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Establishment of a Human Cell Line Persistently Infected with Sendai Virus
持续感染仙台病毒的人细胞系的建立   

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Abstract

Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is independent of its transcriptional activity. RIPA protects against lethal virus infection in cells and mice (Chattopadhyay et al., 2016). In the absence of RIPA, caused by genetic ablation, chemical mutagenesis or inhibition of the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I), Sendai virus (SeV) infection does not trigger cellular apoptosis and become persistently infected (Peters et al., 2008; Chattopadhyay et al., 2013). IRF3-expressing wild type (WT) cells (U4C) undergo SeV-induced apoptosis; however, the P2.1 cells, which are deficient in IRF3 expression are not capable of triggering viral apoptosis (Figure 1). Ectopic expression of human IRF3 restores the apoptotic activity in P2.1 cells (P2.1/IRF3, Figure 1). SeV is used as a model for studying pathogenic human viruses, which are difficult to work with or require BSL3 facility. We have previously reported that both human and mouse cells can establish SeV persistence in the absence of IRF3’s apoptotic activity (Chattopadhyay et al., 2013). Here, we outline a detailed procedure for the development of a persistently SeV-infected human cell line (Figure 2), which continuously expresses viral protein and produces low levels of infectious viral particles.


Figure 1. SeV-induced apoptosis is IRF3-dependent. HT1080-derived cell lines (U4C, P2.1 and P2.1/IRF3) were infected with Sendai virus and three days post infection culture fields were photographed, scale bar represents 50 µm.

Keywords: Sendai virus(仙台病毒), Persistence(持续), IRF3(IRF3), Apoptosis(细胞凋亡), P2.1(P2.1)

Background

IRF3 is essential for initiating antiviral defense mechanisms in host cells by way of promoting transcription of antiviral genes (Hiscott, 2007; Chattopadhyay and Sen, 2017). Upon recognition of viral dsRNA by PRRs in the cell, IRF3 becomes phosphorylated, dimerizes, and translocates to the nucleus, where it binds to the interferon-sensitive response element (ISRE), and promotes transcription of type-1 interferons, e.g., IFN-β. IRF3 is also critical for triggering apoptosis via a distinct pathway, which does not require its transcriptional activity. In a series of previous studies, we have discovered the pathway, which we named RIPA that triggers apoptosis in virus-infected cells. In RIPA, IRF3 interacts with BCL-2-Associated X protein (BAX), a pro-apoptotic factor (Chattopadhyay et al., 2010). Upon binding to BAX, IRF3 translocates to the mitochondria, and initiates a signaling cascade that ultimately promotes apoptosis (Chattopadhyay et al., 2010). In the absence of IRF3 or other components of RIPA, the cells establish viral persistence when infected with Sendai virus (SeV) (Peters et al., 2008; Chattopadhyay et al., 2013). These persistent cell lines are useful for studying the full anti-viral mechanisms of cells because the cells do not undergo apoptotic cell death. In the current protocol, we provide a detailed method to create a SeV persistent human cell line, which are defective in IRF3 expression. Viral persistence is common for many viruses, which efficiently antagonize the cell death pathways of the infected cells. An in vitro approach to study persistently infected cells will reveal ways to avoid the establishment of viral persistence. It will also be evaluated in future whether the absence of RIPA can be used as a tool to generate persistently infected cells using viruses of different lifestyles.

Materials and Reagents

  1. Materials
    1. Pipette tips (USA Scientific)
    2. 6 cm tissue culture plates (USA Scientific, Cyto-One, catalog number: CC7672-3359 )
    3. Cryovials (USA Scientific, catalog number: 1412-9100 )
    4. 1.5 ml Eppendorf tubes (USA Scientific)
    5. PVDF membrane (Bio-Rad Laboratories, catalog number: 1620177 )
    6. Autoradiography film (Denville Scientific, catalog number: E3012 )
    7. 6-well plate

  2. Cells
    1. U4C cells: these cells were generated from HT1080 cells and are deficient in IFN signaling
      Note: These cells are maintained in DMEM containing 10% FBS, 100 international units of penicillin, 100 µg/ml streptomycin (complete DMEM).
    2. P2.1 cells: these cells were generated from U4C cells and are deficient in IRF3 expression
      Note: These cells are maintained in DMEM containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin (complete DMEM).
    3. P2.1/IRF3 cells: these cells were generated by stably expressing human IRF3 in P2.1 cells and were selected under puromycin (1 µg/ml)
      Note: These cells are maintained in DMEM containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin and puromycin (1 µg/ml).
    4. LLC-MK2 cells (ATCC, catalog number: CCL-7 ):
      Note: These cells are maintained in Medium 199 containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin.

    Note: U4C, P2.1 and P2.1/IRF3 cells were generated in the authors’ laboratory and were described previously. See Chattopadhyay et al., 2010 and 2016. These cell lines are available from the authors upon request.

  3. Viruses
    Sendai virus (SeV) Cantell strain (Charles River laboratories)–this strain was originally obtained from ATCC (ATCC, catalog number: VR-907 )

  4. Reagents
    1. Dulbecco’s modified Eagle’s medium (DMEM) (Lerner Research Institute Central Cell Services, catalog number: 11-500p )
    2. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
    3. Complete EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 11873580001 )
    4. Cryoprotective medium (Lonza, catalog number: 12-132A )
    5. Phosphate-buffered saline (PBS) (Lerner Research Institute Central Cell Services, catalog number: 123-1000p )
    6. SDS-PAGE loading buffer (Laemelli) (Bio-Rad Laboratories, catalog number: 1610737 )
    7. 10x SDS-PAGE running buffer (AMRESCO, catalog number: 0783 )
    8. Protein assay dye (Bio-Rad Laboratories, catalog number: 5000006 )
    9. 10x transfer buffer (AMRESCO, catalog number: 0307 )
    10. Nonfat dry milk (Bio-Rad Laboratories, catalog number: 1706404XTU )
    11. Tris buffered saline with Tween-20 (TBST) (AMRESCO, catalog number: K873 )
    12. Antibodies
      1. Sendai Virus C antibody (generated in author’s laboratory) (Chattopadhyay, 2016)
        Note: Another anti-SeV antibody from Abcam, catalog number: ab33988 may be used to detect the presence of SeV.
      2. Goat-anti-rabbit secondary antibody conjugated with horseradish peroxidase was obtained from Rockland Lab (Rockland, catalog number: 611-103-122 )
    13. ECL plus solution (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 80196 )
    14. Medium 199 (Thermo Fisher Scientific, GibcoTM, catalog number: 11150059 )
    15. Agar (BD, DifcoTM, catalog number: 214050 )
    16. Guinea pig red blood cells (Colorado Serum Company, catalog number: 30100 )
    17. Tris buffer (pH 7.4) (Affymetrix, catalog number: 22639 )
    18. 5 M sodium chloride solution (NaCl) (Affymetrix, USB, catalog number: 75888 )
    19. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
    20. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508-10G )
    21. Sodium fluoride (Sigma-Aldrich, catalog number: S6521 )
      Note: This product has been discontinued.
    22. β-Glycerophosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G9422 )
    23. Sodium pyrophosphate (Sigma-Aldrich, catalog number: S6422 )
    24. Cell lysis buffer (see Recipes)

Equipment

  1. Micropipettes (10 μl, 200 μl, 1,000 μl) (Eppendorf)
  2. SDS-PAGE and transfer apparatus (Bio-Rad Laboratories, model: Mini PROTEAN-II )
    Note: This equipment is no longer available at manufacturer (also use the same machine for agarose gel).
  3. Vortex (Thermo Fisher Scientific)
  4. Tissue culture incubator (at 37 °C with 5% CO2) (Thermo Fisher Scientific)
  5. Biosafety cabinet (Baker SterilGARD)
  6. Table top centrifuge (Eppendorf)
  7. Heating block (at 95 °C) (Benchmark)
  8. Rocker (Benchmark)
  9. Rotator (Labnet)
  10. Refrigerator (4 °C) and freezers (-20 °C and -80 °C)
  11. Autoradiography film processor (Kodak)
  12. Spectrophotometer (Benchmark)
  13. Liquid Nitrogen Tank Cryosafe CM2 (D.A.I. Scientific Equipment)

Procedure

  1. Infection of P2.1 cells with SeV
    1. Seed 500,000 P2.1 cells into 6 cm tissue culture dishes (Figure 2).


      Figure 2. Development of SeV-persistent cell lines using P2.1 parental cells. A flow chart of generation and maintenance of SeV-persistently infected cells using P2.1 cell line. The Western blot shows the consistent levels of viral protein (SeV C) expression at various passage numbers (indicated by P), and actin was used as a loading control.

    2. After cells have adhered (about 16-18 h post seeding), check for 80% confluence, wash cells two times with DMEM containing 2% FBS (virus infection medium), then add a minimum amount (1 ml) of virus infection medium.
    3. Add SeV at a concentration of 80 hemagglutinating units/ml. Incubate the cells with virus for 1 h with gentle physical agitation every 10 min.
    4. Remove the virus-containing medium, and wash cells two times with DMEM containing 10% FBS.
    5. Continue culturing cells in DMEM containing 10% FBS, passaging cells every 3 days, setting aside 10% for detection of SeV and 50% for freezing.
    6. After each passage, reserve some of the cells for future analysis.
      1. Combine 0.5 ml cell suspension in DMEM containing 10% FBS with 0.5 ml cryoprotective medium in a labeled cryovial.
      2. Immediately place in a freezer kept at -80 °C.
      3. For long-term storage, store cells in liquid nitrogen tank.
    7. To determine the presence of virus, harvested cells are analyzed by Western blot for viral protein detection, as described below.

  2. SDS-PAGE and Western blot to confirm SeV infection
    1. Seed P2.1 cells and SeV infected P2.1 cells into separate 6 cm tissue culture dishes.
    2. After 24 h or when the cells are confluent, harvest cell lysates.
      1. Remove media, and wash the plates once with cold PBS.
      2. Lyse cells in 50 µl of cold lysis buffer (see Recipes). Gently vortex and keep cell lysates on ice for 30 min.
      3. Centrifuge cell lysates at 16,200 x g (Rcf) for 20 min in a cold (4 °C) centrifuge.
    3. Load equal amounts of protein on a 20% SDS-PAGE gel.
      Note: Prior to loading, combine cell lysates with equal volumes of 2x SDS-PAGE loading buffer.
    4. Electrophorese the proteins at 100 V for 2 h, or until adequate separation of proteins in the 10-100 kDa region is achieved.
    5. Transfer proteins onto a PVDF membrane using a Bio-Rad semi-dry transfer apparatus at 0.06 mA for 1.5 h.
    6. Block the membrane in TBST containing 5% nonfat dry milk on a rocker at room temperature for 30 min.
    7. Add the anti-Sendai Virus C antibody at 1:5,000 dilution in blocking buffer. Incubate on a rocker at room temperature for 1 h or 12-16 h at 4 °C.
    8. Wash the membrane three times in TBST, and add the secondary (HRP-conjugated) antibody, diluted to 1:5,000 dilution in blocking buffer, incubating for 1 h at room temperature.
    9. Wash the membrane three times in TBST and incubate in ECL solution for 1-3 min.
    10. Develop on autoradiograph film using an autoradiograph film processor.

  3. Quantification of infectious virus particles in persistently infected cells
    The infectious SeV titers in the culture supernatants were determined by previously described procedure (Peters et al., 2008) and was represented as pfu/ml. Briefly, the following procedure is performed:
    1. Seed LLC-MK2 cells in 6-well plate at 100,000 cells per well in Medium 199 and allow the cells to become confluent (in about three days).
    2. The culture supernatants from the persistently infected cell lines are serial diluted and then used to infect confluent LLC-MK2 monolayer, using the virus infection protocol (as described in Procedure A).
    3. After infection, the cells are washed and overlaid with Medium 199 containing 0.5% agar.
    4. After 3 days, the agar layer is removed and the cells are washed with PBS.
    5. Virus colonies are visualized by incubating the monolayer with a 0.1% suspension of guinea pig red blood cells for 20-30 min. The monolayer is then washed with PBS and the hemabsorbed plaques are counted.

Data analysis

To confirm that viral persistence has been achieved, a few relatively simple assays may be performed. Sendai virus expresses a non-structural protein (C), also known as SeV C, which can be detected by Western blotting of the persistently infected P2.1 cells, the procedure for which is outlined in the previous section. To confirm the presence, or rather, the absence of clearance of SeV in P2.1 cells, an anti-SeV C antibody can be applied to the Western blot, which, if a band of approximately 25 kDa is elucidated, can confirm persistence (Figure 2).

Notes

Western blot for detection of SeV C protein should be carefully evaluated because the antibody also cross reacts with other viral and cellular proteins which are of similar sizes. Careful separation of these proteins on a 20% SDS gels will facilitate the analyses (Figure 3).


Figure 3. A representative Western blot for SeV C protein expression. Human cells either mock infected or infected with SeV for 16 h, when cell lysates were analyzed by Western blot. NS, non specific.

Recipes

  1. Cell lysis buffer
    50 mM Tris buffer, pH 7.4
    150 mM NaCl
    0.1% Triton X-100
    1 mM sodium orthovanadate
    10 mM sodium fluoride
    10 mM β-glycerophosphate
    5 mM sodium pyrophosphate

Acknowledgments

Our work was supported by the American Heart Association Scientist Development grants 15SDG25090212 (SC) and The University of Toledo College of Medicine and Life Sciences startup funds (SC). The protocol and some representative results were adapted from our previous work (Peters et al., 2008).

References

  1. Chattopadhyay, S., Fensterl, V., Zhang, Y., Veleeparambil, M., Yamashita, M. and Sen, G. C. (2013). Role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by Sendai virus. J Virol 87(1): 16-24.
  2. Chattopadhyay, S., Kuzmanovic, T., Zhang, Y., Wetzel, J. L. and Sen, G. C. (2016). Ubiquitination of the transcription factor IRF-3 activates RIPA, the apoptotic pathway that protects mice from viral pathogenesis. Immunity 44(5): 1151-1161.
  3. Chattopadhyay, S., Marques, J. T., Yamashita, M., Peters, K. L., Smith, K., Desai, A., Williams, B. R. and Sen, G. C. (2010). Viral apoptosis is induced by IRF-3-mediated activation of Bax. EMBO J 29(10): 1762-1773.
  4. Chattopadhyay, S. and Sen, G. C. (2017). RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA): a new antiviral pathway. Protein Cell 8(3): 165-168.
  5. Hiscott, J. (2007). Triggering the innate antiviral response through IRF-3 activation. J Biol Chem 282(21): 15325-15329.
  6. Peters, K., Chattopadhyay, S. and Sen, G. C. (2008). IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence. J Virol 82(7): 3500-3508.

简介

干扰素调节转录因子3(IRF3)是一种通过病毒感染活化促进抗病毒基因如干扰素合成的转录因子(Hiscott,2007)。除了诱导基因外,IRF3通过RIG-I样受体诱导的IRF3介导的凋亡途径(RIPA)引发抗病毒凋亡,其与其转录活性无关。 RIPA可防止细胞和小鼠的致死病毒感染(Chattopadhyay et al。,2016)。在不存在RIPA的情况下,遗传消融引起的化学诱变或模式识别受体(PRR)视黄酸诱导型基因I(RIG-1)的抑制,仙台病毒(SeV)感染不会引发细胞凋亡并持续感染(Peters等人,2008; Chattopadhyay等人,2013)。表达IRF3的野生型(WT)细胞(U4C)经历SeV诱导的凋亡;然而,缺乏IRF3表达的P2.1细胞不能引发病毒凋亡(图1)。人类IRF3的异位表达恢复了P2.1细胞的凋亡活性(P2.1 / IRF3,图1)。 SeV用作研究致病性人类病毒的模型,难以与BSL3设施配合使用。我们以前曾报道,人和小鼠细胞都可以在没有IRF3的凋亡活性的情况下建立SeV持久性(Chattopadhyay et al。,2013)。在这里,我们概述了持续的SeV感染的人类细胞系(图2)的开发的详细程序,其连续表达病毒蛋白并产生低水平的感染性病毒颗粒。
【背景】IRF3对于通过促进抗病毒基因的转录来启动宿主细胞中的抗病毒防御机制至关重要(Hiscott,2007; Chattopadhyay和Sen,2017)。在细胞中通过PRR识别病毒dsRNA后,IRF3变得磷酸化,二聚化和转位到细胞核,其中它结合干扰素敏感反应元件(ISRE),并促进1型干扰素的转录,例如IFN- β。 IRF3对于通过不需要其转录活性的不同途径引发凋亡也是至关重要的。在一系列以前的研究中,我们发现了通路,我们命名为RIPA,其触发病毒感染细胞的细胞凋亡。在RIPA中,IRF3与BCL-2相关X蛋白(BAX),一种促凋亡因子(Chattopadhyay等,2010)相互作用。结合BAX后,IRF3转运到线粒体,并启动最终促进细胞凋亡的信号级联(Chattopadhyay et al。,2010)。在没有IRF3或RIPA的其他组分的情况下,当感染仙台病毒(Sev)时,细胞建立病毒持久性(Peters等人,2008; Chattopadhyay等,2013)。这些持续性细胞系可用于研究细胞的全部抗病毒机制,因为细胞不经历凋亡性细胞死亡。在当前的协议中,我们提供了一种创建SeV持续人类细胞系的详细方法,它们在IRF3表达中是有缺陷的。病毒持久性对于许多病毒是常见的,其有效地拮抗感染细胞的细胞死亡途径。研究持续感染细胞的体外方法将揭示避免病毒持久性建立的方法。将来还将评估RIPA的缺失是否可以用作使用不同生活方式的病毒产生持续感染细胞的工具。

关键字:仙台病毒, 持续, IRF3, 细胞凋亡, P2.1

材料和试剂

  1. Materials
    1. 移液器提示(美国科学)
    2. 6厘米的组织培养板(USA Scientific,Cyto-One,目录号:CC7672-3359)
    3. Cryovials(USA Scientific,目录号:1412-9100)
    4. 1.5 ml Eppendorf管(USA Scientific)
    5. PVDF膜(Bio-Rad Laboratories,目录号:1620177)
    6. 放射自显影片(Denville Scientific,目录号:E3012)
    7. 6孔板

  2. 细胞
    1. U4C细胞:这些细胞是从HT1080细胞产生的,并且缺乏IFN信号传递 注意:这些细胞维持在含有10%FBS,100个国际青霉素单位,100μg/ ml链霉素(完全DMEM)的DMEM中。
    2. P2.1细胞:这些细胞由U4C细胞产生,并且缺乏IRF3表达
      注意:这些细胞维持在含有10%FBS,100μg/ ml青霉素,100μg/ ml链霉素(完全DMEM)的DMEM中。
    3. P2.1 / IRF3细胞:通过在P2.1细胞中稳定表达人IRF3产生这些细胞,并在嘌呤霉素(1μg/ ml)下选择
      注意:这些细胞维持在含有10%FBS,100μg/ ml青霉素,100μg/ ml链霉素和嘌呤霉素(1μg/ ml)的DMEM中。
    4. LLC-MK2细胞(ATCC,目录号:CCL-7):
      注意:这些细胞维持在含有10%FBS,100μg/ ml青霉素,100μg/ ml链霉素的培养基199中。

    注意:U4C,P2.1和P2.1 / IRF3细胞在作者的实验室中产生并且在之前描述。请参阅Chattopadhyay等人,2010年和2016年。这些细胞系可根据要求从作者获得。

  3. 病毒
    仙台病毒(SeV)Cantell菌株(Charles River实验室) - 该菌株最初从ATCC(ATCC,目录号:VR-907)获得

  4. 试剂
    1. Dulbecco改良Eagle's培养基(DMEM)(Lerner Research Institute Central Cell Services,目录编号:11-500p)
    2. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11550)
    3. 完全无EDTA蛋白酶抑制剂(Roche Diagnostics,目录号:11873580001)
    4. 冷冻保护介质(Lonza,目录号:12-132A)
    5. 磷酸盐缓冲盐水(PBS)(Lerner Research Institute Central Cell Services,目录号:123-1000p)
    6. SDS-PAGE加载缓冲液(Laemelli)(Bio-Rad Laboratories,目录号:1610737)
    7. 10x SDS-PAGE运行缓冲液(AMRESCO,目录号:0783)
    8. 蛋白质测定染料(Bio-Rad Laboratories,目录号:5000006)
    9. 10x传输缓冲区(AMRESCO,目录号:0307)
    10. 脱脂奶粉(Bio-Rad Laboratories,目录号:1706404XTU)
    11. 使用Tween-20(TBST)的Tris缓冲盐水(AMRESCO,目录号:K873)
    12. 抗体
      1. 仙台病毒C抗体(在作者实验室产生)(Chattopadhyay,2016)
        注意:来自Abcam的另一种抗SeV抗体,目录号:ab33988可用于检测SeV的存在。
      2. 与Rockland Lab(Rockland,目录号:611-103-122)得到的与辣根过氧化物酶缀合的山羊抗兔二抗获得了
    13. ECL plus溶液(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:80196)
    14. 培养基199(Thermo Fisher Scientific,Gibco TM,目录号:11150059)
    15. 琼脂(BD,Difco TM ,目录号:214050)
    16. 豚鼠红细胞(科罗拉多血清公司,目录号:30100)
    17. Tris缓冲液(pH 7.4)(Affymetrix,目录号:22639)
    18. 5M氯化钠溶液(NaCl)(Affymetrix,USB,目录号:75888)
    19. Triton X-100(Sigma-Aldrich,目录号:T9284)
    20. 原钒酸钠(Sigma-Aldrich,目录号:S6508-10G)
    21. 氟化钠(Sigma-Aldrich,目录号:S6521)
      注意:本产品已停产。
    22. β-甘油磷酸二钠盐水合物(Sigma-Aldrich,目录号:G9422)
    23. 焦磷酸钠(Sigma-Aldrich,目录号:S6422)
    24. 细胞裂解缓冲液(参见食谱)

设备

  1. 微量移液管(10μl,200μl,1,000μl)(Eppendorf)
  2. SDS-PAGE和转移装置(Bio-Rad Laboratories,型号:Mini PROTEAN-II)
    注意:制造商不再提供此设备(也可使用相同的琼脂糖凝胶机)。
  3. 涡旋(Thermo Fisher Scientific)
  4. 组织培养箱(37℃,5%CO 2)(Thermo Fisher Scientific)
  5. 生物安全柜(Baker SterilGARD)
  6. 台式离心机(Eppendorf)
  7. 加热块(95°C)(基准)
  8. 摇杆(基准)
  9. 旋转器(Labnet)
  10. 冰箱(4°C)和冷柜(-20°C和-80°C)
  11. 放射自显影胶片处理器(柯达)
  12. 分光光度计(基准)
  13. 液氮罐冷冻机CM2(D.A.I.科学仪器)

程序

  1. 用SeV感染P2.1细胞
    1. 将500,000个P2.1细胞种植到6厘米的组织培养皿中(图2)

      图2.使用P2.1亲本细胞开发SeV持久性细胞系使用P2.1细胞系产生和维持SeV持续感染细胞的流程图。蛋白质印迹显示在不同通道数(由P表示)下病毒蛋白(SeV C)表达水平一致,肌动蛋白用作加载对照。

    2. 细胞粘附后(接种后约16-18小时),检查80%汇合,用含有2%FBS(病毒感染培养基)的DMEM洗涤细胞两次,然后加入最少量(1ml)的病毒感染培养基。
    3. 以80个凝血单位/ ml的浓度添加SeV。使用病毒孵育细胞1小时,每10分钟轻轻搅动身体
    4. 取出含病毒的培养基,并用含有10%FBS的DMEM洗涤细胞两次。
    5. 继续培养含有10%FBS的DMEM细胞,每3天传代细胞,放置10%用于检测SeV,50%用于冷冻。
    6. 每次通过后,保留一些细胞用于将来的分析。
      1. 将含有10%FBS的DMEM中的0.5ml细胞悬浮液与0.5ml冷冻保护培养基结合在标记的冷冻管中。
      2. 立即放入保存在-80°C的冷藏库。
      3. 对于长期储存,将电池存放在液氮罐中
    7. 为了确定病毒的存在,收集的细胞通过蛋白质印迹分析进行病毒蛋白检测,如下所述
  2. SDS-PAGE和Western blot检测SeV感染
    1. 种子P2.1细胞和SeV感染的P2.1细胞分成6厘米的组织培养皿
    2. 24小时后或细胞汇合时,收获细胞裂解物。
      1. 取出培养基,用冷PBS清洗板一次。
      2. 在50μl冷裂解缓冲液中溶解细胞(参见食谱)。轻轻涡旋并将细胞裂解物在冰上保持30分钟
      3. 在冷(4℃)离心机中离心细胞裂解物16,200×g(Rcf)20分钟。
    3. 在20%SDS-PAGE凝胶上加载等量的蛋白质。
      注意:加载前,将细胞溶解产物与等体积的2x SDS-PAGE加载缓冲液组合。
    4. 将蛋白质在100V电泳2小时,或直到达到10-100kDa区域中蛋白质的充分分离。
    5. 使用Bio-Rad半干式转移装置在0.06 mA下将蛋白质转移到PVDF膜上1.5 h
    6. 将TBST中含有5%脱脂乳的TBST在室温下封闭30分钟
    7. 在阻塞缓冲液中加入1:5,000稀释的抗仙台病毒C抗体。在室温下在4℃下在摇床上孵育1小时或12-16小时。
    8. 在TBST中洗涤膜三次,并加入次级(HRP缀合的)抗体,在封闭缓冲液中稀释至1:5000稀释,在室温下孵育1小时。
    9. 在TBST中洗涤膜三次,并在ECL溶液中孵育1-3分钟
    10. 使用放射自显影胶片处理器制作放射自显影胶片。

  3. 持续感染细胞中感染性病毒颗粒的定量
    培养上清液中的感染性SeV滴度通过先前描述的方法(Peters等人,2008)测定,并以pfu / ml表示。简而言之,执行以下过程:
    1. 种子LLC-MK2细胞在培养基199中每孔100,000细胞的6孔板中,并使细胞变得融合(大约三天)。
    2. 来自持续感染的细胞系的培养物上清液被连续稀释,然后使用病毒感染方案(如方法A所述)用于感染融合的LLC-MK2单层。
    3. 感染后,将细胞洗涤并用含有0.5%琼脂的培养基199覆盖
    4. 3天后,除去琼脂层,细胞用PBS洗涤
    5. 通过将单层与0.1%的豚鼠红细胞悬浮液孵育20-30分钟来显现病毒菌落。然后用PBS洗涤单层,计数血液吸收的噬菌斑。

数据分析

为了证实病毒持久性已经实现,可以进行一些相对简单的测定。仙台病毒表达非结构蛋白(C),也称为SeV C,可以通过Western印迹法检测持续感染的P2.1细胞,其方法在上一节中概述。为了证实存在,或者说,在P2.1细胞中SeV不存在,可以将抗SeV C抗体应用于Western印迹,如果阐明了约25kDa的条带,则可以确认持久性(图2)。

笔记

应仔细评估用于检测SeV C蛋白的蛋白质印迹,因为抗体还与具有相似大小的其他病毒和细胞蛋白质交叉反应。在20%SDS凝胶上仔细分离这些蛋白质将有助于分析(图3)

图3. SeV C蛋白表达的代表性Western印迹。当通过Western印迹分析细胞裂解物时,人类细胞模拟感染或用SeV感染16小时。 NS,非特异性。

食谱

  1. 细胞裂解缓冲液
    50mM Tris缓冲液,pH 7.4 150 mM NaCl
    0.1%Triton X-100
    1mM原钒酸钠
    10 mM氟化钠
    10mMβ-甘油磷酸盐
    5mM焦磷酸钠

致谢

我们的工作得到美国心脏协会科学家发展资助15SDG25090212(SC)和托莱多大学医学与生命科学学院启动基金(SC)的支持。该协议和一些代表性的结果是从我们以前的工作(Peters等人,2008)中改编而成。

参考

  1. Chattopadhyay,S.,Fensterl,V.,Zhang,Y.,Veleeparambil,M.,Yamashita,M.and Sen,GC(2013)。  干扰素调节因子3介导的细胞凋亡在仙台病毒持续感染的建立和维持中的作用。 87(1):16-24。
  2. Chattopadhyay,S.,Kuzmanovic,T.,Zhang,Y.,Wetzel,JL和Sen,GC(2016)。转录因子IRF-3的泛素化激活RIPA,保护小鼠免受病毒发病的凋亡途径。免疫性 44(5 ):1151-1161。
  3. Chattopadhyay,S.,Marques,JT,Yamashita,M.,Peters,KL,Smith,K.,Desai,A.,Williams,BR and Sen,GC(2010)。通过IRF-3介导的Bax激活诱导病毒凋亡。 29(10):1762-1773。
  4. Chattopadhyay,S.和Sen,GC(2017)。 RIG-I样受体诱导的IRF3介导的细胞凋亡途径(RIPA):一种新的抗病毒途径。蛋白质细胞 8(3):165-168。
  5. Hiscott,J.(2007)。触发天生的抗病毒药物通过IRF-3激活的反应。 J Biol Chem 282(21):15325-15329。
  6. Peters,K.,Chattopadhyay,S. and Sen,GC(2008)。  仙台病毒感染的IRF-3激活是细胞凋亡和避免持久性所必需的。 82(7):3500-3508。
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引用:Coakley, C., Peter, C., Fabry, S. and Chattopadhyay, S. (2017). Establishment of a Human Cell Line Persistently Infected with Sendai Virus. Bio-protocol 7(16): e2512. DOI: 10.21769/BioProtoc.2512.
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