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HIV-1 Virus-like Particle Budding Assay
HIV-1病毒样颗粒芽殖试验   

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Abstract

Viral replication culminates with the egress of the mature virion from the host cell. This step of the viral life cycle has recently garnered increased attention with the discovery of the cellular restriction factor, Tetherin, which tethers budded virions to the surface of infected cells and inhibits viral spread. The importance of this block in viral infections has been suggested by the discovery of viral antagonists, such as HIV-1 Vpu, which counteract Tetherin. This protocol describes a system to study HIV-1 budding under BSL-2 safety conditions. It takes advantage of the ability of many viral matrix/capsid proteins to generate non-infectious virus-like particles (VLPs) with the expression of a single viral protein (i.e. HIV-1 p24 Gag). This protocol was recently used to characterize the effect of Tetherin isoforms on VLP release in the presence of HIV-1 Vpu (Cocka and Bates, 2012). Simultaneous expression of Tetherin and other viral antagonists can be used to study Tetherin-mediated restriction on viral budding.

Keywords: HIV-1(HIV-1), Virus-like(病毒样), Budding(萌芽), Virus release(病毒的释放)

Materials and Reagents

  1. HEK 293T cell line (ATCC, catalog number: CRL-11268 )
  2. Antibody, HIV-1 p24 Gag (NIH AIDS Reagent Program, catalog number: 24-3 )
  3. Antibody, HIV-1 Vpu (optional) (NIH AIDS Reagent Program, catalog number: 969 )
  4. Antibody, human Tetherin (optional) (NIH AIDS Reagent Program, catalog number: 11721 )
  5. Bromophenol Blue (Sigma-Aldrich, catalog number: B-5525 )
  6. Complete Mini protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693132001 )
  7. Criterion Precast Gels, 4-15% Tris-HCl (Bio-Rad Laboratories, catalog number: 345-0027 )
  8. Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: BP172-5 )
  9. Dulbecco’s Modified Eagle Media (DMEM) (Life Technologies, InvitrogenTM, catalog number: 11965-084 )
  10. Dulbecco’s Phosphate-buffered saline (DPBS) (Life Technologies, InvitrogenTM, catalog number: 14190-136 )
  11. 0.5 M EDTA solution (Life Technologies, InvitrogenTM, catalog number: 15575-038 )
  12. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F2442-500ML )
  13. Glycerol (Thermo Fisher Scientific, catalog number: BP229-1 )
  14. Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: A144-212 )
  15. Instant Nonfat Dry Milk (Nestle, catalog number: 050000033188 )
  16. Lipofectamine 2000 (Life Technologies, InvitrogenTM, catalog number: 11668-019 )
  17. NaCl (Thermo Fisher Scientific, catalog number: BP 358-212 )
  18. Opti-MEM (Life Technologies, InvitrogenTM, catalog number: 31985-070 )
  19. Plasmid, human Tetherin cDNA (optional) (OpenBiosystems, catalog number: 5217945 )
  20. Plasmid, psPAX2 HIV Gag (Addgene, catalog number: 12260 )
  21. Plasmid, Tetherin antagonist (HIV-1 Vpu) (optional)
  22. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: BP166-500 )
  23. D-Sucrose (Thermo Fisher Scientific, catalog number: BP220-1 )
  24. Tris Base (Thermo Fisher Scientific, catalog number: BP152-500 )
  25. Triton X-100 (Thermo Fisher Scientific, catalog number: BP151-500 )
  26. Tween 20 Thermo Fisher Scientific, catalog number: BP337-500 )
  27. Cell culture media (see Recipes)
  28. 20% sucrose solution (see Recipes)
  29. Triton X-100 lysis buffer (see Recipes)
  30. Blocking solution (see Recipes)
  31. 6x protein gel loading buffer (see Recipes)

Equipment

  1. 24 well plate, cell culture treated (Corning, catalog number: 3524 )
  2. 1.7 ml Microcentrifuge tubes (GeneMate, catalog number: C-3260-1X )
  3. 8 x 34 mm Ultracentrifuge tubes (Beckman Coulter, catalog number: 343776 )
  4. Tabletop Centrifuge, refrigerated (Eppendorf Centrifuge, model: 5417R )
  5. TLA 120.1 rotor (Beckman Coulter, model: 357655 )
  6. Ultracentrifuge, refrigerated (Beckman Coulter Optima, model: TLX-IM-3 )

Procedure

  1. Plate 293T cells in 500 μl of Cell Culture Media at a concentration of 2.5 x 105 cells/well in a 24 well plate. Cells should grow to 70-80% confluence 18-24 h post-seeding.
  2. For the following transfection, the amounts of plasmid DNA have been optimized for this protocol. Transfect seeded cells with 50 ng of psPAX2, an HIV gag expression vector, using Lipofectamine 2000. Follow the manufacturer’s protocol using a ratio of Lipofectamine (μl):DNA (μg) of 2.5:1 diluted in OPTI-MEM. Aspirate the transfection reagent from the transfected cells 5 hours post transfection and add 500 μl fresh Cell Culture Media. Incubate at 37 °C and 5% CO2 for 36-48 h.
    1. Alternatively cells can be transfected with any other vector expressing a viral matrix/capsid protein, such as Ebola VP40 (Kaletsky, 2009) that can produce virus-like particles (VLPs).
    2. The effect of the cellular restriction factor Tetherin can be assessed by co-transfecting increasing amounts (10-200 ng) of the pCMV-SPORT6 Tetherin expression vector with a VLP producing plasmid.
    3. Viral antagonists of Tetherin can be studied by co-transfection of increasing amounts (25-100 ng) of a Tetherin antagonist such as pCAGGS HIV-1 Vpu along with a Tetherin and VLP expression plasmid.
  3. Harvest cellular proteins and purify VLPs.
    1. VLP analysis. 36-48 h post-trasnfection, pipet all of the media from each transfected well into individual 1.7 ml microcentrifuge tubes. Centrifuge at 1,700 x g for 2 min at 4 °C to clear media and pellet cell debris. Transfer 450 μl of cleared media by pipetting into individual 8 x 34 mm ultracentrifuge tubes and underlay with 100 μl of the 20% Sucrose Solution. Place ultracentrifuge tubes in a pre-chilled TLA 120.1 rotor and centrifuge at 40,000 rpm for 30 min at 4 °C in a refrigerated ultracentrifuge. Carefully remove and discard all of the media and sucrose underlay by pipetting. Be very careful, as the protein pellet is not clearly visible. Add 50 μl PBS to the protein pellet on ice and incubate for 1-24 h (1 h minimum).
    2. Cellular Lysate Analysis. After removing the VLP containing media, add 80 μl Triton X-100 Lysis Buffer to each transfected well in the 24-well plate and incubate for 5 min at room temperature. Transfer all of the lysate to a 1.7 ml microcentrifuge tube. Centrifuge at 17,900 x g for 3 min at 4 °C to pellet insoluble cell debris. Transfer 50 μl of the cleared lysate to new microcentrifuge tube for immunoblot analysis.
  4. Immunoblot Analysis. For each sample, either sucrose pelleted VLPs or cellular lysates, transfer 25 μl into a new 1.7 ml microcentrifuge tube. Add 5 μl of 6x protein gel loading buffer to each sample. Heat the samples to 95 °C for 10 min. Analyze the VLPs and cellular lysates on separate Criterion Precast 4-15% SDS-PAGE gels. Transfer gel to Western blot membrane and continue to immunoblot using the Blocking Solution. Immunoblot with an anti-p24 Gag antibody (1:10,000 dilution in Blocking Solution) to detect the VLPs and cellular p24 gag. Membranes can also be stripped and immunoblotted for other transfected proteins (Tetherin, Vpu) or housekeeping genes as loading controls (GAPDH). For example, Tetherin can be used to restrict VLP release as shown in Figure 1. Samples containing VLPs co-transfected with Tetherin have less detectable p24 Gag expression compared to VLPs co-transfected without Tetherin. In contrast, the cellular lysates exhibit detectable p24 Gag across all samples, irrespective of Tetherin expression. Rescue of VLP release can be observed in samples expressing increasing amounts of a Tetherin antagonist.


    Figure 1. An immunoblot showing the effects of Tetherin on p24 Gag VLP production and release. In the top image, release of VLPs into the culture media is significantly decreased in the presence of Tetherin. However, production of p24 Gag in the cellular lysates is unaffected by increased Tetherin expression.

Recipes

  1. Cell Culture Media (1 L solution)
    1 L DMEM
    10% FBS
  2. 20% sucrose solution (100 ml solution)
    20 g sucrose
    100 ml DPBS
  3. Triton X-100 lysis buffer
    50 mM Tris-HCl
    150 mM NaCl
    Buffer the solution to pH 7.5 with HCl
    5 mM EDTA
    1% Triton X-100
    Immediately prior to use, add complete Mini protease inhibitor to Triton X-100 Buffer at concentration suggested by manufacturer.
  4. Blocking Solution (1 L solution)
    50 mM Tris-HCl
    150 mM NaCl
    Buffer the solution to pH 7.5 with HCl
    50 g Instant Nonfat Dry Milk
    1 ml Tween-20
  5. 6x protein gel loading buffer
    350 mM Tris-HCl buffered to pH 6.8
    0.6 M DTT
    10% SDS
    30% glycerol
    0.012% Bromophenol Blue

Acknowledgments

This protocol was adapted from and recently used in Cocka and Bates (2012).

References

  1. Cocka, L. J. and Bates, P. (2012). Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities. PLoS Pathog 8(9): e1002931.
  2. Kaletsky, R. L., Francica, J. R., Agrawal-Gamse, C. and Bates, P. (2009). Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein. Proc Natl Acad Sci U S A 106(8): 2886-2891.

简介

病毒复制随着来自宿主细胞的成熟病毒体的流出而达到高潮。病毒生命周期的这个步骤最近通过发现细胞限制因子Tetherin而发现增加的注意力,Tetherin将暴露的病毒粒子束缚于感染细胞的表面并抑制病毒扩散。这种阻断在病毒感染中的重要性已经通过发现病毒拮抗剂如HIV-1 Vpu(其抵抗了细胞因子)而提出。该协议描述了在BSL-2安全条件下研究HIV-1芽生的系统。它利用了许多病毒基质/衣壳蛋白产生具有单个病毒蛋白(即HIV-1p24Gag)表达的非感染性病毒样颗粒(VLPs)的能力。这个协议最近用于表征在HIV-1 Vpu存在下,Tetherin亚型对VLP释放的影响(Cocka和Bates,2012)。同时表达的Tetherin和其他病毒拮抗剂可用于研究Tetherin介导的病毒芽出的限制。

关键字:HIV-1, 病毒样, 萌芽, 病毒的释放

材料和试剂

  1. HEK 293T细胞系(ATCC,目录号:CRL-11268)
  2. 抗体,HIV-1 p24 Gag(NIH AIDS Reagent Program,目录号:24-3)
  3. 抗体,HIV-1 Vpu(可选)(NIH AIDS Reagent Program,目录号:969)
  4. 抗体,人类Tetherin(可选)(NIH AIDS Reagent Program,目录号:11721)
  5. 溴酚蓝(Sigma-Aldrich,目录号:B-5525)
  6. 完全微型蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:04693132001)
  7. Criterion Precast Gel,4-15%Tris-HCl(Bio-Rad Laboratories,目录号:345-0027)
  8. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,目录号:BP172-5)
  9. Dulbecco's Modified Eagle Media(DMEM)(Life Technologies,Invitrogen TM ,目录号:11965-084)
  10. Dulbecco磷酸盐缓冲盐水(DPBS)(Life Technologies,Invitrogen TM ,目录号:14190-136)
  11. 0.5M EDTA溶液(Life Technologies,Invitrogen TM,目录号:15575-038)
  12. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442-500ML)
  13. 甘油(Thermo Fisher Scientific,目录号:BP229-1)
  14. 盐酸(HCl)(Thermo Fisher Scientific,目录号:A144-212)
  15. 速溶脱脂牛奶(Nestle,目录号:050000033188)
  16. Lipofectamine 2000(Life Technologies,Invitrogen TM ,目录号:11668-019)
  17. NaCl(Thermo Fisher Scientific,目录号:BP 358-212)
  18. Opti-MEM(Life Technologies,Invitrogen TM,目录号:31985-070)
  19. 质粒,人Tetherin cDNA(可选)(OpenBiosystems,目录号:5217945)
  20. 质粒,psPAX2 HIV Gag(Addgene,目录号:12260)
  21. 质粒,Tetherin拮抗剂(HIV-1 Vpu)(可选)
  22. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientific,目录号:BP166-500)
  23. D-蔗糖(Thermo Fisher Scientific,目录号:BP220-1)
  24. Tris碱(Thermo Fisher Scientific,目录号:BP152-500)
  25. Triton X-100(Thermo Fisher Scientific,目录号:BP151-500)
  26. Tween 20 Thermo Fisher Scientific,目录号:BP337-500)
  27. 细胞培养基(参见配方)
  28. 20%蔗糖溶液(见配方)
  29. Triton X-100裂解缓冲液(参见配方)
  30. 阻止解决方案(参见配方)
  31. 6x蛋白凝胶上样缓冲液(参见配方)

设备

  1. 24孔板,细胞培养处理(Corning,目录号:3524)
  2. 1.7ml微量离心管(GeneMate,目录号:C-3260-1X)
  3. 8×34mm超速离心管(Beckman Coulter,目录号:343776)
  4. 台式离心机,冷藏(Eppendorf离心机,型号:5417R)
  5. TLA 120.1转子(Beckman Coulter,型号:357655)
  6. 超速离心机,冷藏(Beckman Coulter Optima,型号:TLX-IM-3)

程序

  1. 将293T细胞在500μl细胞培养基中以2.5×10 5个细胞/孔的浓度在24孔板中铺板。 在接种后18-24小时,细胞应生长至70-80%汇合
  2. 对于以下转染,已经为该方案优化了质粒DNA的量。 使用Lipofectamine 2000,用50ng psPAX2(一种HIV gag表达载体)转染接种的细胞。遵循制造商的方案,使用在OPTI-MEM中稀释的2.5:1的Lipofectamine(μl):DNA(μg)的比率。 转染后5小时从转染细胞吸出转染试剂,加入500μl新鲜细胞培养基。 在37℃和5%CO 2孵育36-48小时
    1. 或者,细胞可以用表达病毒基质/衣壳蛋白的任何其它载体转染,例如可以产生病毒样颗粒(VLP)的埃博拉病毒VP40(Kaletsky,2009)。
    2. 可以通过用产生VLP的质粒共转染增加量(10-200ng)的pCMV-SPORT6表达载体来评估细胞限制因子Tetherin的作用。
    3. 可以通过共同转染增加量(25-100ng)的Tetherin拮抗剂如pCAGGS HIV-1 Vpu以及Tetherin和VLP表达质粒来研究Tetherin的病毒拮抗剂。
  3. 收获细胞蛋白和纯化病毒样颗粒。
    1. VLP分析。转染后36-48小时,将来自每个转染孔的所有培养基吸移到单独的1.7ml微量离心管中。在4℃下以1,700×g离心2分钟,以澄清培养基和沉淀细胞碎片。通过吸液转移450微升清除的介质到个别8×34毫米超速离心管和底层与100微升的20%蔗糖溶液。将超速离心管置于预冷的TLA 120.1转子中,并在4℃下,在冷冻超速离心机中以40,000rpm离心30分钟。小心地通过移液除去并丢弃所有的培养基和蔗糖底层。要非常小心,因为蛋白质沉淀不清晰可见。在冰上加入50微升PBS蛋白质沉淀,孵育1-24小时(至少1小时)
    2. 细胞裂解物分析。去除含VLP的培养基后,向24孔板中的每个转染孔中加入80μlTriton X-100裂解缓冲液,并在室温下孵育5分钟。将所有裂解物转移至1.7ml微量离心管中。在4℃下在17,900×g离心3分钟以沉淀不溶性细胞碎片。将50μl澄清的裂解液转移到新的微量离心管中进行免疫印迹分析
  4. 免疫印迹分析。对于每个样品,蔗糖粒化的VLP或细胞裂解物,转移25μl到新的1.7ml微量离心管中。向每个样品中加入5μl6x蛋白凝胶上样缓冲液。将样品加热至95℃10分钟。在单独的Criterion Precast 4-15%SDS-PAGE凝胶上分析VLP和细胞裂解物。将凝胶转移至蛋白质印迹膜,并使用封闭溶液继续免疫印迹。用抗p24Gag抗体(在封闭溶液中1:10,000稀释)进行免疫印迹以检测VLP和细胞p24 gag。还可以剥离膜并对其它转染的蛋白(Tetherin,Vpu)或持家基因作为加样对照(GAPDH)进行免疫印迹。例如,如图1所示,可以使用Tetherin来限制VLP的释放。与不具有Tetherin的共转染的VLP相比,含有与Tetherin共转染的VLP的样品具有较少的可检测的p24 Gag表达。相反,细胞裂解物在所有样品中显示出可检测的p24 Gag,而不管组织蛋白酶表达。在表达增加量的凝集素拮抗剂的样品中可以观察到VLP释放的救援

    图1.显示了Tetherin对p24 Gag VLP产生和释放的影响的免疫印迹。 在顶部图像中,在存在Tetherin的情况下,VLP向培养基中的释放显着降低。然而,细胞裂解物中p24 Gag的产生不受增加的组蛋白表达的影响

食谱

  1. 细胞培养基(1L溶液)
    1 L DMEM
    10%FBS
  2. 20%蔗糖溶液(100ml溶液) 20克蔗糖 100 ml DPBS
  3. Triton X-100裂解缓冲液 50mM Tris-HCl
    150mM NaCl 用HCl缓冲溶液至pH 7.5 5 mM EDTA
    1%Triton X-100 在使用前,在制造商建议的浓度下,向Triton X-100缓冲液中加入完全Mini蛋白酶抑制剂
  4. 阻断溶液(1 L溶液)
    50mM Tris-HCl
    150mM NaCl 用HCl缓冲溶液至pH 7.5 50克速溶脱脂牛奶
    1ml Tween-20
  5. 6x蛋白质凝胶上样缓冲液
    缓冲至pH 6.8的350mM Tris-HCl 0.6 M DTT
    10%SDS
    30%甘油 0.012%溴酚蓝

致谢

该协议改编自并最近在Cocka和Bates(2012)中使用。

参考文献

  1. Cocka,L.J。和Bates,P。(2012)。 鉴定具有不同抗病毒和信号传导活性的可选翻译的细胞外基质同工型。 PLoS Pathog 8(9):e1002931。
  2. Kaletsky,R.L.,Francica,J.R.,Agrawal-Gamse,C.and Bates,P。(2009)。 纤维细胞介导的纤毛病毒芽殖限制受到埃博拉糖蛋白的拮抗。 Proc Natl Acad Sci USA 106(8):2886-2891。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vande Burgt, N. H., Cocka, L. J. and Bates, P. (2013). HIV-1 Virus-like Particle Budding Assay. Bio-protocol 3(14): e830. DOI: 10.21769/BioProtoc.830.
  2. Cocka, L. J. and Bates, P. (2012). Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities. PLoS Pathog 8(9): e1002931.
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