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Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus
哺乳动物正呼肠孤病毒传染性亚病毒颗粒诱导的溶血试验   

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Journal of Virology
Mar 2016

Abstract

Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.

Keywords: Virology (病毒学), Reoviridae (呼肠孤病毒科), Mammalian orthoreovirus (哺乳动物正呼肠弧病毒), Viral entry (病毒进入), Membrane penetration (膜穿透), Hemolysis (溶血)

Background

Reoviruses are nonenveloped, double-stranded RNA viruses that are composed of two concentric protein shells: the inner capsid (core) and the outer capsid (Dryden et al., 1993; Zhang et al., 2005; Dermody et al., 2013). Following attachment, virions are endocytosed (Borsa et al., 1979; Ehrlich et al., 2004; Maginnis et al., 2006; Maginnis et al., 2008) and host cathepsin proteases degrade the σ3 outer capsid protein (Chang and Zweerink, 1971; Silverstein et al., 1972; Borsa et al., 1981; Sturzenbecker et al., 1987; Dermody et al., 1993; Baer and Dermody, 1997; Ebert et al., 2002). This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the cell penetration protein, µ1, is exposed (Dryden et al., 1993). Reovirus ISVPs undergo a second conformational change to deposit the genome- containing core into the host cell cytoplasm. The altered particle is called ISVP* (Chandran et al., 2002). ISVP-to-ISVP* conversion culminates in the release of µ1-derived pore forming peptides (Nibert et al., 1991; Zhang et al., 2005; Chandran et al., 2002; Odegard et al., 2004; Nibert et al., 2005; Agosto et al., 2006; Ivanovic et al., 2008). The released peptides form pores within endosomal membranes, which are thought to mediate core delivery (Agosto et al., 2006; Ivanovic et al., 2008; Zhang et al., 2009).

Many of the conformational changes that define reovirus entry can be recapitulated in vitro: (i) ISVPs are produced by digesting purified virions with chymotrypsin (Joklik, 1972; Borsa et al., 1973a), and (ii) ISVP* formation can be induced using heat (Middleton et al., 2002), large monovalent cations (Borsa et al., 1973b), µ1-derived peptides (Agosto et al., 2008), red blood cells (Chandran et al., 2002; Sarkar and Danthi, 2010), or lipids (Snyder and Danthi, 2015 and 2016). Thus, questions related to reovirus entry are studied using biochemical and cell-based approaches. In this protocol, we describe an in vitro assay that recapitulates ISVP-to-ISVP* conversion and subsequent pore formation.

Materials and Reagents

  1. Pipette tips
    1. 0.1-10 µl capacity (USA Scientific, catalog number: 1111-3700 )
    2. 1-200 µl capacity (VWR, catalog number: 89079-474 )
    3. 100-1,250 µl capacity (VWR, catalog number: 53508-924 )
  2. PCR 8-well tube strips (VWR, catalog number: 20170-004 )
  3. 50 ml centrifuge tube (VWR, catalog number: 89039-660 )
  4. 1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
  5. Vacuum driven and disposable bottle top 0.22 µm filter (Merck, catalog number: SCGPT05RE )
  6. Flat bottom, 96-well plate (Greiner Bio One International, catalog number: 655180 )
  7. Purified reovirus stocks (see Berard and Coombs, 2009; Kobayashi et al., 2010 for propagation and purification procedures)
  8. Crushed ice
  9. Standard SDS-PAGE materials and reagents (e.g., 10% SDS-polyacrylamide mini gels)
  10. Coomassie Brilliant Blue stain and destain solutions (Bio-Rad Laboratories, catalog number: 1610435 )
  11. Citrated bovine calf blood (Colorado Serum Company, catalog number: 31023 )
  12. Bleach (Biz4USA, Janitorial Supplies, catalog number: CLO30966CT )
  13. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) (MP Biomedicals, catalog number: 02103133 )
  14. Sodium chloride (NaCl) (Merck, catalog number: SX0420-3 )
  15. 0.1 N hydrochloric acid (Sigma-Aldrich, catalog number: 2104 )
  16. 0.1 N sodium hydroxide (Sigma-Aldrich, catalog number: 2105 )
  17. Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (Worthington Biochemical, catalog number: LS001432 )
  18. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  19. Isopropyl alcohol (Avantor Performance Materials, Macron, catalog number: 3032-02 )
  20. Dulbecco’s phosphate buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 21600044 )
  21. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
  22. Ultrapure DNase/RNase-free distilled H2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  23. Triton X-100 (TX-100) (Sigma-Aldrich, catalog number: X100 )
  24. 50% bleach (see Recipes)
  25. Virus storage buffer (VB) (see Recipes)
  26. 2 mg/ml TLCK-treated chymotrypsin (see Recipes)
  27. 100 mM phenylmethylsulfonyl fluoride (PMSF) (see Recipes)
  28. Phosphate buffered saline supplemented with 2 mM MgCl2 (PBSMg) (see Recipes)
  29. 10% Triton X-100 (TX-100) (see Recipes)

Equipment

  1. Personal protective equipment (PPE)
    1. Laboratory coat
    2. Gloves
    3. Eye protection
  2. Biosafety level 2 (BSL-2) laboratory facility
  3. BSL-2 certified tissue culture hood
  4. Solid and liquid waste containers
  5. Autoclave
  6. Vacuum pump and aspirator
  7. Ice bucket
  8. -20 °C freezer
  9. Micropipettes
    1. 0.1-2.5 µl capacity (Eppendorf, catalog number: 3123000012 )
    2. 2-20 µl capacity (Eppendorf, catalog number: 3123000039 )
    3. 20-200 µl capacity (Eppendorf, catalog number: 3123000055 )
    4. 100-1,000 µl capacity (Eppendorf, catalog number: 3123000063 )
  10. Digital pH meter (VWR, model: SB70P )
  11. Digital laboratory balance (Mettler Toledo, model: PB1502-S )
  12. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: ND-1000 )
  13. Hot plate stirrer (VWR, catalog number: 12365-382 )
  14. Magnetic stir bar (VWR, catalog number: 58948-273 )
  15. Microcentrifuge (Eppendorf, model: 5424 )
  16. Thermal cycler (Bio-Rad Laboratories, model: S1000TM )
  17. Temperature controlled water bath (VWR, catalog number: 89501-466 )
  18. Gel imaging system (LI-COR, model: Odyssey® Classic )
  19. Microplate reader (Molecular Devices, model: FilterMax F5 Multi-Mode )
  20. 250 ml glass beaker (VWR, catalog number: 89000-204 )
  21. 1,000 ml glass beaker (VWR, catalog number: 89000-212 )
  22. 100 ml graduated cylinder (VWR, catalog number: 65000-006 )
  23. 1,000 ml graduated cylinder (VWR, catalog number: 65000-012 )
  24. 100 ml storage bottle (VWR, catalog number: 89000-234 )
  25. 1,000 ml storage bottle (VWR, catalog number: 89000-240 )
    Note: This product has been discontinued.

Software

  1. Image Studio Lite (LI-COR)
  2. SoftMax Pro (Molecular Devices)

Procedure

  1. Generation of infectious subviral particles (ISVPs)
    1. Propagate and purify reovirus virions as previously described (Berard and Coombs, 2009; Kobayashi et al., 2010). Using a NanoDrop spectrophotometer, determine particle concentration by measuring the optical density of the purified virus stocks at 260 nm (OD260; 1 unit at OD260 = 2.1 x 1012 particles/ml) (Smith et al., 1969).
    2. In 1 tube of an 8-well tube strip, dilute 2 x 1011 virions into 90 µl of ice cold VB (see Recipes).
    3. Add 10 µl of ice cold 2 mg/ml TLCK-treated chymotrypsin (see Recipes) to the diluted virus. Mix by pipetting up and down 3-4 times.
      Note: For an undigested control, substitute 10 µl of ice cold VB for 10 µl of TLCK-treated chymotrypsin.
    4. Incubate the reaction for 20 min at 32 °C in a thermal cycler.
      Note: Under these conditions, σ3 is degraded (Joklik, 1972; Borsa et al., 1973a) and µ1 is cleaved (Nibert and Fields, 1992; Chandran et al., 1999).
    5. Following digestion, quench chymotrypsin activity by the addition of 1 µl of 100 mM PMSF (see Recipes). Mix by pipetting up and down 3-4 times.
    6. Incubate the reaction for 20 min on ice.
    7. To confirm that ISVPs are generated, run 2 x 1010 particles per lane on a 10% SDS-polyacrylamide mini gel. Run the gel for 40-45 min at 200 V constant.
    8. Visualize the protein bands by Coomassie Brilliant Blue staining (see Data analysis, Figure 1).
    9. Store ISVPs on ice, and use within 2-3 h for hemolysis experiments.

  2. Preparation of bovine red blood cells (RBCs)
    1. Perform all steps on ice or at 4 °C.
    2. Transfer 1 ml of citrated bovine calf blood to a microcentrifuge tube.
      Note: Citrated bovine calf blood should be used within 2 weeks of the draw date.
    3. Pellet the RBCs by centrifugation at 500 x g for 5 min.
      Note: RBCs are the source of membranes for hemolysis experiments.
    4. Aspirate and discard the supernatant.
    5. Resuspend the RBCs in 1 ml of ice cold PBSMg (see Recipes). Mix by gently pipetting up and down.
    6. Repeat Steps B3-B5 until the supernatant remains clear after pelleting.
    7. Resuspend the washed RBCs in ice cold PBSMg at a 30% (vol/vol) concentration. Mix by gently flicking the side of the tube.
      Note: Estimate the RBC pellet volume by using the volume markers on the microcentrifuge tube.
    8. Store RBCs on ice, and use immediately for hemolysis experiments

  3. ISVP-induced hemolysis assay
    1. In separate microcentrifuge tubes, assemble the following reactions on ice:
      1. 33.3 µl VB + 3.7 µl 30% RBCs (0% hemolysis control)
      2. 30.3 µl VB + 3.7 µl 30% RBCs + 3 µl 10% TX-100 (100% hemolysis control, see Recipes)
      3. 3.3 µl VB + 3.7 µl 30% RBCs + 30 µl ISVPs
    2. Mix the reactions by gently flicking the side of the tubes.
    3. Incubate the reactions for 1 h (T3D reovirus) or for 2 h (T1L reovirus) at 37 °C in a water bath.
      Note: Under these conditions, ISVP-to-ISVP* conversion is induced (Chandran et al., 2002; Sarkar and Danthi, 2010) and the µ1-derived pore forming peptides are released (Nibert et al., 1991; Chandran et al., 2002; Odegard et al., 2004; Nibert et al., 2005; Zhang et al., 2005; Agosto et al., 2006; Ivanovic et al., 2008).
    4. Place the reactions on ice for 20 min.
    5. Pellet intact RBCs by centrifugation at 500 x g for 5 min.
      Note: This step should be performed at 4 °C.
    6. Transfer 20 µl of each supernatant to individual wells of a 96-well plate.
    7. Dilute each transferred supernatant with 80 µl of VB. Mix by pipetting up and down 3-4 times.
    8. To quantify the amount of hemoglobin released (i.e., RBC lysis), measure the absorbance (A) of the diluted supernatants at 405 nm using a microplate reader. A values are recorded on SoftMax Pro software.
    9. Calculate the percent hemolysis (see Data analysis).

Data analysis

  1. Generation of infectious subviral particles (ISVPs)
    1. Record and analyze the results using a gel imaging system and Image Studio Lite software (Figure 1).
      1. Virions contain λ1,2,3, µ1C, σ2, and σ3.
      2. ISVPs contain λ1,2,3, µ1C, δ, and σ2.
      Note: The appearance of δ, the loss of µ1C, and the loss of σ3 indicate the transition from virions to ISVPs. λ1,2,3 and σ2 should remain unchanged.


      Figure 1. SDS-PAGE gel of reovirus virions and ISVPs

  2. ISVP-induced hemolysis assay
    1. All hemolysis experiments should be repeated for at least three independent replicates.
    2. Calculate the percent hemolysis using the following formula:

      [(Asample - Abuffer)/(ATX-100 - Abuffer)] x 100

      1. Abuffer represents the supernatant derived from VB and RBCs.
      2. ATX-100 represents the supernatant derived from VB, RBCs, and TX-100.
      3. Asample represents the supernatant derived from VB, RBCs, and ISVPs.
    3. When comparing the hemolytic capacity of different reovirus strains, calculate P values using Student’s t-test.
    4. Use graphing software to plot percent hemolysis.
      Note: For T3D reovirus, 40-60% hemolysis is typically observed after 1 h incubation at 37 °C.

Notes

  1. When possible, all procedures are performed in a BSL-2 certified tissue culture hood.
  2. Laboratory personnel should use appropriate PPE.
  3. All solid waste is autoclaved prior to disposal.
  4. All liquid waste is inactivated with 50% bleach prior to disposal.

Recipes

  1. 50% bleach
    In a storage bottle, dilute 50 ml of 100% bleach into 50 ml of ultrapure H2
  2. Virus storage buffer (VB) (10 mM Tris, pH 7.4, 15 mM MgCl2, and 150 mM NaCl)
    1. In a glass beaker, dissolve the following into 900 ml of ultrapure H2O:
      1.21 g Tris
      3.05 g MgCl2·6H2O
      8.77 g NaCl
    2. Mix at room temperature using a magnetic stir bar on a stir plate
    3. Adjust to pH 7.4 with 0.1 N hydrochloric acid
    4. In a graduated cylinder, bring the final volume up to 1,000 ml with ultrapure water
    5. Transfer the solution to a storage bottle
    6. Sterilize by autoclaving
    7. Store at room temperature3.
  3. 2 mg/ml Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin
    1. In a centrifuge tube, dissolve 100 mg of TLCK-treated chymotrypsin into 50 ml of ultrapure H2O
    2. Mix at room temperature by gently inverting the tube until the solution becomes clear
    3. Transfer 1 ml aliquots to microcentrifuge tubes
    4. Store at -20 °C
  4. 100 mM phenylmethylsulfonyl fluoride (PMSF)
    1. In a microcentrifuge tube, dissolve 17.4 mg of PMSF into 1 ml of isopropyl alcohol
    2. Mix at room temperature by gently inverting the tube until the solution becomes clear
    3. Store at -20 °C
  5. Phosphate buffered saline supplemented with 2 mM MgCl2 (PBSMg)
    1. In a glass beaker, dissolve the following into 900 ml of ultrapure H2O:
      9.55 g Dulbecco’s phosphate buffered saline
      0.41 g MgCl2·6H2O
    2. Mix at room temperature using a magnetic stir bar on a stir plate
    3. Adjust to pH 7.4
    4. In a graduated cylinder, bring the final volume up to 1,000 ml with ultrapure water
    5. Sterilize by filtering through a 0.22 µm bottle top filter
    6. Store at room temperature
  6. 10% Triton X-100 (TX-100)
    1. In a glass beaker, dissolve the following into 80 ml of ultrapure H2O:
      0.12 g Tris
      0.31 g MgCl2·6H2O
      0.88 g NaCl
      10 ml of 100% TX-100
    2. Mix at room temperature using a magnetic stir bar on a stir plate
    3. Adjust to pH 7.4
    4. In a graduated cylinder, bring the final volume up to 100 ml with ultrapure water
    5. Transfer the solution to a storage bottle
    6. Sterilize by autoclaving
    7. Store at room temperature

Acknowledgments

This protocol was adapted from previously published studies (Chandran et al., 2002; Sarkar and Danthi, 2010). Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers 1R01AI110637 (to P.D.) and F32AI126643 (to A.J.S.) and by Indiana University Bloomington. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders. The authors declare no conflict of interest.

References

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  8. Borsa, J., Sargent, M. D., Long, D. G. and Chapman, J. D. (1973b). Extraordinary effects of specific monovalent cations on activation of reovirus transcriptase by chymotrypsin in vitro. J Virol 11(2): 207-217.
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  10. Chandran, K., Walker, S. B., Chen, Y., Contreras, C. M., Schiff, L. A., Baker, T. S. and Nibert, M. L. (1999). In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3. J Virol 73(5): 3941-3950.
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简介

哺乳动物正面病毒(呼肠孤病毒)外壳在病毒进入之前或过程中经历一系列构象变化。 这些转换对于跨宿主细胞膜递送含基因组的核心是必要的。 该协议描述了用于监测向膜渗透活性形式(即,ISVP *)的转变的体外试验。

【背景】呼肠孤病毒是无包膜的双链RNA病毒,其由两个同心蛋白质壳组成:内衣壳(核心)和外衣壳(Dryden等人,1993; Zhang等人, / ,2005; Dermody et al ,2013)。在附着之后,病毒颗粒被内吞(Borsa et al。,1979; Ehrlich et al。,2004; Maginnis et al。,2006; Maginnis和宿主组织蛋白酶蛋白酶降解σ3外壳蛋白(Chang和Zweerink,1971; Silverstein等人,1972; Borsa等人,et al。 1981; Sturzenbecker等人,1987; Dermody等人,1993; Baer和Dermody,1997; Ebert等人, 2002年)。这个过程产生一个亚稳中间体,称为感染性亚病毒颗粒(ISVP),其中细胞穿透蛋白μ1被暴露(Dryden等人,1993)。 ISVPs是通过用糜蛋白酶处理纯化的病毒体而在体外产生的(Joklik,1972; Borsa等人,1973a)。外壳经历第二构象变化以将含基因组的核心沉积到宿主细胞的细胞质中。结果,μ1的中心δ片段采取蛋白酶敏感构象。被改变的粒子被称为ISVP *(Chandran et al。,2002)。 ISVP-to-ISVP *转换可以通过使用热(Middleton等人,2002),大的单价阳离子(Borsa等人 ,1973b),μ1衍生肽(Agosto et al。,2008),红细胞(Chandran等人,2002; Sarkar和Danthi,2010) ,或血脂(Snyder,和Danthi,2015年和2016年)。因此,使用生物化学和基于细胞的方法研究与呼肠孤病毒进入有关的问题(例如,粒子稳定性和感染性之间的关系)。在这个协议中,我们描述了一个体外试验,概括了ISVP到ISVP *的转换。

关键字:病毒学, 呼肠孤病毒科, 哺乳动物正呼肠弧病毒, 病毒进入, 膜穿透, 溶血

材料和试剂

  1. 移液器吸头
    1. 0.1-10μl容量(USA Scientific,目录号:1111-3700)
    2. 1-200μl容量(VWR,目录号:89079-474)
    3. 容量100-1,250μl(VWR,目录号:53508-924)
  2. PCR 8孔管条(VWR,目录号:20170-004)
  3. 50毫升离心管(VWR,目录号:89039-660)
  4. 1.7 ml微量离心管(MIDSCI,目录号:AVSS1700)
  5. 纯化的呼肠孤病毒储液(参见[Berard and Coombs,2009; Kobayashi et al。,2010],用于繁殖和纯化过程)
  6. 碎冰
  7. 标准SDS-PAGE材料和试剂(例如,10%SDS-聚丙烯酰胺微型凝胶)
  8. 考马斯亮蓝染色和脱色溶液(Bio-Rad Laboratories,目录号:1610435)
  9. 漂白剂(Biz4USA,Janitorial Supplies,目录号:CLO30966CT)
  10. 2-氨基-2-(羟甲基)-1,3-丙二醇(Tris)(MP Biomedicals,目录号:02103133)
  11. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M9272)
  12. 氯化钠(NaCl)(Merck,目录号:SX0420-3)
  13. 0.1N盐酸(Sigma-Aldrich,目录号:2104)
  14. N氢氧化钠(Sigma-Aldrich,目录号:2105)
  15. 4×Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610747)
  16. N-α-甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)处理的胰凝乳蛋白酶(Worthington Biochemical,目录号:LS001432)
  17. 胰蛋白酶(Sigma-Aldrich,目录号:T6567)
  18. 苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  19. 超纯脱氧核糖核酸酶/无RNA酶的蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  20. 异丙醇(Avantor Performance Materials,Macron,目录号:3032-02)
  21. 50%的漂白剂(见食谱)
  22. 病毒存储缓冲区(VB)(请参阅食谱)
  23. 2毫克/毫升α-对甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)处理的胰凝乳蛋白酶(参见食谱)
  24. 100毫米苯甲基磺酰氟(PMSF)(见食谱)
  25. 1毫克/毫升胰蛋白酶(见食谱)

设备

  1. 个人防护设备(PPE)
    1. 实验室外套
    2. 手套
    3. 眼睛保护
  2. 生物安全2级(BSL-2)实验室设施
  3. BSL-2认证的组织培养罩
  4. 固体和液体废物容器
  5. 高压灭菌器
  6. 冰桶
  7. -20°C冷冻机
  8. 微量移液器
    1. 0.1-2.5μl容量(Eppendorf,目录号:3123000012)
    2. 2-20μl容量(Eppendorf,目录号:3123000039)
    3. 20-200μl容量(Eppendorf,目录号:3123000055)
    4. 100-1,000μl容量(Eppendorf,目录号:3123000063)
  9. 数字pH计(VWR,型号:SB70P)
  10. 数字实验室天平(梅特勒 - 托利多,型号:PB1502-S)
  11. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:ND-1000)
  12. 热板搅拌器(VWR,目录号:12365-382)
  13. 磁力搅拌棒(VWR,目录号:58948-273)
  14. 热循环仪(Bio-Rad Laboratories,型号:S1000 TM)
  15. 模拟干式加热器(VWR,目录号:12621-110)
  16. 温度计(VWR,目录号:89095-566)
  17. 凝胶成像系统(LI-COR,型号:Odyssey®Classic)
  18. 1000毫升玻璃烧杯(VWR,目录号:89000-212)
  19. 1000毫升量筒(VWR,目录号:65000-012)
  20. 1000毫升储存瓶(VWR,目录号:89000-240)
    注:此产品已停产。

软件

  1. Image Studio Lite(LI-COR)

程序

  1. 产生感染性亚病毒颗粒(ISVPs)
    1. 如先前所述(Berard和Coombs,2009; Kobayashi等人,2010)繁殖和纯化呼肠孤病毒毒粒。使用NanoDrop分光光度计,通过测量纯化的病毒原液在260nm处的光密度(OD 260 = 1,OD 260 = 2.1×10 -1, > 12颗粒/ ml)(Smith等人,1969)。
    2. 在8孔管条的1根管中,将2×10 11病毒体稀释到90μl冰冷的VB中(见食谱)。
    3. 加10μl冰冷的2mg / ml TLCK处理的胰凝乳蛋白酶(见食谱)到稀释的病毒中。
      上下移液3-4次 注意:对于未消化的对照,用10μl冰冷的VB取代10μlTLCK处理的胰凝乳蛋白酶。

    4. 在32°C的热循环仪孵育反应20分钟 注意:在这些条件下,σ3被降解(Joklik,1972; Borsa等,1973a),而μ1被裂解(Nibert和Fields,1992; Chandran等,1999)。 >
    5. 消化后,通过加入1μl100mM PMSF淬灭糜蛋白酶活性(参见食谱)。
      上下移液3-4次

    6. 在冰上孵育反应20分钟
    7. 为确认产生了ISVP,在10%SDS-聚丙烯酰胺微型凝胶上每泳道运行2×10 10个颗粒。
      在200V恒定温度下运行凝胶40-45分钟
    8. 通过考马斯亮蓝染色显示蛋白质条带(见数据分析,图1)。
    9. 将ISVP储存在冰上,2-3小时内用于ISVP-to-ISVP *转换实验。

  2. ISVP到ISVP *转换测定

    1. 加入10μlISVPs到8孔管冰上的每个管
    2. 在热循环仪中以温度梯度孵育ISVPs 1小时(即,每管在不同的温度下孵育)。
      注:适当的温度范围将根据孵育时间(5分钟至1小时)和正在研究的呼肠孤病毒菌株而变化。温度梯度的每一步应不低于1°C。 ISVP到ISVP *的转换通常发生在30°C和50°C之间。

    3. 在冰上培养热处理ISVPs5分钟
    4. 加入0.9微升1毫克/毫升胰蛋白酶(见食谱),每管热处理ISVPs。
      上下移液3-4次

    5. 在冰上孵育反应30分钟
    注意:由于ISVP到ISVP *的转换,μ1的中心δ片段采用蛋白酶敏感构象(Chandran et al。,2002)。
    1. 消化后,加入3.3微升4×Laemmli样品缓冲液到每个反应。
      上下移液3-4次

    2. 每个反应煮沸5分钟在95°C
    3. 为了确定ISVP到ISVP *转换发生的温度,运行2×10 10个颗粒( ie ,整个ISVP到ISVP *转换反应在给定温度)在10%SDS-聚丙烯酰胺微型凝胶上进行。
      在200V恒定温度下运行凝胶40-45分钟
    4. 通过考马斯亮蓝染色显示蛋白质条带(见数据分析,图2)。

数据分析

  1. 产生感染性亚病毒颗粒(ISVPs)
    1. 使用凝胶成像系统和Image Studio Lite软件记录和分析结果(图1)。
      1. 病毒体含有λ1,2,3,μ1C,σ2和σ3。
      2. ISVPs包含λ1,2,3,μ1C,δ和σ2。
      注意:δ的出现,μ1C的损失以及σ3的损失表明从病毒粒子到ISVP的转变。 λ1,2,3和σ2应保持不变。


      图1.呼肠孤病毒和ISVPs的SDS-PAGE凝胶

    2. ISVP到ISVP *转换测定
      1. 所有的ISVP-ISVP *实验都应该重复进行至少三次独立的重复。
      2. 使用凝胶成像系统和Image Studio Lite软件记录和分析结果(图2)。
        1. 胰蛋白酶处理的ISVPs含有λ1,2,3,μ1C,δ和σ2。
        2. 胰蛋白酶处理的ISVP * s含有λ1,2,3和σ2。
      注意:μ1C和δ的损失表明从ISV到ISVP的转换。 λ1,2,3和σ2应保持不变。


      图2.加热和胰蛋白酶处理的呼肠弧病毒ISVPs的SDS-PAGE凝胶在这里显示的实验中,ISVP-to-ISVP *转换发生在44,45和46℃。

笔记

  1. 如果可能的话,所有的程序都在BSL-2认证的组织培养罩中进行。
  2. 实验室人员应使用适当的PPE。
  3. 所有的固体废物都是在处理之前进行高压灭菌。
  4. 所有的液体废物在处理之前都用50%的漂白剂灭活。

食谱

  1. 50%漂白
    在储存瓶中,将50ml 100%漂白剂稀释到50ml超纯H 2 O中
  2. 病毒储存缓冲液(VB)(10mM Tris,pH 7.4,15mM MgCl 2和150mM NaCl)
    1. 在玻璃烧杯中,将以下成分溶解在900ml超纯H2O2中:
      1.21克Tris
      3.05克MgCl2•6H2O 2 O. 8.77克NaCl

    2. 在搅拌盘上使用磁力搅拌棒在室温下混合
    3. 用0.1N盐酸调pH至7.4
    4. 在一个量筒中,用超纯水将最终体积加到1000ml
    5. 将解决方案转移到存储瓶
    6. 通过高压灭菌来消毒
    7. 在室温下储存
  3. 2毫克/毫升α-甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)处理的胰凝乳蛋白酶
    1. 在离心管中,将100mg TLCK处理的胰凝乳蛋白酶溶解于50ml超纯H2O2中
    2. 在室温下轻轻翻转管,直到溶液变得透明
    3. 将1毫升等分试样转移到微量离心管中
    4. 在-20°C储存
  4. 100毫米苯甲基磺酰氟(PMSF)
    1. 在微量离心管中,将17.4mg PMSF溶解在1ml异丙醇中
    2. 在室温下轻轻翻转管,直到溶液变得透明
    3. 在-20°C储存
  5. 1毫克/毫升胰蛋白酶
    1. 在微量离心管中,将1mg胰蛋白酶溶解在1ml超纯H 2 O中
    2. 在室温下轻轻翻转管,直到溶液变得透明
    3. 在-20°C储存

致谢

该协议是从以前发表的研究(Chandran等人,2002年)改编的。在该出版物中报道的研究得到了国立卫生研究院的过敏和传染病研究所的支持,奖项号码分别为1R01AI110637(至P.D.)和F32AI126643(至A.J.S.),印第安纳大学布卢明顿分校。内容完全是作者的责任,不一定代表出资者的官方观点。作者宣称没有利益冲突。

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引用:Snyder, A. J. and Danthi, P. (2018). Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus. Bio-protocol 8(2): e2701. DOI: 10.21769/BioProtoc.2701.
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