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Aug 2018
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Analysis of Functional Virus-generated PAMP RNAs Using IFNα/β ELISA Assay
利用干扰素α/β酶联免疫吸附试验检测分析病毒产生的功能性PAMP RNAs   

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Abstract

Virus-generated PAMP RNAs are key factors that activate host immune response. The PAMP RNAs are therefore usually closely related with viral disease pathogenesis. Quantitative real time PCR is a conventional method to assess RNA. However, it cannot be used for detecting short dsRNAs generated by viral replicase. This protocol was established to analyze the PAMP RNAs produced by viruses which are able to induce host immune response. Classical viral PAMP RNAs and non-classical viral PAMP RNAs are analyzed separately. Briefly, to access total viral PAMP RNAs, total RNA was extracted from the virus infected cells and then transfected into Cop5 cells. Whereas, to assess non-classical viral PAMP RNAs, the constructs expressing viral replicase are transfected into Cop5 cells. The amount of IFNα/β produced by Cop5 cells, determined by ELISA, is correlated with the total and non-classical viral PAMP RNAs. Since this method is based on type I IFN response, it is therefore suitable for measuring the functional virus-generated PAMP RNAs and also for assessing the efficiency of these PAMP RNAs.

Keywords: Virus (病毒), Infection (感染), PAMP RNAs (病原相关分子模式RNAs), RNA isolation (RNA分离), IFNα/β detection (干扰素α/β检测)

Background

Under virus infection, the activation of host immune system usually starts with the recognition of viral pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) (Medzhitov and Janeway, 2002). Viral PAMPs, associated with virus invasion, include virus surface glycoproteins, viral DNA and RNAs (Mogensen and Paludan, 2005). In case of RNA virus infection, virus replication generated RNAs are essential PAMPs portion which is able to be recognized by the host intracellular PRRs (Alexopoulou et al., 2001). Two possible candidates for the role of such viral-generated PAMP RNAs have been recently reported. First, classical viral PAMP RNAs are dsRNA replication forms that are recognized by RIG-I and MDA5 and non-capped positive-strand RNAs (polyA+) generated by alphaviruses (Sokoloski et al., 2015) that may be recognized by RIG-I (Akhrymuk et al., 2015). Second, non-classical viral PAMP RNAs are short non-polyadenylated dsRNAs with 5’ triphosphate. They are IFN-inducing RNAs generated by viral replicase using cellular RNA templates (Nikonov et al., 2013). In Semliki Forest virus (SFV) infected cells these RNAs represent the majority of PAMP RNAs (Nikonov et al., 2013).

Given the essential role of viral PAMP RNAs to activate host immune system, a robust protocol to analyze different types of virus-generated PAMP RNAs is useful for virus-host interaction study. Quantitative real time PCR could be used to quantify classical viral dsRNAs by targeting negative strands. However, it is hard to differentiate between capped and non-capped positive strands. Furthermore, the approach is neither able to tell if the targeted PAMP RNA is functional nor able to access the PAMP efficiency and cannot detect the non-classical viral PAMP RNAs. To analyze the functional PAMP RNAs that are able to trigger host immune response, we established this protocol to analyze both classical and non-classical viral PAMP RNAs. To assess the total viral PAMP RNAs, total RNA from the infected cells were extracted for the assessment. Since all types of classical alphaviral PAMP RNAs have unpaired polyA tails and they are presumably the biggest proportion in the viral PAMP RNAs, they may overshadow other types of PAMP RNAs. Therefore, in a parallel experiment, we separated and examined the polyA- fraction from the total RNAs (Nikonov et al., 2013). To assess the non-classical viral PAMP RNAs, viral replicase constructs were used for analysis because the system did not have replication competent viral RNA templates and therefore no classical viral PAMP RNAs were produced. Cop5 cells were used as IFNα/β activation host for all PAMP RNAs analyses. The quantity of the IFNα/β produced by the transfected PAMP RNAs can be determined by ELISA assay.

This protocol is a reliable method to analyze alphavirus-generated PAMP RNAs during the virus replication and, with exception of separation of polyA- fraction, applied to viruses that lack unpaired polyA tails. It is not only to measure the level of the PAMP RNAs but also is able to assess the PAMP RNAs efficiency. Therefore, this protocol is suitable for the study involving the interaction between RNA virus invasion and the host immune response.

Materials and Reagents

  1. Materials
    1. 1.5 ml Eppendorf® DNA LoBind microcentrifuge tubes (Eppendorf, catalog number: 0030108051)
    2. 100 mm Corning® tissue-culture treated culture dishes (Corning, catalog number: 430167)
    3. 12-well CorningTM CostarTM flat bottom cell culture plates (Corning, catalog number: 3513)
    4. 15 ml FalconTM conical centrifuge tubes (Corning, catalog number: 352096)
    5. 6-well plate

  2. Cell lines
    1. ATCC® BHK-21[C13] cells (ATCC, catalog number: CCL-10TM)
    2. Cop5 cells (Tyndall et al., 1981)

  3. Viruses of interest
    Note: Prepare the viruses of interest from infectious clones. It is recommended that the virus titer should be prepared between 105 and 107 plaque forming units (PFU)/ml. The viruses listed below are for a demonstration. These viruses are derived from infectious clones, released (passage 0), passaged in BHK-21 cells and stored at -80 °C. 
    1. RRV-T48
    2. RRV-T48A534V

  4. Viral replicase constructs
    Note: Prepare the constructs expressing the viral replicase (non-structural proteins) of the viruses of interest. The constructs listed below are for a demonstration. As shown in the construct map below (Figure 1), the regions encoding the replicase of RRV-T48, RRV-T48A534V and SFV4 (used as positive control [Nikonov et al., 2013]) were cloned into pMC-gtGTU2 vector, which contains cytomegalovirus early promoter, leader sequence from the thymidine kinase of Herpes Simplex virus (HSV) with an inserted synthetic intron and late polyadenylation signal from simian virus 40 (SV40).
    1. RRV-T48
    2. RRV-T48A534V
    3. SFV


      Figure 1. Viral replicase construct map

  5. Reagents
    1. Dulbecco's modified Eagle medium (DMEM) (Sigma-Aldrich, catalog number: D5030)
    2. Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, catalog number: 51471C)
    3. L-Glutamine (Sigma-Aldrich, catalog number: G6392)
    4. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178)
    5. 100x Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333)
    6. Tryptose phosphate broth solution (TPB) (Sigma-Aldrich, catalog number: T8159)
    7. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5493)
    8. 1 M HEPES buffer (Sigma-Aldrich, catalog number: 83264)
    9. 0.25% Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4049)
    10. RNeasy Mini Kit (QIAGEN, catalog number: 74104)
    11. TRIzol® Reagent (Invitrogen, catalog number: 15596026)
    12. DNase I (Roche, catalog number: 04716728001)
    13. PolyATtract® mRNA Isolation Systems (Promega, catalog number: Z5210)
    14. LipofectamineTM 2000 reagent (Thermo Fisher Scientific, catalog number: 11668019)
    15. VeriKine Mouse Interferon Beta ELISA Kit (PBL Assay Science, catalog number: 42400-1)
    16. pMC-gtGTU2 vector (Nikonov et al., 2013) or any mammalian expression vector (such as pcDNA or pCMV series from Thermo Fisher Scientific)

Equipment

  1. -80 °C freezer
  2. 4 °C refrigerator
  3. Vortexer (Bio-Rad)
  4. Pipettes (Bio-Rad, catalog numbers: 166-0506, 166-0507 and 166-0508)
  5. CO2 incubator (Bio-Rad, catalog number: 4110)
  6. Biosafety level 2 (BSL-2) cabinet
  7. Autoclave (Tuttnauer, ELV-D Line)
  8. UVC5000 Ultraviolet Crosslinker (Hoefer, code number: UVC5000-230V)
  9. SunriseTM absorbance microplate reader (Tecan)
  10. Microfuge 16 Centrifuge (Beckman Coulter Life science, catalog number: A46473)

Software

  1. Microsoft Excel or Prism 5

Procedure

The procedure showing the analysis scheme for viral PAMP RNAs is summarized in Figure 2.


Figure 2. Flow chart for viral PAMP RNAs analysis

  1. Analysis for total and classical viral PAMP RNAs
    1. Preparation of total RNA samples from infected cells.
      Day -1
      1. Seed 8 x 106 BHK-21 cells on to 100 mm cell culture dish and culture in DMEM with 10% FBS. Maintain the cells at 37 °C with 5% CO2 overnight.
      Day 0
      1. Discard the BHK-21 cell growth media and wash the cell monolayer once with PBS. Infect the cells with the viruses of interest. As a demonstration, dilute RRV-T48 and RRV-T48A534V separately in serum free DMEM for the infection. Infect the BHK-21 cell monolayer by the viruses at Multiplicity of Infection (MOI) 1.0 with a minimum coverage volume (approximately 400 μl for each well in a 6-well plate). Incubate the cells for 1 h at 37 °C with 5% CO2 and then maintain the cells in DMEM with 10% FBS for another 6 h. 
      2. Extract the total RNA from the infected BHK-21 cells using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen TRIzolTM Reagent USER GUIDE). Use 1 U DNase I per 1 μg total RNA to treat the extracted RNA for 60 min at 37 °C.
      3. Optional (viruses with non-paired polyA tail): Isolation of non-classical PAMPs. Aliquot 100 μg of the treated RNA into a clean Eppendorf® DNA LoBind microcentrifuge tube (RNase free). Remove the polyA(+) fraction from the total RNA using PolyATtract mRNA Isolation Systems Kit according to the manufacturer’s protocol (Promega).
      4. Purify the total, polyA(+) and polyA(-) RNA samples with RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen) and elute the product within 20 μl RNase free water.
      5. UV inactivates the infectious viral RNAs, presented in the obtained samples, for 5 min in a UVC5000 Ultraviolet Crosslinker at 2000 μJ/cm². 
      6. Optional: Store the RNA at -80 °C for longer period if needed.
    2. Analysis of viral PAMP RNAs
      Day -1
      1. Seed 1 x 106 Cop5 cells into 12-well plate and cultured in IMDM with 10% FBS and 2 mM L-glutamine. Maintain the cells at 37 °C with 5% CO2 overnight.
      Day 0
      1. Transfect 1 μg of treated total, polyA(+) or polyA(-) RNAs respectively into confluent cultures of Cop5 cells in 12-well plates using Lipofectamine2000 reagent. Briefly, dilute 3 μl of the Lipofectamine2000 reagent in 100 μl serum free IMDM and incubate for 2-5 min at room temperature. Dilute 1 μg RNA samples in 100 μl serum free IMDM and incubate for 2-5 min at room temperature. Combine the 100 μl diluted Lipofectamine2000 reagent with the 100 μl diluted RNA samples and incubate the RNA-lipid mixture for 5 to 10 min at room temperate to form the RNA-lipid complex. Discard the BHK-21 cell growth media and wash the cell monolayer once with PBS. Add 1 ml serum free IMDM to the cells. Add the RNA-lipid complex drop-wise on top of the medium and gently rock the plate after adding to the mix. Incubate the transfected cells at 37 °C with 5% CO2 for 24 h.
      Day 1
      1. Collect the supernatant from the transfected Cop5 cells to a 1.5 ml tube. Centrifuge samples at 3,000 x g for 5 min at 4 °C to remove the cell debris. Collect the supernatant to a new tube.
      2. Determine the amount of IFN-β produced by the transfected Cop5 cells using VeriKine Mouse IFN Beta ELISA kit according to the manufacturer’s protocol together with standard curve.

  2. Analysis for non-classical viral PAMP RNAs
    Day -1
    1. Seed 1 × 106 Cop5 cells into a 12-well plate and culture in IMDM with 10% FBS and 2 mM L-glutamine. Maintain the cells at 37 °C with 5% CO2 overnight.
    Day 0
    1. Transfect 1 μg of the plasmids encoding for viral replicase of interest into the confluent cultures of Cop5 cells in 12-well plates using Lipofectamine2000 reagent. Use plasmid that expresses inactive replicase (active-site mutant) as a negative control. As a demonstration, transfect RRV-T48, RRV-T48A534V and SFV4 replicase constructs into the Cop5 cells for the following analysis.
    2. Incubate the transfected cells at 37 °C with 5% CO2 for 48 h. 
    Day 2
    1. Collect the supernatant from the transfected Cop5 cells to a 1.5 ml tube. Centrifuge samples at 3,000 x g for 5 min at 4 °C to remove the cell debris. Collect the supernatant to a new tube.
    2. Determine the amount of IFN-β produced by the transfected Cop5 cells using VeriKine Mouse IFN Beta ELISA kit according to the manufacturer’s protocol (pbl Assay science) together with standard curve. For each independent experiment, normalized the IFN-β levels measured in supernatants from cells transfected with RRV replicase expression plasmids to the amount of IFN-β produced by cells transfected by plasmid expressing SFV replicase. 

Data analysis

Collect the data of the amount of IFN-β in Microsoft Excel or Prism 5. Analyze the data using Student’s two-tailed unpaired t-test. As a demonstration, the standard curve for IFN-β ELISA assay was calculated in Table 1. The levels of IFN-β produced by Cop5 cells under the transfection of total viral PAMP RNAs (Figure 3), non-classic viral PAMP RNAs (Figure 4) and the replicase constructs (Figure 5) from RRV-T48 or RRV-T48A534V were plotted in bar graphs. The corresponding plotting raw data were demonstrated in Tables S1, S2 and S3.

Table 1. IFN-β ELISA assay standard curve. Briefly, seven concentrations of the mouse IFN-β standard were prepared according to the following table. One-hundred microliters per well of interferon standard was added to each well and incubate for 1 h at room temperature. The liquid was removed and washed with wash solution for three times. One-hundred microliters of diluted antibody solution was added to each well and incubated for 1 h at room temperature. The contents in the wells were removed and washed with wash solution for three times. One-hundred microliters of diluted HRP Solution was added to the wells and incubated for 1 h at room temperature. The contents in the wells were removed and washed with wash solution for three times. One-hundred microliters of the TMB substrate solution was added to the wells and incubated for 15 min at room temperature in the dark. 100 μl of stop solution was added to each well to stop the reaction. The absorbance at 450 nm was determined within 5 min.



Figure 3. IFN-β produced by Cop5 cells by the transfection of total viral PAMP RNAs from RRV-T48 or RRV-T48A534V. BHK-21 cells were infected with RRV-T48 and RRV-T48A534V separately at MOI 1.0. Cells were lysed and total RNA was isolated at 6 h post infection. One microgram of each RNA sample was used for transfection of Cop5 cells. The amount of IFN-β in the cell supernatant at 24 h post transfection was determined. IFN-β levels were expressed as the means ±SEM from three experiments (*, P < 0.05 using Student’s two-tailed unpaired t-test).


Figure 4. IFN-β produced by Cop5 cells by the transfection of non-classic viral PAMP RNAs from RRV-T48 or RRV-T48A534V. BHK-21 cells were infected with RRV-T48 and RRV-T48A534V separately at MOI 1.0. Cells were lysed and total RNA was isolated at 6 h post infection. Poly(A)- fraction was obtained from isolated RNAs by removal of poly(A)+ RNAs, including viral dsRNA replication forms. 1 μg of each RNA sample was used for transfection of Cop5 cells. The amount of IFN-β in the cell supernatant at 24 h post transfection was determined.


Figure 5. IFN-β produced by Cop5 cells by the transfection of replicase of RRV-T48 or RRV-T48A534V. Cop5 cells were transfected with plasmids designed to express RRV-T48, RRV-T48A534V or SFV4. The amount of IFN-β in supernatant was determined at 48 h post transfection. Values obtained for cells transfected with RRV-T48 or RRV-T48A534V replicase expression plasmids were normalized to values obtained for cells transfected with plasmid expressing SFV4 replicase (taken as 100). Normalized values were shown as the means ±SEM from three experiments (*, P < 0.05 using Student’s two-tailed unpaired t-test).

Acknowledgments

This protocol was originally published in the paper by Nikonov et al., 2013. This study was supported by grants from the Australian National Health and Medical Research Council to SM (APP1031024) and Estonian Research Council Grant to AM (IUT20-27). Suresh Mahalingam is the recipient of the NHMRC Senior Research Fellowship (ID: APP11544347).

Competing interests

All authors declare no competing interests.

References

  1. Akhrymuk, I., Frolov, I. and Frolova, E. I. (2015). Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode. Virology 487: 230-241.
  2. Alexopoulou, L., Holt, A. C., Medzhitov, R. and Flavell, R. A. (2001). Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413(6857): 732-738.
  3. Medzhitov, R. and Janeway, C. A., Jr. (2002). Decoding the patterns of self and nonself by the innate immune system. Science 296: 298-300.
  4. Mogensen, T. H. and Paludan, S. R. (2005). Reading the viral signature by Toll-like receptors and other pattern recognition receptors. J Mol Med (Berl) 83(3): 180-192.
  5. Nikonov, A., Molder, T., Sikut, R., Kiiver, K., Mannik, A., Toots, U., Lulla, A., Lulla, V., Utt, A., Merits, A. and Ustav, M. (2013). RIG-I and MDA-5 detection of viral RNA-dependent RNA polymerase activity restricts positive-strand RNA virus replication. PLoS Pathog 9(9): e1003610.
  6. Tyndall, C., La Mantia, G., Thacker, C.M., Favaloro, J., Kamen, R., (1981). A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication. Nucleic Acids Research 9, 6231-6250.
  7. Sokoloski, K. J., Haist, K. C., Morrison, T. E., Mukhopadhyay, S. and Hardy, R. W. (2015). Noncapped alphavirus genomic RNAs and their role during infection. J Virol 89(11): 6080-6092.

简介

病毒产生的PAMP RNA是激活宿主免疫应答的关键因子。因此,PAMP RNA通常与病毒性疾病发病机理密切相关。定量实时PCR是评估RNA的常规方法。然而,它不能用于检测由病毒复制酶产生的短dsRNA。建立该方案以分析由能够诱导宿主免疫应答的病毒产生的PAMP RNA。分别分析经典病毒PAMP RNA和非经典病毒PAMP RNA。简而言之,为了获得总病毒PAMP RNA,从病毒感染的细胞中提取总RNA,然后转染到Cop5细胞中。然而,为了评估非经典病毒PAMP RNA,将表达病毒复制酶的构建体转染到Cop5细胞中。通过ELISA测定的Cop5细胞产生的IFNα/β的量与总的和非经典的病毒PAMP RNA相关。由于该方法基于I型IFN应答,因此它适用于测量功能性病毒产生的PAMP RNA以及用于评估这些PAMP RNA的效率。
【背景】在病毒感染下,宿主免疫系统的激活通常始于宿主模式识别受体(PRR)识别病毒病原体相关分子模式(PAMP)(Medzhitov和Janeway,2002)。与病毒入侵相关的病毒PAMP包括病毒表面糖蛋白,病毒DNA和RNA(Mogensen和Paludan,2005)。在RNA病毒感染的情况下,病毒复制产生的RNA是必需的PAMP部分,其能够被宿主细胞内PRR识别(Alexopoulou 等人,,2001)。最近报道了两种可能的这种病毒产生的PAMP RNA作用的候选物。首先,经典的病毒PAMP RNA是由RIG-I和MDA5识别的dsRNA复制形式和由α病毒产生的非加帽的正链RNA(polyA +)(Sokoloski et al。,2015)被RIG-I认可(Akhrymuk et al。,2015)。其次,非经典病毒PAMP RNA是具有5'三磷酸的短非多腺苷酸化dsRNA。它们是使用细胞RNA模板由病毒复制酶产生的IFN诱导RNA(Nikonov 等人,,2013)。在Semliki Forest病毒(SFV)感染的细胞中,这些RNA代表了大部分PAMP RNA(Nikonov et al。,2013)。

鉴于病毒PAMP RNA在激活宿主免疫系统中的重要作用,分析不同类型的病毒产生的PAMP RNA的稳健方案可用于病毒 - 宿主相互作用研究。定量实时PCR可用于通过靶向负链来量化经典病毒dsRNA。然而,很难区分上限和非上限正股。此外,该方法既不能判断靶向PAMP RNA是否有功能,也不能获得PAMP效率,也无法检测非经典病毒PAMP RNA。为了分析能够触发宿主免疫应答的功能性PAMP RNA,我们建立了该方案以分析经典和非经典病毒PAMP RNA。为了评估总病毒PAMP RNA,提取来自感染细胞的总RNA用于评估。由于所有类型的经典甲病毒PAMP RNA都具有未配对的polyA尾巴,并且它们可能是病毒PAMP RNA中最大的比例,它们可能会掩盖其他类型的PAMP RNA。因此,在平行实验中,我们从总RNA中分离并检测了polyA-级分(Nikonov et al。,2013)。为了评估非经典病毒PAMP RNA,使用病毒复制酶构建体进行分析,因为该系统不具有可复制的病毒RNA模板,因此不产生经典的病毒PAMP RNA。 Cop5细胞用作所有PAMP RNA分析的IFNα/β激活宿主。由转染的PAMP RNA产生的IFNα/β的量可以通过ELISA测定来确定。

该方案是在病毒复制期间分析甲病毒产生的PAMP RNA的可靠方法,并且除了分离polyA-馏分之外,应用于缺乏未配对的polyA尾的病毒。它不仅可以测量PAMP RNA的水平,还可以评估PAMP RNA的效率。因此,该方案适用于涉及RNA病毒入侵和宿主免疫应答之间相互作用的研究。

关键字:病毒, 感染, 病原相关分子模式RNAs, RNA分离, 干扰素α/β检测

材料和试剂

  1. 材料
    1. 1.5 ml Eppendorf ® DNA LoBind微量离心管(Eppendorf,目录号:0030108051)
    2. 100 mm Corning ®组织培养处理的培养皿(Corning,目录号:430167)
    3. 12孔Corning TM Costar TM 平底细胞培养板(Corning,目录号:3513)
    4. 15毫升Falcon TM 锥形离心管(Corning,目录号:352096)
    5. 6孔板

  2. 细胞系
    1. ATCC ® BHK-21 [C13]细胞(ATCC,目录号:CCL-10 TM )
    2. Cop5细胞(Tyndall et al。,1981)

  3. 感兴趣的病毒
    注意:从感染性克隆中准备感兴趣的病毒。建议病毒滴度应在105和107噬斑形成单位(PFU)/ ml之间制备。下面列出的病毒用于演示。这些病毒来源于感染性克隆,释放(第0代),在BHK-21细胞中传代并储存在-80°C。
    1. RRV-T48
    2. RRV-T48 A534V

  4. 病毒复制酶构建
    注意:准备表达目的病毒的病毒复制酶(非结构蛋白)的构建体。下面列出的结构用于演示。如下面的构建图所示(图1),将编码RRV-T48复制酶,RRV-T48A534V和SFV4(用作阳性对照[Nikonov等,2013])的区域克隆到pMC-gtGTU2载体中,其中含有巨细胞病毒早期启动子,来自单纯疱疹病毒(HSV)胸苷激酶的前导序列,带有插入的合成内含子和来自猿猴病毒40(SV40)的晚期多腺苷酸化信号。
    1. RRV-T48
    2. RRV-T48 <子> A534V
    3. SFV


      图1.病毒复制酶构建图

  5. 试剂
    1. Dulbecco的改良Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D5030)
    2. Iscove的改良Dulbecco's Medium(IMDM)(Sigma-Aldrich,目录号:51471C)
    3. L-谷氨酰胺(Sigma-Aldrich,目录号:G6392)
    4. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178)
    5. 100x青霉素 - 链霉素(Sigma-Aldrich,目录号:P4333)
    6. 胰蛋白胨磷酸盐肉汤溶液(TPB)(西格玛奥德里奇,目录号:T8159)
    7. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P5493)
    8. 1 M HEPES缓冲液(Sigma-Aldrich,目录号:83264)
    9. 0.25%胰蛋白酶-EDTA溶液(Sigma-Aldrich,目录号:T4049)
    10. RNeasy迷你套件(QIAGEN,目录号:74104)
    11. TRIzol ®试剂(Invitrogen,目录号:15596026)
    12. DNase I(罗氏,目录号:04716728001)
    13. PolyATtract ® mRNA分离系统(Promega,目录号:Z5210)
    14. Lipofectamine TM 2000试剂(赛默飞世尔科技,目录号:11668019)
    15. VeriKine Mouse干扰素βELISA试剂盒(PBL Assay Science,目录号:42400-1)
    16. pMC-gtGTU2载体(Nikonov et al。,2013)或任何哺乳动物表达载体(如来自Thermo Fisher Scientific的pcDNA或pCMV系列)

设备

  1. -80°C冰箱
  2. 4°C冰箱
  3. Vortexer(Bio-Rad)
  4. 移液器(Bio-Rad,目录号:166-0506,166-0507和166-0508)
  5. CO 2 培养箱(Bio-Rad,目录号:4110)
  6. 生物安全2级(BSL-2)柜
  7. 高压灭菌器(Tuttnauer,ELV-D系列)
  8. UVC5000紫外线交联剂(Hoefer,代号:UVC5000-230V)
  9. Sunrise TM 吸光度酶标仪(Tecan)
  10. Microfuge 16离心机(Beckman Coulter生命科学,目录号:A46473)

软件

  1. Microsoft Excel或Prism 5

程序

显示病毒PAMP RNA分析方案的程序总结在图2中。


图2.病毒PAMP RNA分析的流程图

<强>

  1. 分析总病毒和经典病毒PAMP RNA
    1. 从感染细胞制备总RNA样品。
      第-1天
      1. 将8×10 6个BHK-21细胞接种到100mm细胞培养皿上,并在含有10%FBS的DMEM中培养。将细胞保持在37℃,5%CO 2 过夜。
      < style =“color:#666666; font-family:Arial,Helvetica,sans-serif; font-size:13.3333px; text-align:justify; white-space:normal;”>第0天
      1. 弃去BHK-21细胞生长培养基并用PBS洗涤细胞单层一次。用感兴趣的病毒感染细胞。作为示例,在无血清DMEM中分别稀释RRV-T48和RRV-T48 A534V 用于感染。通过感染复数(MOI)1.0的病毒感染BHK-21细胞单层,具有最小覆盖体积(对于6孔板中的每个孔,约400μl)。将细胞在37℃,5%CO 2下孵育1小时,然后将细胞维持在含有10%FBS的DMEM中另外6小时。&nbsp;
      2. 根据制造商的方案,使用TRIzol试剂从受感染的BHK-21细胞中提取总RNA (Invitrogen TRIzol TM 试剂用户指南) 。每1μg总RNA使用1U DNase I在37°C下处理提取的RNA 60分钟。
      3. 可选(具有非配对polyA尾的病毒):隔离非经典PAMP。将100μg经处理的RNA等分到干净的Eppendorf®DNALoBind微量离心管(无RNase)中。根据制造商的协议(Promega)。
      4. 使用RNeasy Mini Kit根据制造商的方案(Qiagen)并在20μl无RNase的水中洗脱产物。
      5. UV在UVC5000紫外交联剂中以2000μJ/ cm 2使得到的样品中存在的感染性病毒RNA灭活5分钟。&nbsp;
      6. 可选:如果需要,将RNA保存在-80°C更长时间。

    2. 病毒PAMP RNA的分析
      第-1天
      1. 将1×10 6个 Cop5细胞接种到12孔板中,并在含有10%FBS和2mM L-谷氨酰胺的IMDM中培养。将细胞保持在37℃,5%CO 2 过夜。
      style =“font-size:10pt;”>第0天
      1. 使用Lipofectamine2000试剂将1μg经处理的总polyA(+)或polyA( - )RNA分别转染到12孔板中的Cop5细胞的汇合培养物中。简而言之,在100μl无血清IMDM中稀释3μlLipofectamine2000试剂,并在室温下孵育2-5分钟。在100μl无血清IMDM中稀释1μgRNA样品,并在室温下孵育2-5分钟。将100μl稀释的Lipofectamine2000试剂与100μl稀释的RNA样品混合,并在室温下孵育RNA-脂质混合物5至10分钟以形成RNA-脂质复合物。弃去BHK-21细胞生长培养基并用PBS洗涤细胞单层一次。向细胞中加入1ml无血清IMDM。将RNA-脂质复合物逐滴添加到培养基上,并在添加到混合物后轻轻摇动板。将转染的细胞在37℃,5%CO 2 下孵育24小时。
        第1天
      2. 将转染的Cop5细胞的上清液收集到1.5ml管中。在4℃下以3,000 x g 离心样品5分钟以除去细胞碎片。将上清液收集到新管中。
      3. 根据制造商的方案和标准曲线,使用VeriKine MouseIFNβELISA试剂盒测定转染的Cop5细胞产生的IFN-β的量。

  1. 分析非经典病毒PAMP RNA
    第-1天
    1. 将1×10 6个 Cop5细胞接种到12孔板中,并在含有10%FBS和2mM L-谷氨酰胺的IMDM中培养。将细胞保持在37℃,5%CO 2过夜。
    第0天
    1. 使用Lipofectamine2000试剂将1μg编码目的病毒复制酶的质粒转染到12孔板中的Cop5细胞的汇合培养物中。使用表达无活性复制酶的质粒(活性位点突变体)作为阴性对照。作为示例,将RRV-T48,RRV-T48 A534V 和SFV4复制酶构建体转染到Cop5细胞中用于以下分析。
    2. 将转染的细胞在37°C和5%CO2下孵育48小时。&nbsp;
    第2天
    1. 将转染的Cop5细胞的上清液收集到1.5ml管中。在4℃下以3,000 x g 离心样品5分钟以除去细胞碎片。将上清液收集到新管中。
    2. 根据制造商的协议(pbl Assay science)以及标准曲线。对于每个独立实验,将用RRV复制酶表达质粒转染的细胞的上清液中测量的IFN-β水平标准化为由表达SFV复制酶的质粒转染的细胞产生的IFN-β的量。&nbsp;

数据分析

在Microsoft Excel或Prism 5中收集IFN-β数量的数据。使用学生的双尾未配对 t -test分析数据。作为示例,IFN-βELISA测定的标准曲线计算在表1中.Cop5细胞在转染总病毒PAMP RNA(图3),非经典病毒PAMP RNA时产生的IFN-β水平(图4)将来自RRV-T48或RRV-T48 A534V 的复制酶构建体(图5)绘制在条形图中。相应的绘图原始数据在表S1,S2和S3。

表1.IFN-βELISA测定标准曲线简言之,根据下表制备7种浓度的小鼠IFN-β标准品。每孔加入100微升干扰素标准品,并在室温下孵育1小时。除去液体并用洗涤溶液洗涤三次。向每个孔中加入100微升稀释的抗体溶液,并在室温下温育1小时。除去孔中的内容物并用洗涤溶液洗涤三次。向孔中加入100微升稀释的HRP溶液,并在室温下温育1小时。除去孔中的内容物并用洗涤溶液洗涤三次。将100微升TMB底物溶液加入孔中,并在室温下避光孵育15分钟。向每个孔中加入100μl终止溶液以终止反应。在5分钟内测定450nm处的吸光度。



图3. Cop5细胞通过转染RRV-T48或RRV-T48 A534V 的总病毒PAMP RNA产生的IFN-β。用RRV感染BHK-21细胞-T48和RRV-T48 A534V 分别为MOI 1.0。裂解细胞并在感染后6小时分离总RNA。每克RNA样品1微克用于转染Cop5细胞。测定转染后24小时细胞上清液中IFN-β的量。表达IFN-β水平 作为三次实验的平均值±SEM(*, P <0.05,使用学生的双尾未配对 t - 测试)。


图4. Cop5细胞通过转染来自RRV-T48或RRV-T48 A534V 的非经典病毒PAMP RNA产生的IFN-β。 BHK-21细胞被感染在MOI 1.0处分别使用RRV-T48和RRV-T48 A534V 。裂解细胞并在感染后6小时分离总RNA。通过去除poly(A)+ RNA(包括病毒dsRNA复制形式)从分离的RNA获得Poly(A) - 级分。将1μg每种RNA样品用于转染Cop5细胞。测定转染后24小时细胞上清液中IFN-β的量。


图5.通过转染RRV-T48或RRV-T48 A534V 的复制酶由Cop5细胞产生的IFN-β。用设计用于表达RRV-的质粒转染Cop5细胞。 T48,RRV-T48 A534V 或SFV4。在转染后48小时测定上清液中IFN-β的量。
将用RRV-T48或RRV-T48A534V复制酶表达质粒转染的细胞获得的值标准化为用表达SFV4复制酶的质粒转染的细胞获得的值(取为100)。归一化值显示为来自三次实验的平均值±SEM(*, P <0.05,使用学生的双尾未配对 t - 测试)。

致谢

该协议最初发表在Nikonov et al。,2013年的论文中。该研究得到了澳大利亚国家健康与医学研究委员会向SM(APP1031024)和爱沙尼亚研究理事会授予AM的资助。 (IUT20-27)。 Suresh Mahalingam是NHMRC高级研究奖学金获得者(ID:APP11544347)。

利益争夺

所有作者都声明没有竞争利益。

参考

  1. Akhrymuk,I.,Frolov,I。和Frolova,E。I.(2015)。 RIG-I和MDA5均以浓度依赖模式检测甲病毒复制。 病毒学 487:230-241。
  2. Alexopoulou,L.,Holt,A。C.,Medzhitov,R。和Flavell,R。A.(2001)。 通过Toll样受体3识别双链RNA和激活NF-κB。 自然 413(6857):732-738。
  3. Medzhitov,R。和Janeway,C。A.,Jr。(2002)。 通过先天免疫系统解读自我和非自我的模式。 Science 296:298-300。
  4. Mogensen,T。H.和Paludan,S。R.(2005)。 通过Toll样受体和其他模式识别受体读取病毒特征。 J Mol Med (Berl)83(3):180-192。
  5. Nikonov,A.,Molder,T.,Sikut,R.,Kiiver,K.,Mannik,A.,Toots,U.,Lulla,A.,Lulla,V.,Utt,A.,Merits,A。and Ustav,M。(2013)。 RIG-I和MDA-5检测病毒RNA依赖性RNA聚合酶活性限制正链RNA病毒复制。 P LoS Pa thog 9(9):e1003610。
  6. Tyndall,C.,La Mantia,G.,Thacker,C.M.,Favaloro,J.,Kamen,R。,(1981)。 复制起点和晚期蛋白质编码序列之间的多瘤病毒基因组区域需要顺式早期基因表达和病毒DNA复制。 核酸研究 9,6231-6250。
  7. Sokoloski,K.J.,Haist,K.C.,Morrison,T.E.,Mukhopadhyay,S。和Hardy,R.W。(2015)。 非上限甲病毒基因组RNA及其在感染期间的作用。 J Virol 89(11):6080-6092。

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mutso, M., Liu, X., Merits, A. and Mahalingam, S. (2019). Analysis of Functional Virus-generated PAMP RNAs Using IFNα/β ELISA Assay. Bio-protocol 9(12): e3282. DOI: 10.21769/BioProtoc.3282.
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