBS
    8 mM Na2HPO4
    2 mM NaH2PO4
    140 mM NaCl
    0.27 mM KCl
    0.176 mM KH2PO4
  4. Hypotonic buffer
    10 mM Tris (pH 8)
    1.5mM MgCl2
    10 mM NaAc
  5. Sucrose
    70% dissolve 70 g sucrose in 100 ml hypotonic buffer
    60% dissolve 60 g sucrose in 100 ml hypotonic buffer
    40% dissolve 40 g sucrose in 100 ml hypotonic buffer
    30% dissolve 30 g sucrose in 100 ml hypotonic buffer
  6. 5 M NaCl solution
    Dissolve 1,461 g NaCl in 500 ml water
  7. IP washing buffer I
    Hypotonic buffer containing 40% Sucrose and 150 mM NaCl
  8. IP washing buffer II
    Hypotonic buffer containing 10% Sucrose and 150 mM NaCl
  9. IP elution buffer
    IP washing buffer II containing 250 µg/ml hemagglutinin peptide and 0.01 % NaN3

Acknowledgments

The protocol described here has been published originally in a short form in Paul et al. (2013). Research was supported by the DFG TRR83 TP13.

References

  1. Paul, D., Hoppe, S., Saher, G., Krijnse-Locker, J. and Bartenschlager, R. (2013). Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol 87(19): 10612-10627.

简介

至于所有正链RNA病毒,丙型肝炎病毒(HCV)RNA复制与称为病毒复制工厂的重排宿主细胞膜紧密相关。 然而,到目前为止对病毒复制工厂的病毒和细胞成分了解甚少。 在这里,我们描述一个协议,通过使用功能NS4B HA标记亚基因组复制子和C末端HA标记calnexin过表达细胞系,特异性隔离HCV重塑的宿主细胞膜和内质网(ER)膜的幼稚细胞, 分别。 首先通过密度梯度离心富集核后全细胞膜级分,随后通过HA特异性亲和标签纯化。 在天然条件下洗脱时,纯化的样品可进行多种生物化学和功能测定。

关键字:丙型肝炎病毒, 双膜囊, 亲和纯化, 加链RNA病毒, 复制厂

材料和试剂

  1. Huh7人肝癌细胞,稳定复制wt JFH1亚基因组复制子(NS4B wt)和在NS4B(NS4B )中携带HA表位和适应性突变Q31R的重组复制子细胞系, )(Paul等人,2013)和稳定过表达C末端HA标记的钙联接蛋白(CANX HA )的Huh7肝癌细胞(Paul等人, em>,2013)
  2. 100 U/ml青霉素和链霉素(Life Technologies,Gibco ,目录号:15140122)
  3. 10%(v/v)胎牛血清(Life Technologies,Gibco ,目录号:10270-106)
  4. 1x非必需氨基酸(Life Technologies,目录号:11140-035)
  5. 遗传霉素(Life Technologies,Gibco ,目录号:11811-098)
  6. 杀稻瘟素(Life Technologies,Gibco ,目录号:R210-01)
  7. Bradford试剂
  8. NaN 3(Sigma-Aldrich,目录号:S2002)
  9. 血凝素肽(Sigma-Aldrich,目录号:I2149)
  10. 抗HA琼脂糖珠(Sigma-Aldrich,目录号:A2095)
  11. 1x蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693116001)
  12. Dulbecco's Modified Eagle's Medium(DMEM)(Life Technologies,Gibco ,目录号:41965-039)(参见Recipes)
  13. 牛血清白蛋白(BSA)标准(见Recipes)
  14. PBS(无菌)(参见配方)
  15. 低渗缓冲液(见配方)
  16. 蔗糖(参见食谱)
  17. 5 M NaCl溶液(见配方)
  18. IP洗涤缓冲液I(参见配方)
  19. IP洗涤缓冲液II(参见配方)
  20. IP洗脱缓冲液(参见配方)

设备

  1. 15厘米细胞培养皿
  2. 37℃,5%CO 2细胞培养箱中培养
  3. 细胞刮刀( SARSTEDT AG,目录号:83.1830)
  4. Dounce匀浆器(1ml)(例如WHEATON,目录号:357538)
  5. 具有用于15ml和50ml falcon管(例如Heraeus Multifuge 3-SR)的摆动桶的台式冷却离心机。
  6. 用于SW40转子的敞口超清洁超离心管(Seton Identification Products,目录号:7031)
  7. 超速离心机和SW40转子和桶(例如Beckman Optima LE80K超速离心机和Beckman SW40Ti转子和桶)
  8. 实验室支架和夹具
  9. 19 G针(例如BD,目录号:301500)
  10. 分光光度计(例如Amersham Ultrospec 2100pro)和比色皿(例如SARSTEDT AG,目录号:67.742)
  11. 用于Eppendorf管的旋转轮(例如,Labinco,型号:L28,目录号:2800)
  12. 用于Eppendorf反应管(例如Eppendorf,型号:5417R)的台式冷却离心机
  13. Eppendorf Thermomixer紧凑型

程序

  1. 在37℃,5%CO 2亚环境中,在复制子细胞系的情况下,在0.5mg/ml遗传霉素存在下,在培养箱中的DMEM cplt中培养细胞, 10μg/ml杀稻瘟菌素用于CANX过表达细胞。 实验需要约5-7.5×10 7个细胞,其等于六个15cm细胞培养皿。
    注意:收获当天细胞汇合不应超过90%。
  2. 将细胞在PBS中洗涤两次,随后在每个培养皿中刮5ml PBS,并转移到50ml falcon管中。
  3. 细胞在4℃下以700×g离心10分钟
  4. 除去上清液,将细胞重悬浮于含有1×蛋白酶抑制剂混合物的2ml低渗缓冲液中
  5. 将细胞在冰上孵育30分钟
  6. 用dounce匀浆器通过40次冲击破碎细胞。
    注意:在每个行程中将砂浆室底部的杵转动90°。
  7. 通过在4℃下以700×g离心10分钟使细胞碎片和细胞核沉淀。
  8. 在超离心SW40管中制备不连续蔗糖梯度:
    吸取4毫升70%蔗糖在底部,并连续小心地与3毫升60%蔗糖,2毫升40%蔗糖和2毫升30%蔗糖重叠。
  9. 将2ml后核上清液(参见步骤7)小心地铺在蔗糖梯度的顶部
  10. 通过使用SW40转子在130,000×g下超速离心4小时,在蔗糖梯度中分离膜级分。
  11. 将管放置在实验室支架的夹子中,并使用19 G针在底部小心地做一个洞。收集13个1ml级分,其中级分数1是最低的,数字13表示梯度的顶部。
    注意:渐变的洗脱速度应该是每秒1滴左右,这可以通过底部的孔尺寸进行调整。
  12. 可以分析梯度级分的蛋白质和HCV RNA含量。通常,大部分HCV RNA和蛋白质存在于级分7-11(Paul等人,2013)中,因此随后汇集。
  13. 通过加入合适体积的5M NaCl溶液(例如150μl5M NaCl,5ml级分7-11)将合并的级分7-11平衡至150mM NaCl,并储存少量等分试样(〜 50μl作为输入样品)。
  14. 使用Bradford测定法和BSA标准曲线确定合并级分中的蛋白质浓度
  15. 用500μlIP洗涤缓冲液I在2ml Eppendorf管(对于每种NS4B ,NS4B 和CANX )中洗涤25μl抗HA琼脂糖珠, sup>)。通过在4℃下以3,800×g离心3分钟沉淀颗粒珠并除去上清液。
  16. 在4℃下在旋转轮上孵育含有总共约1mg蛋白质(约1-1.5ml)的膜部分的珠子4小时。
    注意:三个IP样品的总蛋白输入应相同。
  17. 通过在4℃下以3,800×g离心3分钟来沉淀小珠和结合材料,并除去上清液。
  18. 用1ml IP洗涤缓冲液I洗涤珠子三次,并在每个洗涤步骤之间,如步骤17所述,颗粒珠子
  19. 加入1ml IP洗涤缓冲液I,并将珠子和结合材料转移到新的eppendorf管和颗粒珠子,如步骤17所述。
  20. 用1ml IP洗涤缓冲液II和颗粒珠洗涤珠子,如步骤17所述
  21. 结合物质的洗脱:
    1. 向珠子中加入100μlIP洗脱缓冲液,并在25℃下以1,100rpm将样品摇动15分钟
    2. 通过在25℃下以3,800×g离心3分钟来沉淀颗粒珠
    3. 收集并保留上清液。
    4. 向珠子中加入100μlIP洗脱缓冲液,并在25℃下以1,100rpm将样品摇动15分钟
    5. 通过在25℃下以3,800×g离心3分钟来沉淀颗粒珠
    6. 收集并与来自步骤21c的上清液混合。
  22. IP洗脱级分可以在多种后续测定中进行分析,包括蛋白质印迹,RNA定量以及电子显微镜分析(Paul等人,2013)。

食谱

  1. 完全Dulbecco's改良的Eagle培养基(DMEM subpl) 2mM L-谷氨酰胺 1x非必需氨基酸
    补充100U/ml青霉素和链霉素 10%(v/v)胎牛血清(在56℃热灭活30分钟)
  2. BSA标准
    将0.1g BSA溶于10ml IP洗涤缓冲液I(终浓度10μg/μl)中 在Bradford测定中使用BSA标准品的1,2,3和4μl。
  3. PBS
    8mM Na 2 HPO 4
    2mM NaH 2 PO 4>/
    140mM NaCl 0.27mM KCl
    0.176mM KH 2 PO 4 sub/
  4. 低渗缓冲液
    10mM Tris(pH8)
    1.5mM MgCl 2 v/v 10 mM NaAc
  5. 蔗糖
    70%将70g蔗糖溶解在100ml低渗缓冲液中 60%将60g蔗糖溶于100ml低渗缓冲液中 40%将40g蔗糖溶解在100ml低渗缓冲液中 30%将30g蔗糖溶于100ml低渗缓冲液中
  6. 5 M NaCl溶液
    将1,461g NaCl溶于500ml水中
  7. IP洗涤缓冲液I
    含有40%蔗糖和150mM NaCl的低渗缓冲液
  8. IP洗涤缓冲液II 含有10%蔗糖和150mM NaCl的低渗缓冲液
  9. IP洗脱缓冲液
    含有250μg/ml血凝素肽和0.01%NaN 3的IP洗涤缓冲液II

致谢

这里描述的协议已经在Paul等人的(2013)中以简短的形式出版。 研究得到DFG TRR83 TP13的支持。

参考文献

  1. Paul,D.,Hoppe,S.,Saher,G.,Krijnse-Locker,J.and Bartenschlager,R.(2013)。 膜性丙型肝炎病毒复制区的形态学和生物化学特征。 Virol 87(19):10612-10627。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Paul, D. and Bartenschlager, R. (2014). Purification of HCV-remodeled and Control ER Membranes. Bio-protocol 4(10): e1134. DOI: 10.21769/BioProtoc.1134.
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