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Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
慢病毒敲减白足鼠中转录因子STAT1以评价其在抑制蜱传黄病毒中的作用   

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Abstract

Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein interactions (Duggal and Emerman, 2012). The potency of the IFN signaling response determines the outcome of TBFV infection (Best, 2016; Carletti et al., 2017). Interestingly, data from our lab show that TBFV replication is significantly restricted in cells of the reservoir species Peromyscus leucopus thereby suggesting a potent antiviral response (Izuogu et al., 2017). We assessed the relative contribution of IFN signaling to resistance in P. leucopus by knocking down a major transcription factor in the IFN response pathway. Signal transducer and activator of transcription 1 (STAT1) was specifically targeted in P. leucopus cells by shRNA technology. We further tested the impact of gene knockdown on the ability of cells to respond to IFN and restrict virus replication; the results indicate that when STAT1 expression is altered, P. leucopus cells have a decreased response to IFN stimulation and are significantly more susceptible to TBFV replication.

Keywords: Interferon(干扰素), Flavivirus(黄病毒), Restriction(抑制), Reservoir host(储存宿主), Antiviral response(抗病毒反应), Lentiviral(慢病毒), Knockdown(敲减), shRNA(shRNA), STAT1(STAT1)

Background

IFN signaling is the first line of defense against flaviviruses invading a host cell (Robertson et al., 2009; Lazear and Diamond, 2015). Molecular signatures associated with the virus particles are detected by pattern-recognition receptors (PRRs) which then elicit downstream signaling via transcription factors to release type 1 IFNs from the cell (Kawai and Akira, 2011; Loo and Gale, 2011). Further, the IFN response pathway is initiated by binding of IFN-beta (IFN-β) to its cognate receptor and activation of the JAK-STAT signaling cascade (Loo et al., 2008). STAT1 is a major player in the IFN response pathway and functions within a complex to activate the IFN–stimulated response element (ISRE) promoter in the nucleus (Stark et al., 1998). Ultimately, IFN signaling transcriptionally upregulates numerous host genes that act to curtail infection and limit viral pathogenesis (Liu et al., 2011).

TBFVs contribute to the global burden of disease by causing encephalitis or hemorrhagic fevers in infected individuals. There are about 10,000 cases of TBE annually and disease is endemic to Europe and parts of Asia (Suss, 2008). In recent years, TBFVs have shown emerging nature due to their occurrence in previously unreported regions and re-occurrence in areas of eradication (Robertson et al., 2009; Ebel, 2010). The reason for the increased spread is not known, and treatment for clinically-recognized cases is limited to palliative care (Fernandez-Garcia et al., 2009; Lani et al., 2014). While virus infection in humans and other susceptible species can result in deleterious illness, infection of a natural reservoir host P. leucopus remains asymptomatic (Telford et al., 1997; Santos et al., 2016) suggesting that the host could lack relevant proviral factors or express effective antiviral factors. Data from our lab has explored these possibilities and demonstrated that viral restriction in P. leucopus cells is mediated by an antiviral response occurring via the IFN signaling cascade (Izuogu et al., 2017). Our studies involved resolving the sequence of antiviral gene homologs in P. leucopus and targeting components of the IFN response pathway. Specifically, our study first showed that STAT1 expression was consistently higher in P. leucopus cells compared to the susceptible control M. musculus following IFN treatment and virus infection (Izuogu et al., 2017). Based on these data, we further assessed the relative role of STAT1 in virus restriction by specific gene knockdown. This protocol describes the technique used to design shRNA to target STAT1, packaging into lentiviruses and cellular transduction to establish P. leucopus stable cell lines with diminished STAT1 expression. We further provide details of how these cells were assayed for a loss of restriction following infection with a TBFV, Langat virus (LGTV).

Materials and Reagents

  1. Materials
    1. Tissue culture plates (Greiner Bio One International, catalog numbers: 657160 , 662160 , 677180 –6, 24, and 48 wells respectively)
    2. Tissue culture flasks (Greiner Bio One International, catalog numbers: 658175 and 660175 –75 cm2 and 175 cm2 respectively)
    3. Eppendorf tubes (1.5 ml) (USA Scientific, catalog number: 1615-5510 )
    4. PVDF membranes (Thermo Fisher Scientific, catalog number: 88518 )
    5. Lightcycler 480 96-well plate (Roche Molecular Systems, catalog number: 04729692001 )
    6. Labtek slides (Thermo Fisher Scientific, catalog number: 154534 )
    7. 1.2 ml tubes (USA Scientific, catalog number: 1412-1000 )
    8. Autoradiography film (McKesson Medical-Surgical, catalog number: EBA45 )
    9. Coverslips (Fisher Scientific, catalog number: 12-545-88 )
    10. Reagent reservoir troughs (VistaLab, catalog number: 3054-1001 )
    11. Cell scraper (Fisher Scientific, catalog number: 08-100-241 )
    12. Airtight container (Sterilite, catalog number: 1642 )
    13. XCell4 SureLock Midi SDS-PAGE system (Thermo Fisher Scientific, InvitrogenTM, catalog number: WR0100 )
    14. 20-well Tris-Glycine midi gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: WT0102BOX )

  2. Cells
    1. Human embryonic kidney (HEK)-293T cells (ATCC, catalog number: CRL-3216 )
    2. HEK-293 cells (ATCC, catalog number: CRL-1573 )
    3. African green monkey kidney cells–Vero cells (ATCC, catalog number: CCL-81 )
    4. P. leucopus adult skin fibroblast cells (Coriell Institute, catalog number: AG22353 )
    Note: These cells mammalian cells are maintained in complete DMEM containing 10% fetal bovine serum (FBS, Nalgene, 100 International units of penicillin, 100 µg/ml streptomycin (Thermo Fisher Scientific).
    1. One ShotTM TOP10 Chemically-Competent E. coli cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 )

  3. Viruses
    Langat virus (LGTV) strain TP21 (kindly provided by Dr. Sonja M. Best, NIAID/NIH)
    Note: LGTV is a naturally-attenuated member of the tick-borne flavivirus serocomplex used under biosafety level 2 (BSL2) laboratory conditions. All safety precautions for BSL2 level containment were adhered to.

  4. Reagents
    1. pLENTI X2- DEST vector (Campeau et al., 2009)
    2. Recombinant mouse IFN-β (Pestka Biomedical Laboratories, catalog number: 12405-1 )
    3. Oligonucleotide primer pairs (see Table 1)
    4. RNAi BLOCK-iT U6 RNAi Entry Vector Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K4945-00 )
    5. Agarose (BioExpress,, catalog number: E-3120-500 )
    6. 100 bp DNA ladder (New England Biolabs, catalog number: N3231L )
    7. TAE (10x) buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15558042 )
    8. 10x Tris-glycine SDS-PAGE running buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC2675 )
    9. LR Clonase II Enzyme Mix (Thermo Fisher Scientific, catalog number: 11791100 )
    10. ViraPowerTM Lentiviral Packaging Mix (Thermo Fisher Scientific, catalog number: K497500 )
    11. Lipofectamine 3000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: L3000015 )
    12. Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
    13. Lenti-X GoStix (Takara Bio, Clontech, catalog number: 631244 )
    14. Polybrene (Sigma-Aldrich, catalog number: 107689 )
    15. Hygromycin B (Thermo Fisher Scientific, GibcoTM, catalog number: 10687010 )
    16. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: QVC0508 ) alone or with 0.5% Tween-20 (PBST)
    17. iBlot transfer stacks (Thermo Fisher Scientific, InvitrogenTM, catalog number: IB4010-01 )
    18. Antibodies
      1. α-actin (Sigma-Aldrich, catalog number: A5441 )
      2. α-STAT1 (Cell Signaling Technology, catalog number: 9172S , used at 1:1,000) detects phosphorylation of STAT1 at Tyr701
      3. α-STAT1-P (Cell Signaling Technology, catalog number: 9167S , used at 1:1,000)
      4. α-LGTV E (provided by Dr. C. Schmaljohn, USAMRIID used at 1:1,000)
      5. α-LGTV NS3 (as described in Taylor et al., 2011, used at 1:2,000)
      6. Goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: A28177 , used at 1:3,000 for immunoblotting and at 1:1,000 viral for immunofocus assays)
      7. Rabbit anti-chicken AlexaFluor 594 (Thermo Fisher Scientific, catalog number: A11042 , used at 1:1,000)
      8. Goat anti-mouse AlexaFluor 488 (Thermo Fisher Scientific, catalog number: A11029 , used at 1:1,000)
    19. ECL Plus (Thermo Fisher Scientific, PierceTM, catalog number: 80196 )
    20. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
    21. QuantiTect Reverse Transcription Kit (QIAGEN, catalog number: 205310 )
    22. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9935 )
    23. Gene-specific qRT-PCR primers (see Table 2)
    24. FastStart essential DNA Green master (Roche Molecular Systems, catalog number: 06402712001 )
    25. Paraformaldehyde 16% w/v (Alfa Aesar, catalog number: 43368 )
    26. ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36931 )
    27. Methanol
    28. Opti-MEM reduced serum medium (Thermo Fisher Scientific, catalog number: 31985062 )
    29. Tris (Fisher Scientific, catalog number: BP152-5 )
    30. Sodium chloride (NaCl) (Gentrox, catalog number: 60-037 )
    31. Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
    32. 10x Tris-glycine buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC2672 )
    33. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500 )
    34. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: 542334 )
    35. Magnesium chloride hexahydrate (MgCl2·6H2O) (Avantor Performance Materials, MACRON, catalog number: 5958 )
    36. Calcium chloride (CaCl2) (Acros Organics, catalog number: 192735000 )
    37. 3,3-Diaminobenzidine HCl (Sigma-Aldrich, catalog number: D5637 )
    38. Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: H3410 )
    39. Glycerol (AMRESCO, catalog number: 0854 )
    40. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
    41. β-Mercaptoethanol (Alfa Aesar, catalog number: J61337 )
    42. Non-fat dry milk
    43. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 12100061 )
    44. Methylcellulose (Fisher Scientific, catalog number: M352-500 )
    45. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
    46. Triton X-100 ( Sigma Aldrich, catalog number: X100 )
    47. Sodium citrate (Sigma-Aldrich, catalog number: PHR1416 )
    48. Bovine serum albumin (BSA)
    49. Goat serum
    50. Quibit DNA BR kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 )
    51. Quibit RNA HS kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32852 )
    52. RNase-free DNase set (QIAGEN, catalog number: 79254 )
    53. Complete EDTA-free protease inhibitor cocktail (Thermo Fisher Scientific, PierceTM, catalog number: A32955 )
    54. Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 11873580001 )
    55. DC Protein assay reagents (Bio-Rad Laboratories, catalog number: 5000116 )
    56. Radioimmunoprecipitation assay (RIPA) buffer (see Recipes)
    57. DNase I buffer (10x) (see Recipes)
    58. Peroxidase substrate (prepared fresh) (see Recipes)
    59. 6x SDS-PAGE sample buffer (6x SB) (see Recipes)
    60. Western blot blocking solution (see Recipes)
    61. Culture overlay media (see Recipes)
    62. Permeabilization solution (see Recipes)
    63. Immunofluorescence assay blocking solution (see Recipes)

Equipment

  1. Pipettes (USA Scientific)
  2. Transilluminator (Omega Ultra-Lum)
  3. Tabletop centrifuge (Eppendorf)
  4. iBlot dry blotting transfer machine (Thermo Fisher Scientific)
  5. Rocker (Benchmark)
  6. Rotator (Bellco Biotechnology)
  7. Autoradiography film processor (Kodak)
  8. Thermocycler (Roche Lightcycler)
  9. Tissue culture incubator (at 37 °C with 5% CO2) (Thermo Fisher Scientific)
  10. Biosafety cabinet
  11. Water bath (VWR Scientific)
  12. Heating block (at 95 °C) (Fisher Scientific)
  13. SDS-PAGE apparatus (Pharmacia Biotech)
  14. Refrigerator
  15. Vortex (Fisher Scientific)
  16. Qubit 2.0 fluorometer (Thermo Fisher Scientific, current model: Qubit 3.0 )
  17. Confocal microscope (Olympus Life Science)

Software

  1. GraphPad Prism 6: https://www.graphpad.com/scientific-software/prism/
  2. Roche Lightcycler 96 software: https://lifescience.roche.com/en_us/brands/realtime-pcr
  3. Invitrogen primer design software: https://rnaidesigner.thermofisher.com/rnaiexpress/
  4. MacVector software version 12.7: http://macvector.com/EcoRI/macvector12.7.5installer.html

Procedure

  1. Design and cloning of short hairpin RNA (shRNA) into lentiviral vectors
    Our group recently resolved and published the sequence of STAT1 in the reservoir species P. leucopus (Izuogu et al., 2017) with accession number KY451962. Based on the gene sequence, the oligonucleotides for knockdown were designed and cloned as described below; A flowchart describing the process of oligonucleotide processing and generation of lentiviral vectors is represented in Figure 1.


    Figure 1. Construction of lentiviral plasmids. A. Schematic representation of P. leucopus STAT1 showing the regions targeted for knockdown. B. Workflow of procedure to generate lentiviral expression vectors with shRNA to P. leucopus STAT1.

    1. Upload the complete sequence to the Invitrogen primer design software (or equivalent) and generate oligo candidates for shRNA design. Regions around the beginning, middle and end of the gene are targeted to ensure complete transcript coverage and avoid off-target effects. Candidates selected are shown in Table 1 and Figure 1A.

      Table 1. Oligonucleotide sequences utilized for gene knockdown. shRNA top and bottom strand sequences designed to target 3 distinct regions of the P. leucopus STAT1 gene. No special conditions were specified in the primer order.


    2. As a control, design non-specific oligonucleotides to create lentiviral vectors that will not result in knockdown. An shRNA that targets the GFP sequence was used for this purpose in our study (see Table 1). Since GFP is not expressed in the cells of interest, the control cell lines should be transcriptionally equivalent to the background, non-transduced cells.
    3. Anneal the top and bottom strands by mixing oligonucleotides in equal proportion (200 μM each) and subjecting to high temperature (95 °C) for 4 min according to the manufacturer’s annealing protocol for the BLOCK-iT U6 RNAi Entry Vector Kit (Thermo Fisher scientific). After the heating process, immediately transfer the samples to the bench and allow for cooling to occur slowly. This process will generate double-strand shRNA.
    4. To confirm the annealing step, resolve the samples on a 4% agarose gel (immersed in 1x TAE buffer) alongside a 100 bp ladder by adding equal volume of each sample (10 μl) to single wells and running at 100 V. Transfer the gel to a transilluminator to visualize double-strand oligonucleotides compared to the single-strand samples (Illustrated in Figure 1B).
    5. After the double-strand is confirmed by agarose gel electrophoresis, proceed with the annealed samples and ligate the resultant shRNA into the U6 vector to generate an entry vector for further cloning. The U6 vector is contained within the BLOCK-iT U6 RNAi Entry Vector Kit (Thermo Fisher Scientific) and all manufacturers’ recommendations were adhered to.
    6. Recombine the entry vector into a lentiviral destination vector that can be delivered into mammalian cells. In this study, we utilized the pLENTI X2- DEST vector (Campeau et al., 2009) for Gateway cloning with the Gateway LR Clonase II Enzyme Mix according to the manufacturer’s protocol with the exception that recombination was allowed to proceed overnight.
    7. Transform chemically-competent One ShotTM TOP10 Chemically-Competent E. coli bacteria cells with the recombined DNA and select colonies for DNA extraction to obtain pure plasmid DNA.

  2. Generation of Lentiviruses
    1. Set up 293T cells at 3,000,000 cells per well in a 6-well dish and grow overnight.
    2. Transfect cells with purified lentiviral plasmids targeting the individual regions of the gene of interest and the non-specific (GFP) sequence. Importantly, include a pool of DNA containing 1 μg of lentiviral vectors targeting each region into a single transfection tube. Therefore, prepare a single tube for shRNA targeting each region of interest and an additional tube with all shRNA candidates pooled together in one tube.
    3. Add 1 μg of ViraPower Lentiviral Packaging Mix along with plasmid DNA and complete transfection using Lipofectamine 3000 Transfection Kit according to the manufacturer’s protocol. Pipet slowly onto cells to avoid detachment.
    4. At 24 h post-transfection, replace the transfection media with fresh DMEM containing 10 mM sodium butyrate, 2 ml total volume was suitable for a 6-well dish in our studies.
    5. Incubate the cells for another 48 h without perturbing the culture dishes.
    6. Collect supernatants containing the newly-generated lentiviruses. Confirm the presence of lentiviruses by running a rapid test using Lenti-X GoStix (use 20 µl sample supernatant followed by three drops of the kit running buffer and allow time for bands to develop). Lentiviral supernatants were not filtered.
    7. Store the lentivirus stock at -80 °C until needed.

  3. Transduction of P. leucopus cells with STAT1 shRNA-expressing lentiviruses
    1. Set up P. leucopus fibroblasts at 300,000 cells per well in 6-well dishes and incubate overnight.
    2. Infect the cells with 1 ml of the lentivirus stock and add 8 µg polybrene (diluted from a stock solution prepared in sterile water).
    3. At 1 h post-transduction, add another 1 ml of culture medium with 8 µg polybrene. 24 h later, re-transduce the cells and treat with 8 µg polybrene.
    4. On day 2, remove the lentiviral inoculum and replace with fresh culture medium.
    5. At day 3 post-transduction, add the appropriate antibiotic selection (400 µg/ml hygromycin was used for P. leucopus fibroblasts).
    6. Maintain the cells in selection for 1 week and sub-clone to obtain clonal cells by splitting single cells into each well of 48-well dishes and observe for cell proliferation in individual wells over 1-2 weeks.
    7. Expand clonal cells into large flasks (175 cm2) and proceed to test for gene knockdown.

  4. Assessing gene knockdown in transduced cell lines
    Our studies have demonstrated that STAT1 expression is upregulated in P. leucopus following treatment with IFN-β (Izuogu et al., 2017). Hence, in order to determine if the gene of interest is knocked down, the cells are treated with IFN and probed for gene induction.
    1. Set up the STAT1 knockdown (KD) and NS KD P. leucopus fibroblasts at 1,000,000 cells per well and incubate overnight.
    2. Treat the cells (or not) with 1,000 international units (IU) mouse interferon beta (mIFN-β) for 8 h and harvest samples to test for knockdown as follows:
      By SDS-PAGE and Western blot
      1. Remove the cellular supernatant and wash the cells 2 x with PBS buffer.
      2. Add 500 µl of RIPA buffer (see Recipes) and scrape the cell monolayer gently. Pipet a few times and transfer the sample to Eppendorf tubes.
      3. Incubate the samples with 50 µl 1x DNase I buffer (see Recipes) for 30 min at 37 °C.
      4. Centrifuge at max speed for 10 min, collect supernatant and mix with 6x SB to achieve 1x SB (see Recipes). Load equal amounts of protein on 10% SDS gels and resolve at 150 V for 1.5 h.
      5. After the electrophoresis step, transfer the proteins to PVDF membranes using the iBLOT transfer apparatus for 7 min.
      6. Incubate the membranes with Western blot blocking solution (see Recipes) while rocking for 1 h at room temperature (RT).
      7. Probe the blots with specific antibodies to STAT1 and phosphorylated STAT1 (STAT1-P) for 16 h at 4 °C on a rocker and wash 3 x in PBST for 5 min.
      8. Incubate with secondary antibodies conjugated to HRP while rocking for 1 h at RT. Wash 3 x in PBST for 10 min.
      9. Perform a final wash with PBS for 5 min, incubate membranes with ECL-plus reagents for an additional 5 min and utilize an automated film processor to visualize protein expression. Figure 2A shows Western blot results for STAT1 KD.

      By qRT-PCR
      1. Remove cellular supernatant and wash the cells 2 x in PBS. Harvest cell lysates according to RNA extraction protocol. In this study, cells were collected in 350 µl of cell lysis buffer (RLT buffer included RNeasy mini kit) and RNA extraction was performed using the RNeasy mini kit according to the manufacturer’s protocol, including the DNase treatment step using the RNase-free DNase set (QIAGEN).
      2. Quantify the RNA and normalize the concentration to ensure an equal amount of RNA between all the samples.
      3. Perform cDNA synthesis using an equal amount of RNA template (1 µg was used). Our study utilized the QuantiTect Reverse Transcription Kit according to the manufacturer’s protocol.
      4. Make a 1/8 dilution of the samples using PCR-grade water and add 3 µl of each sample to individual wells of a clean, opaque 96-well plate. Add 1 µl each of Fwd. and Rev. primers for a total of 5 µl; primer sequences used to detect P. leucopus STAT1 are shown in Table 2. In this study, the FastStart Essential Green Master Enzyme Mix was used and 5 µl of this was added to each sample for the reaction. Thus, the total reaction volume was 10 µl.

        Table 2. qRT-PCR primers used to probe for STAT1 and β-actin in P. leucopus cells


      5. Run on Lightcycler for 50 cycles and analyze the resultant data. The fold induction of STAT1 in the NS controls was set to 100% and the fold induction in the KD cells was assessed comparative to the NS cells. All data are normalized to β-actin. Figure 2B shows relative STAT1 mRNA expression.


        Figure 2. Assessing gene knockdown in transduced cell lines. A. Flow chart and data of Western blot showing decreased expression of STAT1 in P. leucopus KD cells expressing shRNA to positions 261 (STAT1KD #1), 630 (STAT1KD #2) and all 3 positions–261, 630 and 1,742 (STAT1KD All). All further experiments were performed with the ‘STAT1 KD All’ cells and they are henceforth named as STAT1 KD cells. Samples were probed with antibodies to STAT1, STAT1-P and β-actin. B. Flow chart and data of qRT-PCR showing percentage relative STAT1 mRNA expression in the STATIKD cells compared to the NS control. Asterisks indicate: **** = P < 0.00001.

  5. Perform a functional assay for IFN response in STAT1 KD cells
    Having observed knockdown, we tested the impact of STAT1 KD on the upregulation of an ISG. Data in our lab has identified a homolog of the TBFV restriction factor-tripartite motif protein 79 (TRIM79) and also demonstrated it to be an ISG in P. leucopus cells (Taylor et al., 2011). We assayed the fold induction of P. leucopus TRIM79 (plTRIM79) in STAT1 KD cells following IFN treatment according to the following protocol:
    1. Set up STAT1 KD and NS control cells as in Procedure D, treat with mIFN-β for 8 h and proceed to harvest RNA as described above in the qRT-PCR section.
    2. Use an equal amount of RNA as template for cDNA synthesis and perform qRT-PCR reaction using gene-specific primers. Fold induction was assessed relative to untreated cells. We also compared plTRIM79 induction in P. leucopus cells with specific lentiviral knockdown to the type 1 IFN receptor (IFNAR1) generated using the same technique outlined above. Data is shown in Figure 3.


      Figure 3. qRT-PCR of an ISG plTRIM79 in STAT1 KD cells relative to the NS control. Cells with altered STAT1 (and IFNAR1) expression show significantly less ISG induction suggesting that knockdown has a functional impact on IFN signaling. Mean ± SD. Asterisks indicate: ** = P < 0.01, *** = P < 0.001.

  6. Test the effect of STAT1 KD on LGTV replication
    By immunofluorescence microscopy
    1. Set up STAT1 KD and NS cells at 50,000 cells per well in 8-well Labtek slides and incubate overnight.
    2. Infect the STAT1 KD and NS cells with LGTV at MOI of 100. Allow a 1 h absorption period and replace the inoculum with 500 µl of fresh culture media.
    3. At 72 h post-infection, remove supernatant, wash cells 2 x with PBS and fix for 20 min in 4% paraformaldehyde solution. Add just enough to cover the slides (about 5 ml).
    4. Wash 2 x with PBST and permeabilize cells for 5 min. Remove permeabilization solution (see Recipes) and add 3 ml immunofluorescence assay blocking solution (see Recipes) for 1 h at RT. Specific antibodies to LGTV envelope (E), and LGTV non-structural protein 3 (NS3) proteins were used in this study at 1:2,000 dilution.
    5. Wash 3 x with PBS and incubate with the appropriate fluorescent secondary antibodies diluted in 3 ml blocking solution for 1 h at RT in the dark. Wash cells 3 x in opaque containers protected from light.
    6. Add mounting media containing DAPI–This study utilized 1 ml of the ProLong Gold Anti-Fade Mountant with DAPI added and slides were imaged on an Olympus confocal microscope. Figure 4A includes the confocal image data showing increased viral protein staining in STAT1 KD cells.

    By virus titration and immunofocus assay
    1. Set up STAT1 KD and NS cells at 100,000 cells per well in 24-well dishes and incubate overnight.
    2. Infect cells with LGTV at a multiplicity of infection (MOI) of 10 such that there would be 10 virion particles to each cell in the monolayer. This is calculated as a product of the cell density, desired MOI and number of wells divided by the viral titer to determine the volume of viral stock needed for infection. Inoculate the cells with 250 µl of the virus solution and allow a 1 h absorption period and replace the inoculum with 500 µl of fresh culture media.
    3. Collect viral supernatants at 24, 48, and 72 h post-infection in triplicates and store at -80 °C.
    4. Set up Vero cells at 200,000 cells per well in 24-well dishes and incubate overnight.
    5. Make six 1/10 serial dilutions of each viral supernatant sample in FBS-free DMEM media using the 1.2 ml tubes and add 250 µl of each diluted to fresh Vero cells. Allow a 1 h absorption period and replace the inoculum with 500 µl of fresh culture media containing 2% FBS and 0.8% methylcellulose (culture overlay media).
    6. At 4 d post-infection, perform the immunofocus assay as described previously (Taylor et al., 2011; Baker et al., 2013). Briefly, remove the culture overlay media (see Recipes) and wash the cells 2 x with PBS. Fix with 100% methanol for 30 min and wash again with PBS.
    7. Incubate for 20 min in Opti-MEM medium and follow up with a virus-specific antibody. In this study, an antibody to detect the LGTV envelope (E) protein was used at 1:1,000. Incubate with primary antibody for 1 h at 37 °C in an airtight container.
    8. Wash cells 2 x with PBS and incubate with the appropriate secondary antibody. Goat anti-mouse IgG antibody was used in this study at 1:1,000. Incubate as in primary antibody and follow with 2 PBS washes.
    9. Add the substrate and allow 15 min for foci to develop. Determine the number of focus-forming units (FFU) per ml of viral supernatant and compare virus release in each cell type and condition. Results of STAT1 KD on LGTV infection are shown in Figure 4B.


      Figure 4. Determining the impact of STAT1 KD on LGTV infection of P. leucopus. A. Visualizing LGTV protein staining in STAT1 KD P. leucopus cell lines by immunofluorescence microscopy. Images were captured on an Olympus microscope at 40x magnification showing LGTV E (green), NS3 (red) and nuclei are stained with DAPI (blue). B. Quantifying LGTV replication in P. leucopus STAT1 KD cells assessed by performing an immunofocus assay. This assay involves titrating supernatants from the infected clonal cells onto fresh Vero cells and quantifying the resultant foci. Viral titer is indicated as focus forming units (ffu) per ml. Mean ± SD; Data are from three independent experiments performed in triplicate. Asterisks indicate: * = P < 0.05, *** = P < 0.001, **** = P < 0.00001.

Data analysis

All titration experiments were performed three times in triplicates. Data were analyzed by an unpaired t-test or Mann-Whitney U test using GraphPad Prism 6 software as described in Izuogu et al., 2017 (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179781).

Notes

  1. Depending on the application, cDNA samples can be used at higher dilutions for qRT-PCR.
  2. When annealing oligonucleotides, rather than transferring the sample tubes to the bench, transfer the entire heat block from the heating system–this will allow cooling to occur at a very slow rate.
  3. Vero cells are African green monkey cells which have a defect in interferon secretion thereby making them suitable to count viruses by titration. They also form foci and plaques relatively well.

Recipes

  1. Radioimmunoprecipitation assay (RIPA) buffer
    50 mM Tris pH 7.5 (100 ml 1 M)
    150 mM NaCl (60 ml 5 M)
    0.5% deoxycholate (20 g)
    0.1% SDS (10 ml 20%)
    1% NP-40 (20 ml IGEPAL CA-630)
    dH2O (up to 2 L)
  2. DNase I buffer (10x)
    100 mM Tris-HCl pH 7.5
    25 mM MgCl2
    5 mM CaCl2
  3. Peroxidase substrate (prepared fresh)
    0.4 mg/ml 3,3-diaminobenzidine HCl
    0.45 μl/ml 30% H2O2
    1x PBS
  4. 6x SDS-PAGE sample buffer (6x SB)
    375 mM Tris-HCl, pH 6.8
    9% SDS
    50% glycerol
    0.03% bromophenol blue
    9% β-mercaptoethanol, fresh
  5. Western blot blocking solution
    5% non-fat dry milk in PBST
  6. Culture overlay media
    Complete DMEM supplemented with 0.8% methylcellulose and 2% FBS
  7. Permeabilization solution
    0.1% Triton X-100 and 0.1% sodium citrate in PBS
  8. Immunofluorescence assay blocking solution
    0.5% BSA and 1% goat serum in PBS

Acknowledgments

This work is supported by a pilot grant funding from the Lyme Disease Association and the University of Toledo College of Medicine and Life Sciences startup funds (RTT). The protocol and the representative results shown herein were adapted from our published work (Taylor et al., 2011; Izuogu et al., 2017). We thank Dr. Jason Munshi-South and Dr. Stephen Harris for their help in generating the initial nucleotide hits that were used to resolve the STAT1 sequence in P. leucopus. We declare no conflicting or competing interests.

References

  1. Baker, D. G., Woods T. A., Butchi, N. B., Morgan, T.M., Taylor, R.T., Sunyakumthorn, P., Mukherjee, P., Lubick, K. J., Best, S. M. and Peterson K. E. (2013). Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection. J Gen Virol 94(2): 336-47
  2. Best, S. M. (2016). The many faces of the flavivirus NS5 protein in antagonism of type I interferon signaling. J Virol 91(3).
  3. Campeau, E., Ruhl, V. E., Rodier, F., Smith, C. L., Rahmberg, B. L., Fuss, J. O., Campisi, J., Yaswen, P., Cooper, P. K. and Kaufman, P. D. (2009). A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS One 4(8): e6529.
  4. Carletti, T., Zakaria, M. K. and Marcello, A. (2017). The host cell response to tick-borne encephalitis virus. Biochem Biophys Res Commun.
  5. Duggal, N. K. and Emerman, M. (2012). Evolutionary conflicts between viruses and restriction factors shape immunity. Nat Rev Immunol 12(10): 687-695.
  6. Ebel, G. D. (2010). Update on Powassan virus: emergence of a North American tick-borne flavivirus. Annu Rev Entomol 55: 95-110.
  7. Fernandez-Garcia, M. D., Mazzon, M., Jacobs, M. and Amara, A. (2009). Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5(4): 318-328.
  8. Kawai, T. and Akira, S. (2011). Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34(5): 637-650.
  9. Lani, R., Moghaddam, E., Haghani, A., Chang, L. Y., AbuBakar, S. and Zandi, K. (2014). Tick-borne viruses: a review from the perspective of therapeutic approaches. Ticks Tick Borne Dis 5(5): 457-465.
  10. Lazear, H. M. and Diamond, M. S. (2015). New insights into innate immune restriction of West Nile virus infection. Curr Opin Virol 11: 1-6.
  11. Liu, J., Elmore, J. M., Lin, Z. J. and Coaker, G. (2011). A receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor. Cell Host Microbe 9(2): 137-146.
  12. Loo, Y. M., Fornek, J., Crochet, N., Bajwa, G., Perwitasari, O., Martinez-Sobrido, L., Akira, S., Gill, M. A., Garcia-Sastre, A., Katze, M. G. and Gale, M., Jr. (2008). Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol 82(1): 335-345.
  13. Loo, Y. M. and Gale, M., Jr. (2011). Immune signaling by RIG-I-like receptors. Immunity 34(5): 680-692.
  14. Izuogu, A. O., McNally, K. L., Harris, S. E., Youseff, B. H., Presloid, J. B., Burlak, C., Munshi-South, J., Best, S. M. and Taylor, R. T. (2017). Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses. PLoS One 12(6): e0179781.
  15. Robertson, S. J., Mitzel, D. N., Taylor, R. T., Best, S. M. and Bloom, M. E. (2009). Tick-borne flaviviruses: dissecting host immune responses and virus countermeasures. Immunol Res 43(1-3): 172-186.
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  18. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. and Schreiber, R. D. (1998). How cells respond to interferons. Annu Rev Biochem 67: 227-264.
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简介

蜱传黄热病病毒(TBFV)的细胞感染导致干扰素(IFN)信号传导途径的激活和随后称为IFN刺激基因(ISG)(Schoggins等人,2011)的众多基因的上调。许多ISG通过蛋白质 - 蛋白质相互作用以广泛或特定的方式起作用来防止病毒发病(Duggal和Emerman,2012)。 IFN信号反应的效力决定了TBFV感染的结果(Best,2016; Carletti等人,2017)。有趣的是,我们实验室的数据显示TBFV复制在储库物种Peromyscus leucopus的细胞中显着受到限制,从而表明有效的抗病毒应答(Izuogu等人,2017)。我们评估干扰素信号对抗性的相对贡献。通过敲低IFN反应途径中的主要转录因子来抑制白血病。信号转导和转录激活因子1(STAT1)是专门针对在P。 leucopus细胞通过shRNA技术。我们进一步测试了基因敲低对细胞对IFN反应和限制病毒复制的能力的影响;结果表明当STAT1表达被改变时,leucopus细胞对IFN刺激的反应降低,并且对TBFV复制显着更敏感。

【背景】IFN信号是抵抗侵入宿主细胞的黄病毒的第一道防线(Robertson等人,2009; Lazear和Diamond,2015)。通过模式识别受体(PRR)检测与病毒颗粒相关的分子标记,然后通过转录因子引发下游信号从细胞释放1型IFN(Kawai and Akira,2011; Loo and Gale,2011)。此外,IFN应答途径通过IFN-β(IFN-β)与其同源受体的结合和JAK-STAT信号传导级联的激活而启动(Loo et al。,2008)。 STAT1是IFN应答途径中的主要参与者,并且在复合体内起作用以激活细胞核中的IFN-刺激的应答元件(ISRE)启动子(Stark等人,1998)。最终,IFN信号转录上调许多宿主基因,其起到减少感染和限制病毒发病的作用(Liu等人,2011)。

TBFV通过在感染者中引起脑炎或出血热而导致全球疾病负担。每年大约有1万例TBE,疾病在欧洲和亚洲部分地区流行(Suss,2008)。近年来,TBFV由于其在先前未报告的地区的发生以及在根除地区的再发现而显示出新兴性质(Robertson等人,2009; Ebel,2010)。传播增加的原因尚不清楚,临床认可的病例的治疗仅限于姑息治疗(Fernandez-Garcia等人,2009; Lani等人 ,2014年)。虽然人类和其他易感物种的病毒感染可能导致有害的疾病,但感染天然的水库宿主P. leucopus仍无症状(Telford等人,1997; Santos等人,2016),表明宿主可缺乏相关的原病毒因子或表达有效的抗病毒因子。来自我们实验室的数据已经探索了这些可能性,并证明了病毒的限制。白血病细胞是由经由IFN信号级联发生的抗病毒应答介导的(Izuogu等人,2017)。我们的研究涉及解析抗病毒基因同系物的序列。 leucopus 和靶向IFN应答通路的成分。具体而言,我们的研究首先表明,STAT1表达一贯高于P。 leucopus细胞相比,敏感的控制M。 IFN治疗和病毒感染后的小鼠(Izuogu et al。,2017)。基于这些数据,我们进一步评估了STAT1在特定基因敲低病毒限制中的相对作用。该协议描述了用于设计shRNA以靶向STAT1的技术,包装成慢病毒和细胞转导以建立。 leucopus稳定的细胞系STAT1表达减少。我们进一步提供了这些细胞如何在用TBFV,兰加特病毒(LGTV)感染后测定丧失限制的细节。

关键字:干扰素, 黄病毒, 抑制, 储存宿主, 抗病毒反应, 慢病毒, 敲减, shRNA, STAT1

材料和试剂

  1. 物料
    1. 组织培养板(Greiner Bio One International,目录号分别为657160,662160,677180-6,24和48孔)
    2. 组织培养瓶(Greiner Bio One International,目录号分别为658175和660175-75cm 2和175cm 2)。
    3. Eppendorf管(1.5ml)(USA Scientific,目录号:1615-5510)
    4. PVDF膜(Thermo Fisher Scientific,目录号:88518)
    5. Lightcycler 480 96孔板(Roche Molecular Systems,目录号:04729692001)
    6. Labtek载玻片(Thermo Fisher Scientific,目录编号:154534)
    7. 1.2毫升管(USA Scientific,目录号:1412-1000)
    8. 放射自显影(McKesson Medical-Surgical,目录号:EBA45)
    9. 盖帽(Fisher Scientific,目录号:12-545-88)
    10. 试剂槽(VistaLab,目录号:3054-1001)
    11. 细胞刮刀(Fisher Scientific,目录号:08-100-241)
    12. 密封容器(Sterilite,目录号:1642)
    13. XCell4 SureLock Midi SDS-PAGE系统(Thermo Fisher Scientific,Invitrogen TM,目录号:WR0100)
    14. 20孔Tris-甘氨酸midi凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:WT0102BOX)

  2. 细胞
    1. 人胚胎肾(HEK)-293T细胞(ATCC,目录号:CRL-3216)
    2. HEK-293细胞(ATCC,目录号:CRL-1573)
    3. 非洲绿猴肾细胞-Vero细胞(ATCC,目录号:CCL-81)
    4. P上。 leucopus成人皮肤成纤维细胞(Coriell Institute,目录号:AG22353)
    注:这些细胞哺乳动物细胞维持在含有10%胎牛血清(FBS,Nalgene,100国际单位青霉素,100μg/ ml链霉素(Thermo Fisher Scientific))的完全DMEM中。
    1. One Shot TM TOP10化学能力 (Thermo Fisher Scientific,Invitrogen TM,目录号:C404010)。

  3. 病毒
    朗格病毒(LGTV)菌株TP21(由Sonja M.Best博士,NIAID / NIH提供)
    注:LGTV是在生物安全级别2(BSL2)实验室条件下使用的蜱传黄病毒血清型复合物的天然减毒成员。所有关于BSL2级遏制的安全措施都得到了遵守。

  4. 试剂
    1. pLENTI X2-DEST载体(Campeau等人,2009)
    2. 重组小鼠IFN-β(Pestka生物医学实验室,目录号:12405-1)
    3. 寡核苷酸引物对(见表1)
    4. RNAi BLOCK-iT U6 RNAi入门载体试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K4945-00)
    5. 琼脂糖(BioExpress,目录号:E-3120-500)
    6. 100 bp DNA梯(新英格兰生物实验室,目录号:N3231L)
    7. TAE(10x)缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:15558042)
    8. 10x Tris-甘氨酸SDS-PAGE运行缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:LC2675)
    9. LR Clonase II酶混合物(Thermo Fisher Scientific,目录号:11791100)
    10. ViraPower TM Lentiviral Packaging Mix(Thermo Fisher Scientific,目录号:K497500)
    11. Lipofectamine 3000(Thermo Fisher Scientific,Invitrogen TM,目录号:L3000015)
    12. 丁酸钠(Sigma-Aldrich,目录号:B5887)
    13. Lenti-X GoStix(Takara Bio,Clontech,目录编号:631244)
    14. 聚凝胺(Sigma-Aldrich,目录号:107689)
    15. 潮霉素B(Thermo Fisher Scientific,Gibco TM,目录号:10687010)
    16. 单独使用磷酸盐缓冲液(PBS)(Thermo Fisher Scientific,Invitrogen TM,目录号:QVC0508)或使用0.5%Tween-20(PBST)
    17. iBlot转移叠层(Thermo Fisher Scientific,Invitrogen TM,目录号:IB4010-01)
    18. 抗体
      1. α-肌动蛋白(Sigma-Aldrich,目录号:A5441)
      2. α-STAT1(Cell Signaling Technology,目录号:9172S,以1:1,000使用)检测Tyr701处STAT1的磷酸化。
      3. (Cell Signaling Technology,目录号:9167S,以1:1,000使用)
      4. α-LGTV E(由C.Schmaljohn博士提供,USAMRIID以1:1,000使用)
      5. α-LGTV NS3(如泰勒(Taylor)等人2011所述,以1:2,000使用)。
      6. 山羊抗小鼠IgG(Thermo Fisher Scientific,目录号:A28177,以1:3,000进行免疫印迹,在1:1000病毒进行免疫灶实验)
      7. 兔抗鸡AlexaFluor 594(赛默飞世尔科技,产品目录号:A11042,1:1,000使用)
      8. 山羊抗小鼠AlexaFluor 488(Thermo Fisher Scientific,目录号:A11029,以1:1,000使用)
    19. ECL Plus(Thermo Fisher Scientific,Pierce TM,目录号:80196)
    20. RNeasy迷你试剂盒(QIAGEN,目录号:74104)
    21. QuantiTect反转录试剂盒(QIAGEN,目录号:205310)
    22. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9935)
    23. 基因特异性qRT-PCR引物(见表2)
    24. 快速启动基本的DNA绿色大师(罗氏分子系统,目录号:06402712001)
    25. 多聚甲醛16%w / v(Alfa Aesar,目录号:43368)
    26. 具有DAPI的ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM,目录号:P36931)
    27. 甲醇
    28. Opti-MEM降低血清培养基(Thermo Fisher Scientific,目录号:31985062)
    29. Tris(Fisher Scientific,目录号:BP152-5)
    30. 氯化钠(NaCl)(Gentrox,目录号:60-037)
    31. 脱氧胆酸盐(Sigma-Aldrich,目录号:D6750)
    32. 10x Tris-甘氨酸缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:LC2672)
    33. 十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:BP166-500)
    34. NP-40(IGEPAL CA-630)(Sigma-Aldrich,目录号:542334)
    35. 氯化镁六水合物(MgCl 2•6H 2 O)(Avantor Performance Materials,MACRON,目录号:5958)
    36. 氯化钙(CaCl 2 2)(Acros Organics,目录号:192735000)
    37. 3,3-二氨基联苯胺HCl(Sigma-Aldrich,目录号:D5637)
    38. 过氧化氢(H 2 O 2)(Sigma-Aldrich,目录号:H3410)
    39. 甘油(AMRESCO,目录号:0854)
    40. 溴酚蓝(Bio-Rad Laboratories,目录号:1610404)
    41. β-巯基乙醇(Alfa Aesar,目录号:J61337)
    42. 脱脂奶粉
    43. DMEM(Thermo Fisher Scientific,Gibco TM,目录号:12100061)
    44. 甲基纤维素(Fisher Scientific,目录号:M352-500)
    45. 胎牛血清(FBS)(亚特兰大生物公司,目录号:S11550)
    46. Triton X-100(Sigma Aldrich,目录号:X100)
    47. 柠檬酸钠(Sigma-Aldrich,目录号:PHR1416)
    48. 牛血清白蛋白(BSA)
    49. 山羊血清
    50. Quibit DNA BR试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32850)
    51. Quibit RNA HS试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32852)
    52. 无RNA酶的DNA酶(QIAGEN,目录号:79254)
    53. 完全无EDTA的蛋白酶抑制剂混合物(Thermo Fisher Scientific,Pierce TM,目录号:A32955)
    54. 完全不含EDTA的蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11873580001)
    55. DC蛋白质测定试剂(Bio-Rad Laboratories,目录号:5000116)
    56. 放射免疫沉淀试验(RIPA)缓冲液(见食谱)
    57. DNase I缓冲液(10x)(见食谱)
    58. 过氧化物酶底物(新鲜制备)(见食谱)
    59. 6x SDS-PAGE样品缓冲液(6x SB)(见食谱)
    60. 蛋白质印迹阻断溶液(见食谱)
    61. 文化覆盖媒体(见食谱)
    62. 透化溶液(见食谱)
    63. 免疫荧光试验封闭液(见食谱)

设备

  1. 移液器(美国科学)
  2. Transilluminator(Omega Ultra-Lum)
  3. 台式离心机(Eppendorf)
  4. iBlot干印迹转印机(赛默飞世尔科技)
  5. 摇杆(基准)
  6. 转子(Bellco生物技术)
  7. 放射自显影胶片处理器(柯达)
  8. 热循环仪(Roche Lightcycler)
  9. 组织培养培养箱(37°C,5%CO 2)(赛默飞世尔科技)
  10. 生物安全柜
  11. 水浴(VWR科学)
  12. 加热块(95°C)(Fisher Scientific)
  13. SDS-PAGE装置(Pharmacia Biotech)
  14. 冰箱
  15. 涡旋(费希尔科学)
  16. Qubit 2.0荧光计(Thermo Fisher Scientific,目前的型号:Qubit 3.0)
  17. 共聚焦显微镜(奥林巴斯生命科学)

软件

  1. GraphPad Prism 6: https://www.graphpad.com/scientific-software/prism/
  2. 罗氏Lightcycler 96软件: https://lifescience.roche.com/zh_CN/brands/realtime- pcr
  3. Invitrogen引物设计软件: https://rnaidesigner.thermofisher.com/rnaiexpress/
  4. MacVector软件版本12.7: http://macvector.com/EcoRI/macvector12.7.5installer.html

程序

  1. 将短发夹RNA(shRNA)设计并克隆到慢病毒载体中
    我们的小组最近解决并发布了编号为KY451962的储库物种物种(leucopus)(Izuogu等人,2017)的STAT1序列。基于基因序列,如下所述设计和克隆用于敲低的寡核苷酸;描述寡核苷酸处理和慢病毒载体产生过程的流程图如图1所示。


    图1.慢病毒质粒的构建 A.

    的图示。 leucopus STAT1显示击倒目标区域。 B.用shRNA产生慢病毒表达载体的程序工作流程。 leucopus STAT1。

    1. 将完整序列上传到Invitrogen引物设计软件(或同等产品),并生成shRNA设计的寡核苷酸候选物。基因开始,中间和末端的区域被定位以确保完整的转录物覆盖并避免脱靶效应。选择的候选人见表1和图1A。

      表1.用于基因敲低的寡核苷酸序列设计用于靶向3个不同区域的shRNA顶部和底部链序列。 leucopus STAT1基因。
      在引物顺序中没有指定特殊条件

    2. 作为对照,设计非特异性寡核苷酸以产生不会导致敲低的慢病毒载体。在我们的研究中使用了靶向GFP序列的shRNA用于此目的(参见表1)。由于GFP在感兴趣的细胞中不表达,所以对照细胞系应该与背景非转导细胞在转录上相当。
    3. 通过混合等比例的寡核苷酸(各200μM)并根据制造商的BLOCK-iT U6 RNAi进入载体试剂盒(赛默飞世尔科学(Thermo Fisher Scientific)的退火程序,在高温(95℃)下进行4分钟退火,从而退火顶部和底部链)。加热过程结束后,立即将样品转移到工作台上,使其冷却缓慢。这个过程会产生双链shRNA。
    4. 为了确认退火步骤,通过将等体积的每个样品(10μl)加入到单孔中并在100V下运行,将样品在4%琼脂糖凝胶(浸入1xTAE缓冲液)中以及100bp梯状物上解析。转移凝胶与单链样品相比较,透射仪显示双链寡核苷酸(见图1B)。
    5. 双链通过琼脂糖凝胶电泳确认后,进行退火的样品,并将产生的shRNA连接到U6载体中以产生用于进一步克隆的进入载体。 U6载体包含在BLOCK-iT U6 RNAi入门载体试剂盒(赛默飞世尔科技)
    6. 将入门载体重组到慢病毒目的载体中,可将载体送入哺乳动物细胞。在该研究中,根据制造商的方案,我们利用Gateway LR克隆酶II酶混合物进行Gateway克隆的pLENTI X2-DEST载体(Campeau等人,2009),除了允许重组过夜。
    7. 化学胜任的One Shot TM TOP10化学胜任力E用重组DNA提取大肠杆菌细胞并选择菌落进行DNA提取以获得纯的质粒DNA。

  2. 慢病毒的产生
    1. 在6孔培养皿中以每孔300万个细胞的量培养293T细胞,过夜培养。
    2. 用纯化的慢病毒质粒转染靶向感兴趣基因的个别区域和非特异性(GFP)序列的细胞。重要的是,将包含1μg靶向每个区域的慢病毒载体的DNA池包含在单个转染管中。因此,准备一个shRNA针对每个感兴趣的区域和一个管所有的shRNA候选人汇集在一起的一个管。
    3. 添加1微克ViraPower慢病毒包装混合物与质粒DNA,并使用Lipofectamine 3000转染试剂盒根据制造商的协议完成转染。慢慢地吸到细胞上以避免分离。
    4. 在转染后24小时,用含有10mM丁酸钠的新鲜DMEM替换转染培养基,在我们的研究中,2ml总体积适用于6孔培养皿。
    5. 将细胞再孵育48小时,不扰动培养皿。
    6. 收集含有新产生的慢病毒的上清液。使用Lenti-X GoStix进行快速检测(使用20μl样品上清液,然后使用3滴试剂盒运行缓冲液并留出足够的条带),以确认慢病毒的存在。慢病毒上清液没有过滤。
    7. 将慢病毒储存在-80°C直到需要。

  3. P的转导leucopus细胞与表达STAT1 shRNA的慢病毒
    1. 设置 P。 leucopus成纤维细胞每孔300,000个细胞在6孔培养皿中孵育过夜。
    2. 用1ml慢病毒原液感染细胞,加入8μgpolybrene(用无菌水配制的原液稀释)。
    3. 在转导后1小时,再加入1ml含有8μg聚凝胺的培养基。 24小时后,重新转导细胞,并用8μgpolybrene治疗。
    4. 第2天,取出慢病毒接种物,并用新鲜培养基替换。
    5. 在转导后第3天,加入适当的抗生素选择物(400μg/ ml潮霉素用于白色念珠菌成纤维细胞)。
    6. 维持细胞选择1周,然后进行亚克隆,通过将单细胞分裂到48孔培养皿的每个孔中来获得克隆细胞,并在1-2周内观察单个细胞的细胞增殖情况。
    7. 将克隆细胞扩大成大瓶(175 cm 2),然后进行基因敲低试验。

  4. 在转导的细胞系中评估基因敲低
    我们的研究已经证明STAT1表达在上调。用IFN-β处理后的白血病(Izuogu等人,2017)。因此,为了确定感兴趣的基因是否被敲除,将细胞用IFN处理并探测基因诱导。
    1. 设置STAT1击倒(KD)和NS KD leucopus成纤维细胞每孔1,000,000个细胞并孵育过夜。
    2. 用1,000国际单位(IU)小鼠干扰素β(mIFN-β)处理细胞(或不),8小时,收获样品以测试敲低,如下所示:
      通过SDS-PAGE和蛋白质印迹
      1. 去除细胞上清液,并用PBS缓冲液洗涤细胞2次。
      2. 加入500μL的RIPA缓冲液(见食谱),并轻轻地刮细胞单层。吸取几次,并将样品转移到Eppendorf管。
      3. 用50μl1x DNA酶I缓冲液(见配方)在37°C孵育30分钟。
      4. 以最大速度离心10分钟,收集上清液并与6x SB混合以达到1x SB(参见食谱)。在10%SDS凝胶上加载等量的蛋白质,并在150V下解析1.5小时。
      5. 电泳步骤后,使用iBLOT转移装置将蛋白转移至PVDF膜7分钟。
      6. 用蛋白质印迹封闭液孵育膜(见食谱),同时在室温(RT)下摇动1小时。
      7. 在摇床上用4℃的特异性抗体对STAT1和磷酸化STAT1(STAT1-P)进行16小时探针印迹并在PBST中洗涤3次5分钟。
      8. 用与HRP结合的二抗孵育,同时在室温摇动1小时。在PBST中洗涤3次10分钟。
      9. 用PBS进行最后的洗涤5分钟,用ECL-plus试剂孵育膜另外5分钟,并利用自动化的膜处理器来显现蛋白质表达。图2A显示了STAT1KD的蛋白质印迹结果。

      通过qRT-PCR
      1. 除去细胞上清液,并在PBS中洗涤细胞2次。根据RNA提取方案收获细胞裂解物。在本研究中,将细胞收集在350μl细胞裂解缓冲液(RLT缓冲液包括RNeasy微型试剂盒)中,并使用RNeasy微型试剂盒根据制造商的方案进行RNA提取,包括使用不含RNA酶的DNA酶组的DNase处理步骤(QIAGEN)。
      2. 量化RNA并使浓度标准化以确保所有样品之间的RNA量相等。
      3. 使用等量的RNA模板进行cDNA合成(使用1μg)。我们的研究根据制造商的协议使用QuantiTect反转录试剂盒。
      4. 用PCR级别的水稀释1/8的样品,并将3μl的每个样品加到干净,不透明的96孔板的各个孔中。每个Fwd加1μl。和Rev.引物总共5μl;用于检测P的引物序列。 Leucopus STAT1显示在表2中。在该研究中,使用FastStart Essential Green Master酶混合物,将5μl加入到每个反应样品中。因此,总反应体积为10μl。

        表2.用于探测STAT中的STAT1和β-肌动蛋白的qRT-PCR引物。 leucopus 细胞


      5. 在Lightcycler上运行50个周期并分析结果数据。将NS对照中STAT1的倍数诱导设定为100%,并且与NS细胞相比,评估KD细胞中的倍数诱导。所有的数据都标准化为β-肌动蛋白。图2B显示了相对的STAT1 mRNA表达。


        图2.在转导的细胞系中评估基因敲低A. Western印迹的流程图和数据显示在STAT中STAT1的表达减少。表达shRNA至位置261(STAT1KD#1),630(STAT1KD#2)和全部3个位置-261,630和1,742(STAT1KD全部)的Leucopus KD细胞。所有进一步的实验用“STAT1 KD All”细胞进行,并且以后称为STAT1 KD细胞。用抗STAT1,STAT1-P和β-肌动蛋白的抗体探测样品。 B.qRT-PCR的流程图和数据显示与NS对照相比,STATIKD细胞中相对STAT1 mRNA表达的百分比。星号表示:**** = P 0.00001。

  5. 在STAT1 KD细胞中进行干扰素应答的功能检测
    观察到敲低后,我们测试了STAT1KD对ISG上调的影响。我们实验室的数据已经鉴定了TBFV限制性因子 - 三联基序蛋白79(TRIM79)的同系物,并且也证明它是一种ISG。 (Leucopus)细胞(Taylor等人,2011)。我们测定了P的诱导倍数。根据以下方案在IFN处理后的STAT1KD细胞中的TRIM79(plTRIM79)
    1. 按照程序D设置STAT1KD和NS对照细胞,用mIFN-β处理8小时,然后按照qRT-PCR部分所述进行收获RNA。
    2. 使用等量的RNA作为cDNA合成的模板,并使用基因特异性引物进行qRT-PCR反应。相对于未处理的细胞评估倍数诱导。我们还比较了pTRIM79在

      中的诱导。使用与上述相同的技术产生的具有特异性慢病毒敲低的I型干扰素受体(IFNAR1)的白细胞介素(leucopus)细胞。数据显示在图3中。


      图3. STAT1 KD细胞中的ISG plTRIM79相对于NS对照的qRT-PCR。具有改变的STAT1(和IFNAR1)表达的细胞显示出显着更少的ISG诱导,表明敲减对IFN信号传导具有功能性影响。平均值±SD。星号表示:** = 0.01,*** = 0.001。

  6. 测试STAT1 KD对LGTV复制的影响
    <免疫荧光显微镜
    1. 在8孔Labtek载玻片上以每孔50000个细胞建立STAT1 KD和NS细胞并孵育过夜。
    2. 用LGTV以100的MOI感染STAT1 KD和NS细胞。允许1小时的吸收期并用500μl新鲜培养基代替接种物。
    3. 在感染72小时后,取出上清液,用PBS洗涤细胞2次,并在4%多聚甲醛溶液中固定20分钟。添加刚刚足以覆盖幻灯片(约5毫升)。
    4. 用PBST洗2次,透化细胞5分钟。去除透化溶液(见食谱),并在室温下加入3毫升免疫荧光测定封闭液(见食谱)1小时。本研究使用LGTV包膜(E)和LGTV非结构蛋白3(NS3)蛋白的特异性抗体,稀释度为1:2,000。
    5. 用PBS洗涤3次,并在室温下在黑暗中与用3ml封闭溶液稀释的合适的荧光二次抗体孵育1小时。将细胞清洗3次,置于不透光的容器中避光。
    6. 添加含有DAPI的封片 - 本研究使用了1ml含有DAPI的ProLong金防褪色剂,并在Olympus共聚焦显微镜上成像。图4A包括显示STAT1KD细胞中增加的病毒蛋白染色的共焦图像数据。

    通过病毒滴定和免疫焦点测定

    1. 在24孔培养皿中以每孔10万个细胞建立STAT1 KD和NS细胞并孵育过夜
    2. LGTV以10的感染复数(MOI)感染细胞,使得单层中的每个细胞将有10个病毒粒子。这是作为细胞密度,期望的MOI和孔数量除以病毒滴度的乘积来计算的,以确定感染所需的病毒储存量。用250μl的病毒溶液接种细胞,并允许1小时的吸收期,并用500μl的新鲜培养基代替接种物。
    3. 收集感染后24,48和72小时的病毒上清液一式三份,并储存在-80°C。
    4. 在24孔培养皿中以每孔200,000个细胞建立Vero细胞并孵育过夜。
    5. 使用1.2ml试管,在无FBS的DMEM培养基中制备6个1/10系列稀释的每种病毒上清液样品,并将250μl稀释至新鲜的Vero细胞。允许1小时的吸收期,并用500μl含有2%FBS和0.8%甲基纤维素(培养覆盖培养基)的新鲜培养基代替接种物。
    6. 在感染后4天,进行如前所述的免疫焦点测定(Taylor等人,2011; Baker等人,2013)。简而言之,删除文化覆盖媒体(见食谱),并用PBS洗细胞2次。用100%甲醇固定30分钟,再用PBS清洗。
    7. 在Opti-MEM培养基中孵育20分钟,并随后使用病毒特异性抗体。在这项研究中,用于检测LGTV包膜(E)蛋白的抗体以1:1,000使用。在密封容器中37°C孵育1小时。
    8. 用PBS清洗细胞2次,并与适当的二抗孵育。本研究中使用山羊抗小鼠IgG抗体为1:1,000。在第一抗体中孵育,然后用2次PBS洗涤。
    9. 添加底物,让15分钟的焦点发展。确定每毫升病毒上清液中聚焦形成单位(FFU)的数量,并比较每种细胞类型和条件下的病毒释放。
      对LGTV感染的STAT1 KD结果如图4B所示
      “”
      图4.确定STAT1KD对于白纹伊蚊LGTV感染的影响A.可视化LG1蛋白在STAT1 KD白斑白斑中的染色细胞系通过免疫荧光显微镜检查。在放大40倍的Olympus显微镜上捕获图像,显示LGTV E(绿色),NS3(红色)和细胞核用DAPI(蓝色)染色。 B.在 P中量化LGTV复制。通过进行免疫焦点测定评估白血病STAT1 KD细胞。该测定包括将感染的克隆细胞的上清液滴定到新鲜的Vero细胞上,并定量所得到的病灶。病毒滴度表示为每ml的焦点形成单位(ffu)。平均值±SD;数据来自三次独立实验,一式三份。星号表示:* = P 0.05,*** = 0.001,**** = P 0.00001。

数据分析

所有滴定实验一式三份进行三次。使用GraphPad Prism 6软件通过非配对t检验或Mann-Whitney U检验对数据进行分析,如Izuogu et al。,2017( http://journals.plos.org/plosone/article?id=10.1371/journal.pone 0.0179781 )。

笔记

  1. 取决于应用,cDNA样品可以以更高的稀释度用于qRT-PCR。
  2. 当退火寡核苷酸,而不是将样品管转移到工作台,从加热系统转移整个热块 - 这将允许以非常低的速度进行冷却。
  3. Vero细胞是具有干扰素分泌缺陷的非洲绿猴细胞,因此适合通过滴定计数病毒。
    他们也形成了焦点和斑块

食谱

  1. 放射免疫沉淀试验(RIPA)缓冲液
    50mM Tris pH 7.5(100ml 1M)
    150 mM NaCl(60 ml 5 M)
    0.5%脱氧胆酸(20克)
    0.1%SDS(10ml 20%)
    1%NP-40(20毫升IGEPAL CA-630)
    dH 2 O(最多2升)
  2. DNase I缓冲液(10x)
    100mM Tris-HCl pH 7.5
    25mM MgCl 2•/ 2 5mM CaCl 2 2/2
  3. 过氧化物酶底物(新鲜制备)
    0.4毫克/毫升3,3-二氨基联苯胺盐酸盐
    0.45μl/ ml 30%H 2 O 2/2 1x PBS
  4. 6x SDS-PAGE样品缓冲液(6x SB)
    375mM Tris-HCl,pH 6.8 9%SDS
    50%甘油
    0.03%溴酚蓝
    9%β-巯基乙醇,新鲜
  5. Western blot阻断解决方案
    PBST的5%脱脂奶粉
  6. 文化覆盖媒体
    补充有0.8%甲基纤维素和2%FBS的完全DMEM
  7. 透化解决方案
    0.1%Triton X-100和0.1%柠檬酸钠在PBS中
  8. 免疫荧光试验封闭液
    PBS中的0.5%BSA和1%山羊血清

致谢

这项工作得到了莱姆病协会和托莱多大学医学与生命科学启动基金(RTT)的试点资助。本文所示的方案和代表性结果是根据我们公开的工作(Taylor等人,2011; Izuogu等人,2017)改编的。我们感谢Jason Munshi-South博士和Stephen Harris博士在产生用于解析leucopus 中STAT1序列的初始核苷酸命中的帮助。我们宣布没有冲突或竞争的利益。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Izuogu, A. O. and Taylor, R. T. (2017). Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses. Bio-protocol 7(23): e2643. DOI: 10.21769/BioProtoc.2643.
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