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Mar 2018

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Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry
分支DNA FISH技术联合共聚焦显微镜或流式细胞术检测和鉴别多样的病毒RNA    

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Abstract

Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be achieved through dominant suppression of drug-resistant viruses by their drug-susceptible parents. We have explored the existence of dominant drug targets in poliovirus, dengue virus and hepatitis C virus (HCV). The low replication capacity of HCV required the development of novel strategies for identifying cells co-infected with drug-susceptible and drug-resistant strains. To monitor co-infected cell populations, we generated codon-altered versions of the JFH1 strain of HCV. Then, we could differentiate the codon-altered and wild-type strains using a novel type of RNA fluorescent in situ hybridization (FISH) coupled with flow cytometry or confocal microscopy. Both of these techniques can be used in conjunction with standard antibody-protein detection methods. Here, we describe a detailed protocol for both RNA FISH flow cytometry and confocal microscopy.

Keywords: RNA flow cytometry (RNA流式细胞术), RNA FISH (RNA荧光原位杂交), Branched DNAs (分支DNA), HCV (HCV), Drug Resistance (耐药性), Genetic selection (遗传选择), Viral evolution (病毒进化)

Background

The barriers to development of antiviral drug resistance vary greatly depending on the compound used and the host or viral target chosen. RNA viruses have particularly low genetic barriers to the development of drug resistance as their polymerases have error rates as high as 10-4 to 10-5 misincorporations per nucleotide synthesized. This leads to exceptionally high genetic variability amongst progeny. However, the high level of diversity observed in RNA virus progeny does not always lead to high rates of genetic selection for progeny with increased fitness. This is often due to genetic dominance of drug-susceptible viruses that are present in the same cell as newly synthesized drug-resistant variants. Drug-resistant viral RNA must first be amplified and translated in its cell of origin, making newly synthesized drug-resistant viruses susceptible to dominant suppression by their drug-susceptible parents and cousins. We have coined the term “dominant drug targets” to describe viral targets with higher barriers to the development of antiviral drug resistance due to genetic dominance of drug-susceptible viruses. Study of genetic interactions and physical location of distinct viral genomes in the same cell required the development of the new technology described here.

To identify dominant drug targets for which tool antiviral compounds are available, we first generated drug-resistant viruses and built the mutations into an infectious cDNA clone. To test whether drug-resistant or drug-susceptible viruses were genetically dominant, we generated cells co-infected with drug-susceptible and drug-resistant viruses and then monitored selection from within them. In studies using poliovirus (Crowder and Kirkegaard, 2005; Tanner et al., 2014) and Dengue virus (Mateo et al., 2015) we were able to generate sufficiently high-titer virus stocks to perform coinfections at high multiplicities of infection and thus ensure that all cells in our cultures were coinfected. Recently, we expanded tests for dominance to hepatitis C virus (HCV) (van Buuren et al., 2018), for which high-titer stocks are often difficult to obtain, especially for drug-resistant variants that have reduced fitness. Therefore, when we co-infected Huh7.5.1 cells with two strains of HCV at multiplicities of infection of less than 1 PFU/cell, we generated four cell populations: co-infected cells, two types of singly infected cells and a significant population of uninfected cells. We needed to differentiate co-infected cells from the two types of singly infected cells and learn about genetic selection while doing so. To accomplish this, we were early adopters of the branched DNA (bDNA) technology originally developed by Affymetrix (now Thermo Fisher Scientific). This technology uses tiered DNA oligos to build a network of up to 8,000 fluorophores on each target RNA. This unique type of RNA fluorescent in situ hybridization (FISH) can be coupled with protein detection using standard antibody conjugation and detected using confocal microscopy (ViewRNA® Cell Plus Assay) and flow cytometry (PrimeFlowTM RNA Assay).

These bDNA FISH techniques first generate a series of target probes that bind the RNA of interest at adjacent sequences, but leave 3’ extensions of unique sequence to bind the pre-amplifier DNA that is complementary to two different probes. Cooperative binding of the pre-amplifier DNA to two probes increases the signal-to-noise ratio because any individual mistargeted probe cannot be amplified. Typically, twenty pairs of target probes are designed to bind the RNA of interest; this requires roughly 1,000 nucleotides of sequence space. Each of the twenty pre-amplifier DNAs is then bound by a series of amplifier DNAs, and then subsequently by a series of oligonucleotide-conjugated fluorophores. This process leads to the labeling of each individual target RNA with up to 8,000 fluorophores, sufficient to visualize individual RNAs by confocal microscopy. The PrimeFlow RNA Assay and ViewRNA Cell Plus Assay kits allow for simultaneous detection of three target RNAs. The available fluorophores for PrimeFlow are Alexa Fluor® 488, Alexa Fluor® 647 and Alexa Fluor® 750 and for ViewRNA are Alexa Fluor® 488, Alexa Fluor® 546 and Alexa Fluor® 647.

To apply this technology to dominant drug targeting in HCV, we needed to generate a strain of HCV with sufficient dissimilarity in its RNA sequence that we could differentiate it from wild-type viral RNA. To accomplish this, we generated three codon-altered versions of the JFH1 strain of HCV. Codon optimization algorithms available through GeneArt (Thermo Fisher Scientific) were used to design three approximately 1,000-nucleotide regions of the JFH1 genome that had altered codon usage but retained the same protein sequence. These codon-altered JFH1 strains all contained 200-250 synonymous mutations over the 1,000-nucleotide regions. Of these three strains, two demonstrated decreased fitness, likely due to disruption of RNA secondary structures required for viral replication (Pirakitikulr et al., 2016). The third strain, however, displayed growth kinetics that mimicked wild-type virus and could be used in co-infection experiments and differentiated from wild-type JFH1 using both RNA FISH and flow cytometry.

Materials and Reagents

  1. Pipette tips (with or without filter tips)
  2. Micro slides (VWR, catalog number: 48311-702)
  3. Micro cover glass (VWR, catalog number: 48380-046)
  4. GenePulser cuvettes, 4 mm (Bio-Rad Laboratories, catalog number: 1652088)
  5. BD FACS tubes (BD Falcon, catalog number: 352054)
  6. 12-well cell culture dish (e.g., Corning, Costar, catalog number: 3513)
  7. 10 cm tissue culture dish (e.g., Corning, catalog number: 430167)
  8. T150 tissue culture flask (e.g., Corning, catalog number: 430825)
  9. 15 ml conical centrifuge tube (e.g., AccuFlow, catalog number: EK-4020)
  10. 500 ml Rapid-Flow Filter Unit, 0.2 μm (Thermo Fisher Scientific, catalog number: 566-0020)
  11. Huh7.5.1 cells (Gift from Dr. Michael Gale Jr., University of Washington)
  12. PrimeFlowTM RNA Assay Kit (Thermo Fisher Scientific, catalog number: 88-18005-210) contains:
    1. Flow Cytometry Staining Buffer
    2. Fixation Buffer 1
    3. Permeabilization Buffer with RNase Inhibitors
    4. Fixation Buffer 2
    5. Wash Buffer
    6. Target Probe Diluent
    7. PreAmp Mix
    8. Amp Mix
    9. Label Probe Diluent
    10. 100x Label Probes
  13. ViewRNA® Cell Plus Assay Kit (Thermo Fisher Scientific, catalog number: 88-19000) contains:
    1. Fixation/Permeabilization Buffer
    2. Blocking/Antibody Diluent
    3. Fixative
    4. Probe Set Diluent
    5. Amplifier Diluent along with Pre-Amplifiers and Amplifiers
    6. Label Probe Diluent and Label Probes
    7. Wash Buffer
    8. PBS
    9. DAPI
  14. Target Probes (Thermo Fisher Scientific)
    1. Wild-type JFH1 (VF1-14301)
    2. Codon altered JFH1 (VF4-6000723)
  15. Permafluor Mounting Reagent (Thermo Fisher Scientific, catalog number: TA-030-FM)
  16. 0.05% Trypsin-EDTA (Thermo Fisher Scientific, Gibco, catalog number: 25300-054)
  17. XbaI and CutSmart Buffer (New England Biolabs, catalog number: R0145L)
  18. MEGAscript T7 Kit (Thermo Fisher Scientific, Invitrogen, catalog number: AMB1334-5)
  19. Trizol® Reagent (Thermo Fisher Scientific, Ambion, catalog number: 15596018)
  20. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106)
  21. Human AB Serum (Omega, catalog number: HS-20)
  22. Pen/Strep (Thermo Fisher Scientific, catalog number: 15140-122)
  23. Glutamax (Thermo Fisher Scientific, catalog number: 35050-061)
  24. Non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-050)
  25. DMEM (GE Healthcare, Hyclone, catalog number: SH30243.01)
  26. Fetal bovine serum (Omega, catalog number: FB-22)
  27. KCl
  28. CaCl2
  29. K2HPO4
  30. HEPES
  31. EDTA
  32. MgCl2
  33. Human serum media (see Recipes)
  34. 10% FBS media (see Recipes)
  35. CytoMix (see Recipes)

Equipment

  1. Pipettes (with or without filter tips)
  2. Ultrafine forceps (e.g., Excelta, catalog number: 5-SN)
  3. Modified BD FACScan (Scanford) or LSRII Flow Cytometer
  4. Bio-Rad GenePulser XCell
  5. Biosafety Cabinet (BSC)
  6. Incubator (VWR, model: Model 1565)
  7. Heat Block (e.g., Anodized Aluminum, see Figure 2)
  8. Leica SP8 Confocal Microscope (Leica Microsystems, model: Leica TCS SP8)
  9. Sorvall Centrifuge (e.g., Thermo Fisher Scientific, model: Legend RT plus)
  10. Heracell 150i CO2 Incubator (Thermo Fisher Scientific, model: HeracellTM 150i)
  11. -20 °C Freezer
  12. Refrigerator

Software

  1. FlowJo® v10.0
  2. Volocity v6.0 (PerkinElmer)
  3. Adobe Photoshop vCS4
  4. GraphPad Prism v7.0
  5. Microsoft Excel v16.0

Procedure

  1. Construction of codon altered sequences
    1. Roughly 1,000 nucleotides of RNA sequence are required to support the hybridization of twenty bDNA trees and 8,000 fluorophores. Targets that contain less than the full complement of bDNAs can still be detected by flow cytometry but require higher copy numbers to achieve the same resolution.
    2. For viral RNAs, when possible, scan the literature for any structural information available to determine which areas of the genome are the least likely to contain essential RNA secondary structures. If possible, also choose a region that has convenient cut sites for insertion of your codon-altered sequence. We chose to clone three codon-altered regions of the JFH1 genome because we anticipated decreased viability from some of the codon-altered strains.
    3. GeneArt is a product offered through Thermo Fisher Scientific and can be used to synthesize genes up to 9,000 bp in length (https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis.html). The GeneArt homepage offers several tools, including the gene optimizer tool. Use the gene optimizer algorithms to design codon-altered sequences with wild-type viral RNA sequence as your template. We submitted three regions of JFH1 that were all roughly 1,000 nucleotides in length and flanked by convenient cut sites. The optimizer tool was able to alter nearly 25% of nucleotides in all three cases.
    4. The newly synthesized sequence will arrive incorporated into a plasmid with a defined antibiotic-resistance marker. At this time, your codon-altered gene fragment can be subcloned into a plasmid that encodes the viral genome using restriction digestion and ligation.
    5. Target Probes that differentiated viral RNA sequences were designed and manufactured by Affymetrix (now Thermo Fisher Scientific) for use with both the ViewRNA and PrimeFlow platforms (Figure 1).


      Figure 1. Detection of target RNAs using branched DNA technology. Branched DNA technology for RNA detection can be coupled with confocal microscopy or flow cytometry. Target RNAs are first bound by pairs of Target Probes. Typically, twenty sets of target probe pairs are designed per target RNA. The Pre-Amplifier DNA only binds target probe pairs that are bound to target RNAs in the correct orientation; this greatly limits the signal to noise ratio. Pre-Amplifier DNAs are then bound by Amplifier DNAs and subsequently by Label Probes. This process results in the labeling of target RNAs by up to 8,000 fluorophores.

  2. Collection of codon-altered JFH1 virus stocks
    1. The pJFH1 plasmid encodes the full-length genome of the JFH1 strain of HCV. The wild-type plasmid and all codon-altered versions contain an XbaI cut site at the 3’ end of the genome. Digest 5 μg of plasmid DNA with 20 U of XbaI in the CutSmart Buffer provided in a final reaction volume of 25 μl. Incubate digestions at 37 °C for 2 h.
    2. Purify linearized DNA using the QIAquick PCR Purification Kit, as per manufacturer’s protocol.
    3. Using 1 μg of linearized plasmid as your template, perform in vitro transcription with the MEGAscript T7 kit to make full-length genomic viral RNA. Incubate in vitro transcription reaction at 37 °C for 6 h. The temperature and duration of this incubation can be altered for optimal yield of individual transcripts.
    4. Isolate synthesized viral RNA using Trizol as per the manufacturer’s protocol. Resuspend vRNA pellet in 50 μl of RNase-free water.
    5. Seed 107 Huh7.5.1 cells into a 10 cm tissue culture plate and incubate overnight.
    6. To electroporate 10 μg vRNA into 107 Huh7.5.1 cells to produce continuous HCV cultures:
      1. Wash Huh7.5.1 cells with 5 ml PBS.
      2. Add 2 ml of Trypsin and incubate at 37 °C for 5 min.
      3. Add 5 ml of 10% FBS media and harvest cell suspension into a 15 ml conical tube.
      4. Centrifuge cells at 400 x g for 4 min.
      5. Resuspend cell pellet in 5 ml of PBS.
      6. Centrifuge cells at 400 x g for 4 min.
      7. Resuspend cell pellet with 5 ml of CytoMix. Cytomix recipe can be found below under “Recipes”.
      8. Centrifuge cells at 400 x g for 4 min.
      9. Resuspend cell pellet in 400 μl of CytoMix and transfer to a 4 mm GenePulser cuvette.
      10. Mix 10 μg of viral RNA into cell suspension inside cuvette and gently pipet up and down to mix.
      11. Electroporate RNA-cell mixture using the Bio-Rad GenePulser XCell. Settings set to 950 μF capacitance, 270 V, ∞ resistance and 4 mm cuvette size.
      12. Allow cells to rest at room temperature for 10 min.
      13. Transfer electroporated cells to a fresh 10 cm culture dish with 10 ml of 10% FBS media (see Recipes).
    7. Culture electroporated cells for up to two weeks in 10% FBS media, passaging every 3-4 days as required. As you passage, expand the culture. Typically, cultures of 107 electroporated cells are expanded into either five or ten T150 flasks. This gives HCV time to spread and generates a culture with a higher percentage of cells that are infected and productively synthesizing progeny virus. Further expansion of cells to larger capacity can be done if needed.
    8. Convert JFH1 cultures to Human Serum Media (Steenbergen et al., 2013). Growth of HCV in human serum has two benefits. First, Huh7.5.1 cells differentiate and cease cell division, therefore trypsinization and biweekly passage are no longer required. Instead, virus containing cell supernatants can simply be collected biweekly and directly replaced with fresh medium. Second, growth in Human Serum Media increases viral yield by 10 to 100-fold.

  3. Simultaneous infection with two HCV strains and detection of co-infected cells with Prime-Flow
    1. The description of this protocol has been adapted from the PrimeFlow Assay user’s manual.
    2. Huh7.5.1 cells are seeded into 12-well plates at a density of 105 cells per well using 1 ml of 10% FBS media.
    3. In our hands, JFH1 cultured in human serum media can produce viral titers of 105-106 focus forming units (FFU) per ml. Infect Huh7.5.1 cells at a multiplicity of infection of one virus particle per cell with both wild-type and codon-altered JFH1. This often equates to roughly 1-2 ml of each virus preparation. A total volume of 4 ml can be used carefully in 12-well plates.
    4. Incubate infected cells in a CO2 incubator at 37 °C for 4-6 h. Following initial incubation, remove virus-containing media by aspiration. Replace media with fresh 10% FBS media and incubate infected cells for 72 h.
    5. Replace 10% FBS media with fresh 10% FBS media that either contains antiviral drugs or vehicle and incubate infected cells for 24-36 h.
    6. Aspirate off media containing antivirals or vehicle and wash cells with 1 ml PBS.
    7. Harvest infected cells by treating cells with 0.5 ml trypsin and incubating at 37 °C with CO2 for 5 min.
    8. Inhibit trypsin by adding 1 ml of 10% FBS media to each well. Harvest all cells and transfer to one of the 1.5 ml microfuge tubes supplied in the PrimeFlow Assay kit.
    9. Spin cells at 400 x g for 5 min.
    10. Aspirate off media and trypsin, being careful not to lose any cells. This is achieved by only aspirating down to the 100 μl marker on the side of the Eppendorf tube. Wash cells with 1 ml of Flow Cytometry Staining Buffer. Vortex and spin at 400 x g for 5 min.
    11. Aspirate Flow Cytometry Staining Buffer and fix cells using 1 ml of Fixation Buffer 1 at 4 °C for 30 min.
    12. Spin cells at 800 x g for 5 min.
    13. Resuspend cells in 1 ml of Permeabilization Buffer. Spin cells at 800 x g for 5 min. Repeat wash with Permeabilization Buffer 3 x.
    14. Aspirate final Permeabilization Buffer wash and resuspend cells in 1 ml of Fixation Buffer 2. Incubate cells in the dark at room temperature for 60 min.
    15. Spin cells at 800 x g for 5 min and resuspend in 1 ml of Wash Buffer.
    16. Repeat wash step.
    17. Dilute Target Probes in Target Probe Diluent at 1:20.
    18. Resuspend cells in 100 μl of the Target Probe mixture. Incubate at 40 ± 1 °C for 2 h. We use a heat block in our 40 °C incubator to increase heat conduction to the tubes and protect from large fluctuation in heat (Figure 2). This incubation can be extended from 2 h to overnight. Longer incubations periods allowed for all amplification steps, flow cytometry and data analysis to be completed the following day.


      Figure 2. 40 ± 1 °C incubator setup. Two heat blocks are stored in the incubator to regulate the temperature of RNA FISH flow cytometry samples. A thermometer is kept inside to confirm the digital temperature readings.

    19. Wash cells by adding 1 ml of Wash Buffer, vortex, and spin at 800 x g for 5 min.
    20. Repeat wash step.
    21. Resuspend cells in 100 μl of PreAmp Mix. Incubate at 40 ± 1 °C for 1.5 h.
    22. Wash cells by adding 1 ml of Wash Buffer, vortex, and spin at 800 x g for 5 min.
    23. Repeat wash step.
    24. Resuspend cells in 100 μl of Amp Mix. Incubate at 40 ± 1 °C for 1.5 h.
    25. Wash cells by adding 1 ml of Wash Buffer, vortex, and spin at 800 x g for 5 min.
    26. Repeat wash step.
    27. Prepare Label Probe mix by diluting Label Probes into the Label Probe Diluent at 1:100.
    28. Resuspend cells in 100 μl of Label Probe mix. Incubate at 40 ± 1 °C for 1 h.
    29. Wash cells by adding 1 ml of Wash Buffer, vortex, and spin at 800 x g for 5 min.
    30. Repeat wash step.
    31. Aspirate Wash Buffer leaving 100 μl of residual liquid to resuspend stained cells. Resuspend cells by pipetting up and down and transfer to a labeled BD FACS tube containing 250 μl of PBS.
    32. Analyze cells using a flow cytometer and FlowJo software (details below).

  4. Quantitation of RNA-protein colocalization using confocal microscopy
    1. Huh7.5.1 cells are plated on Micro Cover Glass inside 12-well tissue culture plates at a density of 105 cells per well one day prior to infection (Figure 3).


      Figure 3. Reagents setup for confocal microscopy. Huh7.5.1 cells are plated onto Micro Cover Glass within a 12-well tissue culture plate. These cells are infected following a 24 h incubation to allow cell adherence to the glass. Following infection, the cells are fixed, stained for protein and RNA using the ViewRNA Cell Plus Assay kit, all within the 12-well plate. The Micro Cover Glass is then carefully transferred to a Microslide spotted with PermaFluor/DAPI using fine forceps.

    2. Co-infect cells with wild type JFH1 and codon altered JFH1 at a multiplicity of infection equal to one virus per cell.
    3. At 6 h post infection, aspirate inoculum and replace with 1 ml of 10% FBS media.
    4. At 24 h post infection, wash cells 2 x with 1 ml of PBS.
    5. Add 400 μl of Fixation/Permeabilization Buffer to each well and incubate for 30 min at room temperature.
    6. Wash cells 3 x each with 800 μl of PBS.
    7. Overlay cells with 400 μl of Blocking/Antibody Diluent and incubate at room temperature for 20 min.
    8. Dilute primary antibody in 400 μl Blocking/Antibody Diluent as required. Overlay cells with antibody mixture and incubate at room temperature for 1 h.
    9. Wash cells three times with PBS.
    10. Dilute secondary antibody in 400 μl Blocking/Antibody Diluent as required. We use anti-mouse AlexaFluor-647 diluted at 1:200 for our experiment with HCV. Overlay cells with antibody mixture and incubate at room temperature for 1 h.
    11. Wash cells 3 x with PBS.
    12. Add 400 μl of Fixation Solution to each well and incubate in the dark at room temperature for 1 h.
    13. Wash cells 3 x with PBS.
    14. Dilute Target Probes1:100 in Target Probe Diluent.
    15. After the final wash, overlay cells with 400 μl of Target Probe mixture. Incubate at 40 ± 1 °C for 2 h.
    16. Wash cells 3 x with 800 μl Wash Buffer at room temperature.
    17. Dilute Pre-Amplifiers 1:25 in Amplifier Diluent.
    18. After the final wash, overlay cells with 400 μl of Pre-Amplifier mixture and incubate at 40 ± 1 °C for 1 h.
    19. Wash cells 3 x with 800 μl Wash Buffer at room temperature.
    20. Dilute Amplifiers 1:25 in Amplifier Diluent.
    21. After the final wash, overlay cells with 400 μl of Amplifier mixture and incubate at 40 ± 1 °C for 1 h.
    22. Wash cells 3 x with 800 μl Wash Buffer at room temperature.
    23. Dilute Label Probes 1:100 in Label Probe Diluent.
    24. After the final wash, overlay cells with 400 μl of Label Probe mixture and incubate at 40 ± 1 °C for 1 h.
    25. Wash cells 3 x with 800 μl Wash Buffer at room temperature.
    26. Dilute DAPI 1:100 in Permafluor mounting reagent.
    27. Spot 12.5 μl of Permafluor/DAPI mixture onto a Micro Slide.
    28. Using forceps, carefully remove stained Micro Cover Glass from the 12-well dish, dab on a Kimwipe to remove excess Wash Buffer and place “cells down” onto the drop of Permafluor/DAPI. Allow to harden for at least 4 h.
    29. Visualize cells using a confocal microscope. We use a Leica SP8 Confocal Microscope fitted with a White Light Laser.

Data analysis

Flow cytometry
We analyze all flow cytometry data using FlowJo software. Data are exported from the flow cytometer as individual .fcs files for each sample as well as a .wsp file for the entire experiment. We use FlowJo to open the .wsp file and can then access all .fcs files in the same analysis window. Once files are open in FlowJo data analysis proceeds as follows:


  1. Open your first sample and select forward scatter versus side scatter to view cells collected. Draw a gate around the healthy cells only so that any debris or dead cells are not included in your analysis.
  2. Within your healthy cell subgate, plot the two viral RNA fluorophores against one another. In our case this was typically Alexa Fluor 488 versus Alexa Fluor 750 which did not require compensation. If you are working with Alexa Fluor 657 and Alexa Fluor 750 you will need to run the compensation algorithm within FlowJo before further analysis.
  3. Once data are plotted and compensated, if needed, reset the axes to biexponential (Biex) which minimizes the uninfected cells and emphasizes the viral RNA-positive populations for clearer resolution.
  4. Draw quadrants that divide uninfected cells from the two singly infected cells and coinfected cells.
  5. The percentages from each population will be used to determine the dominance relationships between viral species. In the absence of drug, four cell populations will be visible. In the presence of drug, the cells singly infected with drug-susceptible virus will become uninfected and shift into the lower left quadrant. The singly infected drug-resistant virus will persist. The genetic outcome of the co-infected cells will determine their fate (Figure 4).


    Figure 4. Identification of co-infected cells by PrimeFlow RNA FISH. Huh7.5.1 cells were infected with JFH1-CA, JFH1-WT or co-infected at multiplicities of infection equal to one virus per cell with each virus. Infected cells were incubated for 72 h before labeling viral RNAs using PrimeFlow. Analysis and compensation was performed using FlowJo.


Confocal microscopy

  1. The Leica SP8 creates a file containing all images as a .lif file. The individual channels are exported as individual .tif files for image processing and figure construction (Figure 5).


    Figure 5. Analysis of viral RNA-protein colocalization using ViewRNA Cell Plus. Huh7.5.1 cells were co-infected with JFH1-WT and JFH1-CA on Micro Cover Glass for 72 h. Cells were stained for HCV core protein and both viral RNAs using the ViewRNA Cell Plus Assay. Quantification of colocalization was performed using Volocity software. Scale bars are 2.5 μm in length.

  2. The .lif file can also be opened using Volocity software created by PerkinElmer.
  3. Volocity has a spot-counting algorithm to determine how many puncta exist within each channel. We define a single punctum as larger than 0.1 μm2 and smaller than 0.25 μm2, and ask Volocity to break larger spots into individual units. Confirm that your size range is appropriate by giving a few cells an eye test. Does the number of puncta counted appear to be the same number that you can count by eye? You may need to adjust your maximum and minimum punctum sizes based on this test.
  4. We then ask Volocity to determine colocalization by counting how many spots on our Red channel shared at least 0.05 μm2 of “Mutual Space” with puncta the Green channel. The result is plotted as the total number of puncta that share mutual space between channels versus the total number of puncta in each channel.
  5. Determining colocalization between RNA and protein requires a separate algorithm as the proteins often not localize into discrete countable puncta. We therefore ask Volocity to determine how many of the viral RNA puncta “Touch” anywhere within the protein signal. We then graph the number of RNA puncta that touched protein versus the total number of RNA puncta in each cell.

Notes

  1. We prefer to use the swinging bucket Sorvall centrifuge for all spins for flow cytometry as the cell pellet accumulates at the bottom of the PrimeFlow assay kit-supplied microfuge tubes, which limits cell loss during the multiple-step procedure. However, it is possible to complete the protocol and limit cell loss using a traditional bench top, fixed-angle centrifuge, with careful supernatant removal.
  2. Simultaneous analysis of the Alexa Fluor 647 and Alexa Fluor 750 channels requires a high degree of compensation. To identify double-positive cells unambiguously, use Alexa Fluor 488 in combination with either of the other two channels.
  3. As PrimeFlow is often coupled with antibody staining, it should be noted that not all fluorophores survive the RNA staining protocol. Specifically, all PerCP fluorophores will be inactivated by this technique and should be avoided in panel design.
  4. Coverglass slips are very delicate and can easily break as they are being lifted out of the 12-well plate and placed onto the microslide. Using anything other than fine forceps makes this challenging. New students in our lab are encouraged to practice this technique using blank Micro Cover Glass in PBS prior to attempting a real experiment.

Recipes

  1. Human serum media
    2% human AB serum
    1x Pen/Strep
    1x glutamax
    1x non-essential amino acids
    DMEM
  2. 10% FBS media
    10% fetal bovine serum
    1x Pen/Strep
    1x glutamax
    1x non-essential amino acids
    DMEM
  3. CytoMix
    120 mM KCl
    0.15 mM CaCl2
    10 mM K2HPO4
    25 mM HEPES
    2 mM EDTA
    5 mM MgCl2
    Adjust pH to 7.6
    Filter through a 0.2 μm Rapid-Flow Filter Unit

Acknowledgments

We thank Drs. Yury Goltsev and Garry Nolan for advice on fluorescent cell sorting-based visualization of RNA, Affymetrix for the design and manufacturing of custom viral RNA probes, and Drs. Michael Gale Jr. and Ralf Bartenschlager for the generous donation of reagents.
  This work was supported by funding to KK from NIH U19AI109662 (Jeffrey Glenn, P.I.), an NIH Director's Pioneer Award and the Alison and Steve Krausz Innovation Fund. NvB was supported by the Canadian Institutes for Health Research NCRTP-HepC training program and the American Liver Foundation. The Cell Sciences Imaging Facility used for confocal microscopy was supported by ARRA award number 1S10OD010580 from the NCRR. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the National Institutes of Health.

Competing interests

The authors have no conflicts of interest or competing interests.

References

  1. Crowder, S. and Kirkegaard, K. (2005). Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat Genet 37(7): 701-709.
  2. Mateo, R., Nagamine, C. M. and Kirkegaard, K. (2015). Suppression of drug resistance in dengue virus. MBio 6(6): e01960-01915. 
  3. Pirakitikulr, N., Kohlway, A., Lindenbach, B. D. and Pyle, A. M. (2016). The coding region of the HCV genome contains a network of regulatory RNA structures. Mol Cell 62(1): 111-120. 
  4. Steenbergen, R. H., Joyce, M. A., Thomas, B. S., Jones, D., Law, J., Russell, R., Houghton, M. and Tyrrell, D. L. (2013). Human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a 1000-fold increase in HCV Japanese fulminant hepatitis type 1 titers. Hepatology 58(6): 1907-1917.
  5. Tanner, E. J., Liu, H. M., Oberste, M. S., Pallansch, M., Collett, M. S. and Kirkegaard, K. (2014). Dominant drug targets suppress the emergence of antiviral resistance. Elife 3: e03803.
  6. van Buuren, N., Tellinghuisen, T. L., Richardson, C. D. and Kirkegaard, K. (2018). Transmission genetics of drug-resistant hepatitis C virus. Elife 7: e32579.

简介

由于RNA依赖性RNA聚合酶的异常高突变率,感染性RNA病毒产生广泛的序列多样性,导致微生物世界中抗病毒药物抗性发展的一些最低障碍。我们以前发现,通过药物敏感的父母对抗药性病毒的显性抑制,可以实现更高的耐药性发展障碍。我们已经探索了脊髓灰质炎病毒,登革热病毒和丙型肝炎病毒(HCV)中主要药物靶标的存在。 HCV的低复制能力需要开发用于鉴定与药物敏感和耐药菌株共感染的细胞的新策略。为了监测共感染的细胞群,我们产生了HCV的JFH1株的密码子改变形式。然后,我们可以使用新型RNA荧光原位>杂交(FISH)与流式细胞术或共聚焦显微镜相结合来区分密码子改变的和野生型菌株。这两种技术都可以与标准抗体 - 蛋白质检测方法结合使用。在这里,我们描述了RNA FISH流式细胞仪和共聚焦显微镜的详细方案。

【背景】抗病毒药物耐药性的发展障碍根据所用化合物和所选宿主或病毒靶标而有很大差异。 RNA病毒对耐药性的发展具有特别低的遗传屏障,因为它们的聚合酶具有高达每个核苷酸合成的10 -4 至10 -5 错误掺入的错误率。这导致后代中极高的遗传变异性。然而,在RNA病毒后代中观察到的高水平多样性并不总是导致后代的遗传选择率高且适应性增加。这通常是由于药物敏感病毒的遗传优势,这些病毒存在于与新合成的耐药变体相同的细胞中。必须首先在其原始细胞中扩增和翻译耐药病毒RNA,使新合成的耐药病毒易受其药物敏感的亲本和表亲的显性抑制。我们创造了术语“显性药物靶标”来描述由于药物敏感病毒的遗传优势而具有更高的抗病毒药物抗性发展障碍的病毒靶标。研究同一细胞中不同病毒基因组的遗传相互作用和物理位置需要开发这里描述的新技术。

为了确定可获得工具抗病毒化合物的主要药物靶标,我们首先产生了耐药病毒并将突变构建到感染性cDNA克隆中。为了测试耐药性或药物敏感性病毒是否具有遗传优势,我们生成的细胞与药物敏感和耐药病毒共同感染,然后监测其中的选择。在使用脊髓灰质炎病毒(Crowder和Kirkegaard,2005; Tanner et al。>,2014)和登革热病毒(Mateo et al。>,2015)的研究中,我们能够产生足够高的-titer病毒库在高多重感染时进行共感染,从而确保我们培养物中的所有细胞都被共感染。最近,我们扩大了对丙型肝炎病毒(HCV)的优势测试(van Buuren et al。>,2018),其中通常很难获得高滴度的种群,特别是对于耐药变种而言健身减少了。因此,当我们用低于1PFU /细胞的多重感染的两种HCV共感染Huh7.5.1细胞时,我们产生了四种细胞群:共感染的细胞,两种单独感染的细胞和一个显着的群体。未感染的细胞。我们需要区分共感染细胞和两种单一感染细胞,并在此过程中了解遗传选择。为实现这一目标,我们是最初由Affymetrix(现为赛默飞世尔科技公司)开发的分支DNA(bDNA)技术的早期采用者。该技术使用分层DNA寡核苷酸在每个靶RNA上构建多达8,000个荧光团的网络。这种独特类型的RNA荧光原位>杂交(FISH)可与标准抗体偶联的蛋白质检测结合使用,并使用共聚焦显微镜检测(ViewRNA ® Cell Plus Assay)和流动细胞计数(PrimeFlow TM RNA测定)。

这些bDNA FISH技术首先产生一系列靶探针,它们在相邻序列上结合目的RNA,但留下3'独特序列的延伸以结合与两种不同探针互补的前置放大DNA。前置放大器DNA与两个探针的协同结合增加了信噪比,因为任何单个错误定位的探针都不能被扩增。通常,设计20对靶探针以结合目的RNA;这需要大约1,000个核苷酸的序列空间。然后,二十个前置放大器DNA中的每一个被一系列放大器DNA结合,然后被一系列寡核苷酸 - 缀合的荧光团结合。该过程导致用多达8,000个荧光团标记每个单独的靶RNA,足以通过共聚焦显微镜观察单个RNA。 PrimeFlow RNA Assay和ViewRNA Cell Plus Assay试剂盒可同时检测三种靶RNA。 PrimeFlow的可用荧光团是Alexa Fluor ® 488,Alexa Fluor ® 647和Alexa Fluor ® 750和ViewRNA是Alexa Fluor ® 488,Alexa Fluor ® 546和Alexa Fluor ® 647。
为了将这项技术应用于HCV中的显性药物靶向,我们需要产生一种HCV病毒株,其RNA序列具有足够的不同性,我们可以将其与野生型病毒RNA区分开来。为此,我们产生了三种密码子改变的HCV JFH1株。通过GeneArt(Thermo Fisher Scientific)获得的密码子优化算法用于设计JFH1基因组的三个大约1,000个核苷酸区域,其具有改变的密码子使用但保留相同的蛋白质序列。这些密码子改变的JFH1菌株在1,000个核苷酸区域中都含有200-250个同义突变。在这三种菌株中,有两种表现出适应性降低,可能是由于病毒复制所需的RNA二级结构的破坏(Pirakitikulr et al。>,2016)。然而,第三种菌株显示出模拟野生型病毒的生长动力学,可用于共感染实验,并使用RNA FISH和流式细胞仪分离野生型JFH1。

关键字:RNA流式细胞术, RNA荧光原位杂交, 分支DNA, HCV, 耐药性, 遗传选择, 病毒进化

材料和试剂

  1. 移液器吸头(带或不带过滤器吸头)
  2. 微型载玻片(VWR,目录号:48311-702)
  3. 微盖玻片(VWR,目录号:48380-046)
  4. GenePulser比色皿,4 mm(Bio-Rad Laboratories,目录号:1652088)
  5. BD FACS灯管(BD Falcon,目录号:352054)
  6. 12孔细胞培养皿(例如>,Corning,Costar,目录号:3513)
  7. 10厘米组织培养皿(例如>,Corning,目录号:430167)
  8. T150组织培养瓶(例如>,Corning,目录号:430825)
  9. 15毫升锥形离心管(例如>,AccuFlow,目录号:EK-4020)
  10. 500 ml快速流量过滤器,0.2μm(Thermo Fisher Scientific,目录号:566-0020)
  11. Huh7.5.1细胞(来自华盛顿大学Michael Gale Jr.博士的礼物)
  12. PrimeFlow TM RNA检测试剂盒(赛默飞世尔科技,目录号:88-18005-210)包含:
    1. 流式细胞仪染色缓冲液
    2. 固定缓冲液1
    3. 具有RNase抑制剂的透化缓冲液
    4. 固定缓冲液2
    5. 洗涤缓冲液
    6. 目标探针稀释剂
    7. PreAmp Mix
    8. Amp Mix
    9. 标签探针稀释液
    10. 100x标签探针
  13. ViewRNA ® Cell Plus检测试剂盒(赛默飞世尔科技,目录号:88-19000)包含:
    1. 固定/透化缓冲液
    2. 阻断/抗体稀释剂
    3. 固色剂
    4. 探针组稀释剂
    5. 放大器稀释剂以及前置放大器和放大器
    6. 标签探针稀释剂和标签探针
    7. 洗涤缓冲液
    8. PBS
    9. DAPI
  14. 目标探针(赛默飞世尔科技)
    1. 野生型JFH1(VF1-14301)
    2. 密码子改变了JFH1(VF4-6000723)
  15. Permafluor安装试剂(Thermo Fisher Scientific,目录号:TA-030-FM)
  16. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco,目录号:25300-054)
  17. XbaI和CutSmart缓冲液(New England Biolabs,目录号:R0145L)
  18. MEGAscript T7试剂盒(Thermo Fisher Scientific,Invitrogen,目录号:AMB1334-5)
  19. Trizol ®试剂(Thermo Fisher Scientific,Ambion,目录号:15596018)
  20. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  21. Human AB Serum(Omega,目录号:HS-20)
  22. 笔/链(Thermo Fisher Scientific,目录号:15140-122)
  23. Glutamax(赛默飞世尔科技,目录号:35050-061)
  24. 非必需氨基酸(赛默飞世尔科技,目录号:11140-050)
  25. DMEM(GE Healthcare,Hyclone,目录号:SH30243.01)
  26. 胎牛血清(Omega,目录号:FB-22)
  27. 氯化钾
  28. 氯化钙<子> 2
  29. ķ<子> 2 HPO <子> 4
  30. HEPES
  31. EDTA
  32. 的MgCl <子> 2
  33. 人血清介质(见食谱)
  34. 10%FBS媒体(见食谱)
  35. CytoMix(见食谱)

设备

  1. 移液器(带或不带过滤嘴)
  2. 超细镊子(例如>,Excelta,目录号:5-SN)
  3. 改良的BD FACScan(Scanford)或LSRII流式细胞仪
  4. Bio-Rad GenePulser XCell
  5. 生物安全柜(BSC)
  6. 孵化器(VWR,型号:型号1565)
  7. 加热块(例如>,阳极氧化铝,参见图2)
  8. Leica SP8共聚焦显微镜(Leica Microsystems,型号:Leica TCS SP8)
  9. Sorvall Centrifuge(例如>,Thermo Fisher Scientific,型号:Legend RT plus)
  10. Heracell 150i CO 2 培养箱(Thermo Fisher Scientific,型号:Heracell TM 150i)
  11. -20°C冰箱
  12. 冰箱

软件

  1. FlowJo ® v10.0
  2. Volocity v6.0(PerkinElmer)
  3. Adobe Photoshop vCS4
  4. GraphPad Prism v7.0
  5. Microsoft Excel v16.0

程序

  1. 密码子改变序列的构建
    1. 需要大约1,000个核苷酸的RNA序列来支持20个bDNA树和8,000个荧光团的杂交。含有少于全部bDNA的靶标仍然可以通过流式细胞术检测,但需要更高的拷贝数以达到相同的分辨率。
    2. 对于病毒RNA,如果可能,扫描文献中可获得的任何结构信息,以确定基因组中哪些区域最不可能含有必需的RNA二级结构。如果可能,还可以选择一个区域,该区域具有方便的切割位点,用于插入密码子改变的序列。我们选择克隆JFH1基因组的三个密码子改变的区域,因为我们预期来自一些密码子改变的菌株的活力降低。
    3. GeneArt是Thermo Fisher Scientific提供的产品,可用于合成长度达9,000 bp的基因( https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-ntnthesis。 HTML )。 GeneArt主页提供了多种工具,包括基因优化工具。使用基因优化算法设计以野生型病毒RNA序列为模板的密码子改变序列。我们提交了三个JFH1区域,它们的长度大约为1,000个核苷酸,并且侧面是方便的切割位点。在所有三种情况下,优化工具能够改变近25%的核苷酸。
    4. 新合成的序列将到达具有确定的抗生素抗性标记的质粒中。此时,可以使用限制性消化和连接将密码子改变的基因片段亚克隆到编码病毒基因组的质粒中。
    5. 目标探针分化的病毒RNA序列由Affymetrix(现为赛默飞世尔科技)设计和制造,用于ViewRNA和PrimeFlow平台(图1)。


      图1.使用分支DNA技术检测目标RNA。用于RNA检测的分支DNA技术可与共聚焦显微镜或流式细胞仪结合使用。靶RNA首先被成对的靶探针结合。通常,每个靶RNA设计20组靶探针对。前置放大器DNA仅以正确的方向结合与靶RNA结合的靶探针对;这极大地限制了信噪比。然后通过放大器DNA结合前置放大器DNA,随后通过标签探针结合。该过程导致靶标RNA被多达8,000个荧光团标记。

  2. 收集密码子改变的JFH1病毒库存
    1. pJFH1质粒编码HCV的JFH1株的全长基因组。野生型质粒和所有密码子改变的版本在基因组的3'末端含有XbaI切割位点。在CutSmart缓冲液中用20U XbaI消化5μg质粒DNA,最终反应体积为25μl。在37°C孵育消化2小时。
    2. 根据制造商的方案,使用QIAquick PCR纯化试剂盒纯化线性化的DNA。
    3. 使用1μg线性化质粒作为模板,用MEGAscript T7试剂盒进行体外>转录,制备全长基因组病毒RNA。在37°C孵育体外>转录反应6小时。可以改变该温育的温度和持续时间以获得单个转录物的最佳产量。
    4. 按照制造商的方案,使用Trizol分离合成的病毒RNA。将vRNA沉淀重悬于50μl不含RNase的水中。
    5. 将10μlHh7.5.1细胞接种到10cm组织培养板中并孵育过夜。
    6. 将10μgvRNA电穿孔到10 7 Huh7.5.1细胞中以产生连续的HCV培养物:
      1. 用5ml PBS洗涤Huh7.5.1细胞。
      2. 加入2ml胰蛋白酶,在37℃下孵育5分钟。
      3. 加入5ml 10%FBS培养基并将细胞悬浮液收集到15ml锥形管中。
      4. 将细胞在400 x g >下离心4分钟。
      5. 将细胞沉淀重悬于5ml PBS中。
      6. 将细胞在400 x g >下离心4分钟。
      7. 用5ml CytoMix重悬细胞沉淀。 Cytomix配方可在“配方”下找到。
      8. 将细胞在400 x g >下离心4分钟。
      9. 将细胞沉淀重悬于400μlCytoMix中,转移至4 mm GenePulser比色杯中。
      10. 将10μg病毒RNA混合到比色杯内的细胞悬液中,轻轻地上下移液混合。
      11. 使用Bio-Rad GenePulser XCell电穿孔RNA细胞混合物。设置设置为950μF电容,270 V,∞电阻和4 mm比色杯尺寸。
      12. 让细胞在室温下静置10分钟。
      13. 将电穿孔的细胞转移到具有10ml 10%FBS培养基的新鲜10cm培养皿中(参见配方)。
    7. 在10%FBS培养基中培养电泳细胞长达两周,根据需要每3-4天传代一次。当你通过时,扩展文化。通常,将10 7个电穿孔细胞的培养物扩增到5或10个T150烧瓶中。这使得HCV有时间传播并产生具有更高百分比的被感染和有效合成子代病毒的细胞的培养物。如果需要,可以进一步将细胞扩增到更大的容量。
    8. 将JFH1培养物转化为人血清培养基(Steenbergen et al。>,2013)。人血清中HCV的生长有两个好处。首先,Huh7.5.1细胞分化并停止细胞分裂,因此不再需要胰蛋白酶消化和每两周一次的通过。相反,可以简单地每两周收集含有病毒的细胞上清液并直接用新鲜培养基替换。其次,人血清培养基的生长使病毒产量增加10至100倍。

  3. 用两种HCV菌株同时感染并用Prime-Flow检测共感染细胞
    1. 该协议的描述已经从PrimeFlow Assay用户手册改编而来。
    2. 使用1ml 10%FBS培养基,将Huh7.5.1细胞以10μl/孔细胞/孔的密度接种到12孔板中。
    3. 在我们的手中,在人血清培养基中培养的JFH1可以产生每毫升10μl/ sup> -10 6 焦点形成单位(FFU)的病毒滴度。用野生型和密码子改变的JFH1以每个细胞一个病毒颗粒的多重感染感染Huh7.5.1细胞。这通常相当于每种病毒制剂大约1-2毫升。在12孔板中可以小心地使用4ml的总体积。
    4. 将感染的细胞在CO 2 培养箱中于37°C孵育4-6小时。初始孵育后,通过抽吸除去含病毒的培养基。用新鲜的10%FBS培养基替换培养基并孵育感染的细胞72小时。
    5. 用含有抗病毒药物或载体的新鲜10%FBS培养基替换10%FBS培养基,并孵育感染细胞24-36小时。
    6. 吸出含有抗病毒药物或载体的培养基,用1 ml PBS洗涤细胞。
    7. 通过用0.5ml胰蛋白酶处理细胞并在37℃下用CO 2 温育5分钟来收获感染的细胞。
    8. 通过向每个孔中加入1ml 10%FBS培养基来抑制胰蛋白酶。收集所有细胞并转移至PrimeFlow Assay试剂盒中提供的1.5 ml微量离心管中的一个。
    9. 在400 x g >下旋转细胞5分钟。
    10. 吸出培养基和胰蛋白酶,注意不要丢失任何细胞。这是通过仅吸入Eppendorf管侧面的100μl标记来实现的。用1ml流式细胞仪染色缓冲液洗涤细胞。涡旋并在400 x g >下旋转5分钟。
    11. 吸出流式细胞仪染色缓冲液并使用1 ml固定缓冲液1在4°C下固定细胞30分钟。
    12. 在800 x g >下旋转细胞5分钟。
    13. 将细胞重悬于1ml Permeabilization Buffer中。在800 x g >下旋转细胞5分钟。用Permeabilization Buffer 3 x重复洗涤。
    14. 吸出最终的透化缓冲液洗涤并将细胞重悬于1ml固定缓冲液2中。在室温下在黑暗中孵育细胞60分钟。
    15. 将细胞在800 x g >旋转5分钟并重悬于1ml洗涤缓冲液中。
    16. 重复洗涤步骤。
    17. 1:20在目标探针稀释液中稀释目标探针。
    18. 将细胞重悬于100μl靶探针混合物中。在40±1°C孵育2小时。我们在40°C培养箱中使用加热块来增加管子的热传导,并防止热量的大幅波动(图2)。该孵育可以从2小时延长至过夜。更长的孵育期允许所有扩增步骤,流式细胞仪和数据分析在第二天完成。


      图2. 40±1°C培养箱设置将两个加热块储存在培养箱中以调节RNA FISH流式细胞仪样品的温度。内置温度计以确认数字温度读数。

    19. 通过加入1ml洗涤缓冲液洗涤细胞,涡旋,并在800 x g下旋转5分钟。
    20. 重复洗涤步骤。
    21. 将细胞重悬于100μlPreAmpMix中。在40±1°C孵育1.5小时。
    22. 通过加入1ml洗涤缓冲液洗涤细胞,涡旋,并在800 x g下旋转5分钟。
    23. 重复洗涤步骤。
    24. 将细胞重悬于100μlAmpMix中。在40±1°C孵育1.5小时。
    25. 通过加入1ml洗涤缓冲液洗涤细胞,涡旋,并在800 x g下旋转5分钟。
    26. 重复洗涤步骤。
    27. 通过将标签探针以1:100稀释到标签探针稀释液中来制备标记探针混合物。
    28. 将细胞重悬于100μl标签探针混合物中。在40±1°C孵育1小时。
    29. 通过加入1ml洗涤缓冲液洗涤细胞,涡旋,并在800 x g下旋转5分钟。
    30. 重复洗涤步骤。
    31. 吸出洗涤缓冲液,留下100μl残留液体重悬细胞。通过上下吸移重悬细胞并转移至含有250μlPBS的标记BD FACS管。
    32. 使用流式细胞仪和FlowJo软件分析细胞(详情如下)。

  4. 使用共聚焦显微镜定量RNA蛋白共定位
    1. 在感染前一天,将Huh7.5.1细胞接种在12孔组织培养板内的Micro Cover Glass上,每孔密度为10 5 细胞(图3)。


      图3.共聚焦显微镜的试剂设置。将Huh7.5.1细胞接种到12孔组织培养板内的Micro Cover Glass上。在孵育24小时后感染这些细胞以使细胞粘附到玻璃上。感染后,使用ViewRNA Cell Plus Assay试剂盒固定细胞,染色蛋白质和RNA,均在12孔板内。然后将Micro Cover Glass小心地转移到使用细镊子用PermaFluor / DAPI点样的Microslide上。

    2. 共感染具有野生型JFH1和密码子改变的JFH1的细胞,感染复数等于每个细胞一个病毒。
    3. 在感染后6小时,吸出接种物并用1ml 10%FBS培养基替换。
    4. 在感染后24小时,用1ml PBS洗涤细胞2次。
    5. 每孔加入400μl固定/透化缓冲液,在室温下孵育30分钟。
    6. 用800μlPBS洗涤细胞各3次。
    7. 用400μl封闭/抗体稀释液覆盖细胞,并在室温下孵育20分钟。
    8. 根据需要在400μl封闭/抗体稀释液中稀释一抗。用抗体混合物覆盖细胞并在室温下孵育1小时。
    9. 用PBS洗涤细胞三次。
    10. 根据需要在400μl阻断/抗体稀释液中稀释二抗。我们使用以1:200稀释的抗小鼠AlexaFluor-647进行HCV实验。用抗体混合物覆盖细胞并在室温下孵育1小时。
    11. 用PBS洗涤细胞3次。
    12. 每孔加入400μl固定液,在室温下避光孵育1小时。
    13. 用PBS洗涤细胞3次。
    14. 在Target Probe Diluent中稀释目标探针1:100。
    15. 最后一次洗涤后,用400μl靶探针混合物覆盖细胞。在40±1°C孵育2小时。
    16. 在室温下用800μl洗涤缓冲液洗涤细胞3次。
    17. 在放大器稀释剂中稀释前置放大器1:25。
    18. 最后一次洗涤后,用400μlPre-Amplifier混合物覆盖细胞,并在40±1℃下孵育1小时。
    19. 在室温下用800μl洗涤缓冲液洗涤细胞3次。
    20. 在Amplifier Diluent中稀释放大器1:25。
    21. 最后一次洗涤后,用400μl放大器混合物覆盖细胞,并在40±1℃下孵育1小时。
    22. 在室温下用800μl洗涤缓冲液洗涤细胞3次。
    23. 在Label Probe Diluent中稀释标签探针1:100。
    24. 最后一次洗涤后,用400μl标记探针混合物覆盖细胞,并在40±1℃下孵育1小时。
    25. 在室温下用800μl洗涤缓冲液洗涤细胞3次。
    26. 在Permafluor安装试剂中稀释DAPI 1:100。
    27. 将12.5μlPermafluor/ DAPI混合物置于微型载玻片上。
    28. 使用镊子,小心地从12孔培养皿中取出染色的Micro Cover Glass,轻拍Kimwipe以除去多余的洗涤缓冲液,并将“细胞”放在Permafluor / DAPI滴上。允许硬化至少4小时。
    29. 使用共聚焦显微镜观察细胞。我们使用配有白光激光的Leica SP8共聚焦显微镜。

数据分析

流式细胞仪>
我们使用FlowJo软件分析所有流式细胞仪数据。数据从流式细胞仪输出,作为每个样品的单个.fcs文件以及整个实验的.wsp文件。我们使用FlowJo打开.wsp文件,然后可以访问同一分析窗口中的所有.fcs文件。在FlowJo中打开文件后,数据分析过程如下:


  1. 打开第一个样本并选择前向散射与侧向散射以查看收集的单元格。仅在健康细胞周围画一个门,以便分析中不包含任何碎片或死细胞。
  2. 在您的健康细胞子门中,将两种病毒RNA荧光团彼此对照。在我们的案例中,这通常是Alexa Fluor 488而不是Alexa Fluor 750,不需要赔偿。如果您正在使用Alexa Fluor 657和Alexa Fluor 750,则需要在进一步分析之前在FlowJo中运行补偿算法。
  3. 绘制并补偿数据后,如果需要,将轴重置为双指数(Biex),使未感染细胞最小化,并强调病毒RNA阳性群体以获得更清晰的分辨率。
  4. 绘制象限,将两个单独感染的细胞和共感染细胞中的未感染细胞分开。
  5. 每个群体的百分比将用于确定病毒物种之间的优势关系。在没有药物的情况下,可以看到四个细胞群。在药物存在下,单独感染药物敏感病毒的细胞将变得未感染并转移到左下象限。单独感染的耐药病毒将持续存在。共感染细胞的遗传结果将决定它们的命运(图4)。


    图4.通过PrimeFlow RNA FISH鉴定共感染细胞。 Huh7.5.1细胞用JFH1-CA,JFH1-WT感染或共感染多次感染,每个细胞等于一个病毒。每种病毒。将感染的细胞孵育72小时,然后使用PrimeFlow标记病毒RNA。使用FlowJo进行分析和补偿。


共聚焦显微镜
>

  1. Leica SP8创建一个包含所有图像的文件作为.lif文件。各个通道作为单独的.tif文件导出,用于图像处理和图形构建(图5)。


    图5.使用ViewRNA Cell Plus分析病毒RNA-蛋白共定位。将Huh7.5.1细胞与微盖玻片上的JFH1-WT和JFH1-CA共感染72小时。使用ViewRNA Cell Plus Assay对细胞进行HCV核心蛋白和两种病毒RNA的染色。使用Volocity软件进行共定位的定量。比例尺长度为2.5微米。

  2. 也可以使用PerkinElmer创建的Volocity软件打开.lif文件。
  3. Volocity有一个点计数算法,用于确定每个通道中存在多少个点。我们将单个泪点定义为大于0.1μm 2 且小于0.25μm 2 ,并要求Volocity将较大的斑点分解为单个单位。通过给几个细胞进行眼睛测试,确认您的尺寸范围是合适的。点数的斑点数量是否与您通过眼睛计算的数量相同?您可能需要根据此测试调整最大和最小泪点大小。
  4. 然后我们通过计算我们的红色通道上有多少斑点与绿色通道的斑点共用至少0.05μm 2 的“相互空间”来要求Volocity确定共定位。将结果绘制为共享通道之间的相互间隔的puncta的总数与每个通道中的puncta的总数。
  5. 确定RNA和蛋白质之间的共定位需要单独的算法,因为蛋白质通常不会定位到离散的可数斑点中。因此,我们要求Volocity确定蛋白质信号中任何地方有多少病毒RNA点“触摸”。然后,我们绘制了接触蛋白质的RNA斑点数量与每个细胞中RNA斑点总数的关系图。

笔记

  1. 我们更倾向于使用摆动式铲斗Sorvall离心机进行流式细胞仪的所有旋转,因为细胞沉淀积聚在PrimeFlow检测试剂盒提供的微量离心管的底部,这可以限制多步骤过程中的细胞损失。然而,可以使用传统的台式固定角度离心机完成方案并限制细胞损失,同时小心去除上清液。
  2. 同时分析Alexa Fluor 647和Alexa Fluor 750通道需要高度补偿。要明确识别双阳性细胞,请将Alexa Fluor 488与其他两个通道中的任何一个结合使用。
  3. 由于PrimeFlow通常与抗体染色结合,应注意并非所有荧光团都能在RNA染色方案中存活。具体而言,所有PerCP荧光团都将通过这种技术失活,并且应该在面板设计中避免。
  4. 盖玻片非常脆弱,当它们被从12孔板中提起并放置在微型滑板上时很容易破裂。使用精细镊子以外的任何东西都会带来挑战。我们鼓励我们实验室的新学生在尝试真正的实验之前使用PBS中的空白微盖玻片来练习这项技术。

食谱

  1. 人体血清介质
    2%人类AB血清
    1x笔/ Strep
    1x glutamax
    1x非必需氨基酸
    DMEM
  2. 10%FBS媒体
    10%胎牛血清
    1x笔/ Strep
    1x glutamax
    1x非必需氨基酸
    DMEM
  3. CytoMix
    120 mM KCl
    0.15mM CaCl 2
    10mM K 2 HPO 4
    25 mM HEPES
    2 mM EDTA
    5mM MgCl 2
    将pH调节至7.6
    通过0.2μm快速流量过滤器单元过滤

致谢

我们感谢Drs。 Yury Goltsev和Garry Nolan就RNA的荧光细胞分选可视化提供建议,Affymetrix负责定制病毒RNA探针的设计和制造,以及Drs。 Michael Gale Jr.和Ralf Bartenschlager慷慨捐赠试剂。
&NBSP;这项工作得到了NIK U19AI109662(Jeffrey Glenn,P.I。),NIH董事先锋奖以及Alison和Steve Krausz创新基金的资助。 NvB得到了加拿大健康研究所NCRTP-HepC培训计划和美国肝脏基金会的支持。用于共聚焦显微镜的Cell Sciences Imaging Facility得到了NCRR的ARRA奖号1S10OD010580的支持。本手稿的内容完全由作者负责,并不一定代表NCRR或国立卫生研究院的官方观点。

利益争夺

作者没有利益冲突或利益冲突。

参考

  1. Crowder,S。和Kirkegaard,K。(2005)。 RNA病毒复制的反式显性抑制可以减缓耐药病毒的生长。 Nat Genet > 37(7):701-709。
  2. Mateo,R.,Nagamine,C。M.和Kirkegaard,K。(2015)。 抑制登革热病毒的耐药性。 MBio > 6 (6):e01960-01915。&nbsp;
  3. Pirakitikulr,N.,Kohlway,A.,Lindenbach,B。D.和Pyle,A。M.(2016)。 HCV基因组的编码区域包含调控RNA结构网络。 Mol Cell > 62(1):111-120。&nbsp;
  4. Steenbergen,R.H.,Joyce,M.A.,Thomas,B.S.,Jones,D.,Law,J.,Russell,R.,Houghton,M。和Tyrrell,D.L。(2013)。 人血清可导致人肝癌细胞分化,恢复极低密度脂蛋白分泌, HCV日本暴发性肝炎1型滴度增加1000倍。 Hepatology > 58(6):1907-1917。
  5. Tanner,E.J.,Liu,H.M.,Oberste,M.S.,Pallansch,M.,Collett,M。S.和Kirkegaard,K。(2014)。 显性药物靶点可抑制抗病毒药物的出现。 Elife > 3:e03803。
  6. van Buuren,N.,Tellinghuisen,T。L.,Richardson,C。D. and Kirkegaard,K。(2018)。 耐药性丙型肝炎病毒的传播遗传学。 Elife > 7:e32579。
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Copyright van Buuren and Kirkegaard. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. van Buuren, N. and Kirkegaard, K. (2018). Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry. Bio-protocol 8(20): e3058. DOI: 10.21769/BioProtoc.3058.
  2. van Buuren, N., Tellinghuisen, T. L., Richardson, C. D. and Kirkegaard, K. (2018). Transmission genetics of drug-resistant hepatitis C virus. Elife 7: e32579.
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