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Hepatitis C virus Cell-to-cell Spread Assay
丙型肝炎病毒在细胞之间的传播试验   

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Abstract

Hepatitis C virus (HCV) can infect naïve cells via entry of “cell-free” extracellular virus or direct “cell-to-cell” transmission. Here, we describe an assay for detecting HCV cell-to-cell transmission, using a non-growing cell culture system that avoids confounding effects of cell growth. The assay consists of infecting a small number of cells in a confluent monolayer and then blocking subsequent cell-free extracellular virions with a neutralizing antibody such that only cell-to-cell transmission may occur. Under these conditions, incubation at 37 °C results in the formation of infected cell foci. The extent of cell-to-cell spread can then be determined by counting the number of cells in each focus. The assay may be modified to assess the effects of inhibitors and/or specific cellular genes on cell-to-cell spread of HCV.

Part I. Main protocol: HCV cell-to-cell spread in non-dividing Huh7 cell cultures

Materials and Reagents

  1. Huh7 cells (from Dr. Francis Chisari, The Scripps Research Institute, La Jolla, CA) (Zhong et al., 2005)
  2. JFH-1 HCVcc (generated as previously described and quantified by limiting dilution titer assay) (Yu and Uprichard, 2010; Knipe and Howley, 2007)
    Note: Other infectious HCVcc clones can be used as well.
  3. Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, catalog number: MT-10-013-CV )
  4. Fetal bovine serum (FBS) (Hyclone, catalog number: SH30910.03 )
  5. Penicillin, streptomycin, L-glutamine (Mediatech, catalog number: MT-30-009-CI )
  6. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540 )
  7. 1x PBS (Mediatech, catalog number: MT- 21-030-CV )
  8. Hydrogen peroxide (H2O2) (Thermo Fisher Scientific, catalog number: H325 )
  9. AEC detection substrate (BD Biosciences, catalog number: 551015 )
  10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  11. Ezetimibe (Sequoia Research Products, catalog number: SRP04000e ) prepared in DMSO at 20 mM
  12. Rabbit anti-human CLDN1 polyclonal antibody (Abcam, catalog number: ab63070 )
  13. Rabbit anti-human NPC1L1 polyclonal antibody (Santa Cruz, catalog number: sc-67236 )
  14. Human anti-HCV E2 monoclonal antibody MAb AR3A (from Dr. Mansun Law, The Scripps Research Institute, La Jolla, CA) (Law et al., 2008)
  15. Mouse anti-HCV NS5A 9E10 monoclonal antibody (from Dr. Charles Rice, Rockefeller University, New York, NY) (Lindenbach et al., 2005)
  16. HRP-conjugated goat anti-human (Thermo Fisher Scientific, catalog number: 31410 ), goat anti-mouse (Thermo Fisher Scientific, catalog number: 31430 ), and goat anti-rabbit (Thermo Fisher Scientific, catalog number: 31460 ).
  17. Mouse IgG (Santa Cruz, catalog number: sc-2025 ), rabbit IgG (Santa Cruz, catalog number: sc-2027 )
  18. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  19. Triton X-100 (Sigma-Aldrich, catalog number: T-8787 )
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
  21. Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog number: 13778 )
  22. OptiMEM (Life Technologies, InvitrogenTM, catalog number: 31985-070 )
  23. 50 ml centrifuge tubes (Dot Scientific, catalog number: 451-PG )
  24. Complete DMEM (cDMEM) (see Recipes)

Equipment

  1. 96-well BioCoat collagen-coated tissue culture plates (Corning, catalog number: 356698 )
  2. BioCoat collagen-coated T75 cm2 flask (Corning, catalog number: 356485 )
  3. Inverted microscope
  4. Hemocytometer
  5. 37 °C, 5% CO2 cell culture incubator
  6. Table top centrifuge (e.g. Sorvall, model: T6000D or Eppendorf, model: 5810 R )

Procedure

  1. Preparation of non-growing Huh7 cell cultures
    1. Seed 6,000 Huh7 cells/well in BioCoat collagen-coated 96-well plate in 200 µl/well 10% FBS cDMEM.
    2. Incubate the plate at 37 °C in 5% CO2 cell culture incubator overnight or until the cells reach 95%-99% confluence.
    3. Aspirate out or decant plating media and replace with 200 µl/well of 10% FBS cDMEM containing 1% DMSO and continue to incubate cells at 37 °C in 5% CO2 cell culture incubator for 10 days changing the 1% DMSO, 10% FBS cDMEM every other day. (Media changes should be performed slowly with the pipette tip angled towards the walls of the wells). During the incubation period the cells remain metabolically active, but stop dividing (Sainz and Chisari, 2006). By day 10, each well contains approximately 65,000 cells.

  2. Initiation of infection
    1. Dilute HCVcc in 2% FBS cDMEM to 50-100 foci forming units (ffu)/100 µl.
    2. Add 100 µl of the virus inoculum to each well and incubate at 37 °C in 5% CO2 cell culture incubator overnight (~16 h) to allow initial virus entry.

  3. Blocking subsequent “cell-free” extracellular virus entry
    This step should be performed no later than 18 h post inoculation to prevent subsequent infection of cells by progeny virus released into the medium from the first round of infected cells.
    1. Remove viral inoculum at 16 h post-infection and rinse gently with 1x PBS.
    2. Add 10% FBS, 1% DMSO cDMEM containing 10 µg/ml of the HCV E2 antibody MAb AR3A to each well in a total volume of 150 µl /well to neutralize cell-free spread by HCVcc (Barretto et al., 2014; Timpe et al., 2008). (Additional factor-specific inhibitors can be added to parallel samples at this point to assess their effect on HCV cell-to-cell spread; see section F below.)
    3. Continue to incubate cultures at 37 °C in 5% CO2 cell culture incubator to allow time for cell-to-cell spread of HCV to occur (e.g. 48- 72 h post-infection).
    4. Before proceeding to Section D, collect all the culture medium from the sample wells and transfer it to a new 96-well plate for analysis of neutralization as described in section E below. (This can be assayed immediately or stored at -80°C until assay can be performed.)

  4. Visualizing HCV cell-to-cell spread via immunostaining
    Under these conditions, multicellular HCV foci can only be formed by cell-to-cell spread. Hence, the number of HCV-positive cells in individual HCV foci can be used as a readout for cell-to-cell transmission. Here immunostaining is described to visualizing HCV infected cells, but other methods can be used (e.g. Supplemental protocol A).
    1. Fix cells by adding an equal volume of 4% PFA to each well for a final concentration is 2% and incubate at RT for 25 min.
    2. Decant and rinse the cells three times with 1x PBS.
    3. Block endogenous peroxidases by incubating with 100 µl/well ice-cold 1x PBS containing 0.3% (v/v) hydrogen peroxide for 5 min at room temperature (RT).
    4. Decant and rinse 3 times with 1x PBS.
    5. Permeabilize and block cells for 1 h with 100 µl/well 1x PBS containing 0.5% (v/v) Triton X-100, 3% (w/v) BSA and 10% (v/v) FBS.
    6. Aspirate off the blocking solution.
    7. Immediately add 50 µl/well of appropriate HCV-specific primary antibody diluted in 1x PBS containing 0.5% (v/v) Triton X-100 and 3% (w/v) BSA for 1 h at RT. (e.g. The human anti-HCV E2 MAb AR3A can be used at 2.3 µg/ml or the mouse polyclonal serum anti-HCV NS5A 9E10 can be used at a 1: 500 dilution.)
    8. Decant or aspirate off the primary antibody and rinse cells with 1x PBS three times.
    9. Incubate the cells with an appropriate HRP-conjugated secondary antibody (50 µl/well) for one hour at RT (e.g. we detect bound MAb AR3A with a 1: 1,000 dilution of HRP-conjugated anti-human antibody and 9E10 with a 1: 500 dilution of HRP-conjugated anti-mouse).
    10. Rinse the cells 3 times with 1x PBS.
    11. Incubate cells with 30 µl/well AEC detection substrate for 30 min at RT.
    12. Wash cells 3 times with dH2O and add 150 µl/well of a solution of dH2O: glycerol (1:1) for storage. The plates may be stored at 4 °C.
    13. Quantify and photograph foci using an appropriate microscope. The number of HCV-positive cells per foci is a read-out of HCV cell-to-cell spread (see Figure 1).

  5. Neutralization control assay
    To ensure that cell-free virus in the medium is neutralized during the assay, the presence of infectious cell-free virus in the medium of experimental cultures should be assayed. These samples are harvested at the end of the spread assay as described in section C4).
    1. Preparation of Huh7 cell cultures.
      1. Seed 4,000 Huh7 cells/well in 96-well cell culture plate in 200 µl/well 10% FBS cDMEM.
      2. Incubate the plate at 37 °C in 5% CO2 cell culture incubator overnight.
    2. Aspirate out plating media and transfer the media samples (~200 µl) onto naïve Huh7 cell monolayers.
    3. At 24h aspirate off viral inoculum and add 200 µl/well fresh 10% FBS cDMEM.
    4. At 72 h post-inoculation, fix the cells and perform immunostaining to detect HCV-infected cells (as above).
      If infectious virus is detected in the supernatants, cell-to-cell spread cannot be accurately determined from the assay.

  6. Assess the effect of different factors on HCV cell-to-cell spread
    Aside from monitoring the kinetics of HCV cell-to-cell spread, this assay can be used to identify cellular factors involved in or antivirals that inhibit this step of the viral lifecycle.
    1. Use of Inhibitors.
      Inhibitors to specific factors or antivirals with unknown mechanisms of action can be added when cell-free virus entry is blocked at step C2. In this case, appropriate controls should also be included as well:
      1. “Mock” treated negative control samples (e.g. diluent or IgG control) should be included to control for non-specific inhibition.
      2. As a positive control for inhibition of cell-to-cell spread, parallel cultures can be treated with an inhibitor that blocks a factor known to be required for HCV cell-to-cell spread (e.g. 20 µg/ml anti-Claudin antibody, 30 µg/ml anti-NPC1L1 antibody or 30 µM of the NPC1L1 inhibitor, Ezetimibe). In the absence of cell division the foci formed in the presence of these HCV spread inhibitors are limited to 1-2 cells.
        Note: When performing this assay in actively dividing cells (Supplemental protocol B), these controls are particularly helpful in determining the number of cell divisions that occurred during the assay, i.e. the size of the foci in these control samples provide an indication of the extent to which cell division may have contributed to in increased foci size.
    2. siRNA knockdown.
      RNAi is an alternative method for assessing the involvement of cellular genes in HCV cell-to-cell spread. In this case, the assay is carried out as described above after silencing the gene of interest (see Supplemental protocol C). In this case, the following controls should be included:
      1. To monitor and control for non-specific effects of the transfection.
        1. Untreated and mock transfected control samples.
        2. Non-specific siRNA transfected control samples (e.g. siScramble or siGFP).
      2. As a positive control for inhibition of cell-to-cell spread parallel cultures can be transfected with siRNA targeting a factor known to be required for HCV cell-to-cell spread (e.g. Claudin 1 or NPC1L1).
      3. During the spread assay, the expression level of the siRNA-targeted gene should be monitored in parallel samples (e.g. RT-qPCR and/or western blot, and/or functional assays, if available).
        Although not described here, this silencing approach requires initial experiments to determine the efficiency and kinetics of specific gene silencing to ensure the assay is performed under optimal silencing conditions. Also, because factors of interest may be involved in cell-free extracellular virus entry, virus inoculation should be performed before the silencing takes effect so that the first round of infection can occur.
        Note: If silencing begins prior to infection initiation partial inhibition of infection may result in reduced numbers of foci being formed, however the readout for cell-to-cell spread is foci size not foci number thus this technical issue should not prevent analysis of cell-to-cell spread unless the initial infection is blocked completely.

  7. Example results and Interpretation
    When cell-to-cell spread is blocked, foci of only 1-2 cells in size are observed. Hence, in non-dividing cell cultures foci consisting of 1-2 cells are classified as exhibiting no spread (Figure 1, right panel), while foci with more than 2 cells are interpreted as resulting from of cell-to-cell spread (Figure 1, middle panel). The data can be expressed semi-quantitatively by calculating the percentage of infection events in each well that did not exhibit cell-to-cell spread (i.e. 1-2 cell foci) versus the percentage of infection events in each well that did exhibit cell-to-cell spread (i.e. >2 cell foci). Alternatively, to more quantitatively measure spread (and the effect of different inhibitors/cell factors), the number of cells in each individual focus within a well can be counted.


    Figure 1. HCV cell-to-cell spread assay. Left: Monolayer of Huh7 cells stained for HCV 72 h post infection. Middle: A typical HCV foci formed when cell-to-cell spread is allowed to proceed. Right: HCV foci observed when spread is blocked.

Recipes

  1. Complete DMEM (cDMEM)
    100 units/ml penicillin
    100 mg/ml streptomycin
    2 mM L-glutamine

Part II. Supplemental protocols

Supplemental protocol A: Alternative strategy for visualizing HCV positive cells
As described above, immunostaining is a convenient method for detecting HCV-positive cells; however other methods may be utilized. For example, if real-time live cell imaging of HCV cell-to-cell spread is desired, the Huh7.5-RFP-NLS-IPS reporter cell line (Jones et al., 2010) can be used. In uninfected cells the RFP-IPS-NLS protein is localized to the mitochondria in the cytoplasm. Upon infection the HCV NS3/4A protease cleaves the RFP-IPS-NLS off the mitochondria allowing the protein to localize to the nucleus. As such, infection of cells can be monitored in real-time based on the relocalization of RFP from the cytoplasm to the nucleus.

Materials and Reagents

Note: The same as in the Main Protocol with the following addition.

  1. Huh7-5.1TRIP-RFP-NLS-IPS (from Dr. Charles Rice, Rockefeller University, New York, NY) (Jones et al., 2010)

Equipment

Note: The same as in the Main Protocol with the following addition.

  1. Inverted fluorescence microscope (e.g. Nikon Corporation, model: TE2000U )

Procedure

Perform experiment as described in the Main protocol except:

  1. Utilize Huh7-5.1TRIP-RFP-NLS-IPS cells instead of the parental Huh7 cells in Main Protocol Section A (Preparation of non-growing Huh7 cell cultures).
  2. Monitor cell-to-cell spread under a microscope based on nuclear localization of RFP signal (instead of immunostaining described in Main protocol Section D) (see Figure 2 example results). Due to increases sensitivity, evidence of cell-to-cell spread in this assay may be detected as early as 12 h post-infection.


    Figure 2. Foci consisting of HCV-infected Huh7.5-RFP-NLS-IPS cells (i.e. nuclear RFP) surrounded by uninfected sells (i.e. cytoplasmic RFP)

Supplemental Protocol B: HCV cell-to-cell spread assay in growing cell cultures
For technical reasons, it may sometimes be preferable to perform the assay in growing Huh7 cells (e.g. limiting inhibitor concentrations, toxic effects of sodium azide when higher concentrations of some commercial antibody preparations are used, or more flexibility in timing of siRNA knockdown). In this case, the assay can be performed as described above with the following modifications:

Materials and Reagents

Note: The same as in the Main protocol.

Equipment

Note: The same as in the Main protocol.

Procedure

  1. Preparation of growing Huh7 cell cultures.
    1. The day before the assay, seed 12,000 Huh7 cells/well of 96-well tissue culture plate in 10% FBS cDMEM. (Include extra wells for counting cells as indicated below.)
    2. Incubate the flask at 37 °C in 5% CO2 cell culture incubator overnight and proceed with assay as described in the Main protocol step B.
  2.  Accounting for cell division. Because cell division can also contribute to an increase in the number of cells per focus, it is critical to monitor the extent of cell division during the assay. This can be done by counting the number of cells present in triplicate wells at the time of infection and at the end of the assay. The ratio of the number of cells in each well at the end of the assay over the number of cells in each well at the time of infection provides an estimate of the number cell divisions that occurred during the assay. In combination with the positive controls described in the Main protocol step F2, directly monitoring cell amplification can establish the size of foci that could have resulted purely from cell division and thus help control for the effects of cell growth.

Supplemental Protocol C: Reverse siRNA transfection of non-growing Huh7 cells

Materials and Reagents

Note: The same as in the Main protocol.

Equipment

Note: The same as in the Main protocol.

Procedure

  1. Preparation of non-growing Huh7 cell cultures.
    1. Seed ~2 x 106 Huh7 cells in a BioCoat T75 cm2 flask in 15 ml of 10% FBS cDMEM.
    2. Incubate the flask at 37 °C in 5% CO2 cell culture incubator overnight or until the cells reach 95%-99% confluence.
    3. Replace the medium with 20 ml of 10% FBS cDMEM containing 1% DMSO and continue to incubate cells at 37 °C in 5% CO2 cell culture incubator changing the 1% DMSO, 10% FBS cDMEM every other day.
  2. RNAiMAX siRNA transfection.
    1. On day 14-20 post DMSO treatment, trypsinize flask with 5 ml trypsin for 10 min at 37 °C. Neutralize with 7 ml serum containing DMEM without antibiotics.
    2. Transfer cells to a 50 ml centrifuge tube and spin at 1,000 x g for 5 min in a table top centrifuge.
    3. Rinse the cells twice in 35 ml OptiMEM, collecting the cell pellet by centrifugation in between. Resuspend cells to 7.8 x 105 cells/ml in OptiMEM.
    4. Preparation of transfection mixes (as master mixes or individual transfections in Eppendorf tubes or plate format, as appropriate).
      For each well of a 96-well plate to be transfected:
      1. Mix 0.84 µl 10 µM siRNA stock with 30 µl OptiMEM.
      2. Add 1.2 µl RNAiMax to the siRNA-OptiMEM and gently mix.
      3. Incubate at RT for 20 min.
      4. During incubation transfer 30 µl of the transfection mix into appropriate well of a waiting 96 well transfection plate.
    5. After the 20 min incubation period, add the 90 µl of the cell suspension to each well containing the transfection mix and pipette gently once to mix. (The goal is to reestablish the non-growing cell monolayer of ~65,000 cells. Based on the above, each well should receive ~70,000 cells in a final volume of 120 µl with final siRNA concentration of 70 nM allowing for some cell loss.)
    6. Incubate at 37 °C in 5% CO2 cell culture incubator overnight.
    7. Proceed with infection as described in the Main protocol step B2. (If the silenced gene may be required for cell-free extracellular virus entry, it is recommended to infect the cultures 20-24 h post-transfection before the targeted gene is efficiently silenced.)

Notes

In each user’s hands it is important to determine the number of infectious units and time of incubation that will result in the formation of a sufficient number of well-separated foci, which are big enough to clearly observe spread. This can be achieved by performing pilot assays with different volumes of virus inoculum and incubating for various periods of time.

Acknowledgments

This work was supported by NIH grants R01-AI078881 and R21-AI097809.

References

  1. Barretto, N., Sainz, B., Jr., Hussain, S. and Uprichard, S. L. (2014). Determining the involvement and therapeutic implications of host cellular factors in hepatitis C virus cell-to-cell spread. J Virol 88(9): 5050-5061.
  2. Jones, C. T., Catanese, M. T., Law, L. M., Khetani, S. R., Syder, A. J., Ploss, A., Oh, T. S., Schoggins, J. W., MacDonald, M. R., Bhatia, S. N. and Rice, C. M. (2010). Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nat Biotechnol 28(2): 167-171.
  3. Knipe, D. and Howley, P. M. (2007). Principles of Virology. In: Williams, L. and Wilkins (eds). 5th edition.
  4. Law, M., Maruyama, T., Lewis, J., Giang, E., Tarr, A. W., Stamataki, Z., Gastaminza, P., Chisari, F. V., Jones, I. M., Fox, R. I., Ball, J. K., McKeating, J. A., Kneteman, N. M. and Burton, D. R. (2008). Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14(1): 25-27.
  5. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, B., Tellinghuisen, T. L., Liu, C. C., Maruyama, T., Hynes, R. O., Burton, D. R., McKeating, J. A. and Rice, C. M. (2005). Complete replication of hepatitis C virus in cell culture. Science 309(5734): 623-626.
  6. Sainz, B., Jr. and Chisari, F. V. (2006). Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol 80(20): 10253-10257.
  7. Timpe, J. M., Stamataki, Z., Jennings, A., Hu, K., Farquhar, M. J., Harris, H. J., Schwarz, A., Desombere, I., Roels, G. L., Balfe, P. and McKeating, J. A. (2008). Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47(1): 17-24.
  8. Yu, X. and Uprichard, S. L. (2010). Cell-based hepatitis C virus infection fluorescence resonance energy transfer (FRET) assay for antiviral compound screening. Curr Protoc Microbiol Chapter 17: Unit 17 15.
  9. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland, S. F., Uprichard, S. L., Wakita, T. and Chisari, F. V. (2005). Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102(26): 9294-9299.

简介

丙型肝炎病毒(HCV)可通过"无细胞"细胞外病毒的进入或直接的"细胞到细胞"传递而感染初始细胞。 在这里,我们描述检测HCV细胞到细胞传播,使用一个非增长的细胞培养系统,避免细胞生长的混杂效应的测定。 该测定法包括感染汇合单层中的少量细胞,然后用中和抗体阻断随后的无细胞的细胞外病毒粒子,使得仅可发生细胞与细胞的传递。 在这些条件下,在37℃下孵育导致感染的细胞病灶的形成。 然后可以通过计数每个焦点中的细胞数量来确定细胞与细胞扩散的程度。 可以修改测定以评估抑制剂和/或特异性细胞基因对HCV的细胞与细胞扩散的影响。

第I部分。主要方案:在不分裂的Huh7细胞培养物中的HCV细胞 - 细胞扩散

材料和试剂

  1. Huh7细胞(来自Francis Chisari博士,The Scripps Research Institute,La Jolla,CA)(Zhong et al。,2005)
  2. JFH-1 HCVcc(如前所述产生并通过有限稀释滴度测定定量)(Yu和Uprichard,2010; Knipe和Howley,2007)
    注意:也可以使用其他感染性HCVcc克隆。
  3. Dulbecco改良Eagle培养基(DMEM)(Mediatech,目录号:MT-10-013-CV)
  4. 胎牛血清(FBS)(Hyclone,目录号:SH30910.03)
  5. 青霉素,链霉素,L-谷氨酰胺(Mediatech,目录号:MT-30-009-CI)
  6. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D4540)
  7. 1x PBS(Mediatech,目录号:MT-21-030-CV)
  8. 过氧化氢(H 2 O 2)(Thermo Fisher Scientific,目录号:H325)
  9. AEC检测底物(BD Biosciences,目录号:551015)
  10. 甘油(Sigma-Aldrich,目录号:G5516)
  11. Ezetimibe(Sequoia Research Products,目录号:SRP04000e)在DMSO中制备,浓度为20mM
  12. 兔抗人CLDN1多克隆抗体(Abcam,目录号:ab63070)
  13. 兔抗人NPC1L1多克隆抗体(Santa Cruz,目录号:sc-67236)
  14. 人抗-HCV E2单克隆抗体MAb AR3A(来自Dr.Mansun Law,The Scripps Research Institute,La Jolla,CA)(Law等人,2008)
  15. 小鼠抗HCV NS5A 9E10单克隆抗体(来自Charles Rice,Rockefeller University,New York,NY)(Lindenbach等人,2005)
  16. HRP缀合的山羊抗人(Thermo Fisher Scientific,目录号:31410),山羊抗小鼠(Thermo Fisher Scientific,目录号:31430)和山羊抗兔(Thermo Fisher Scientific,目录号:31460)。
  17. 小鼠IgG(Santa Cruz,目录号:sc-2025),兔IgG(Santa Cruz,目录号:sc-2027)
  18. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  19. Triton X-100(Sigma-Aldrich,目录号:T-8787)
  20. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
  21. Lipofectamine RNAiMAX转染试剂(Life Technologies,目录号:13778)
  22. OptiMEM(Life Technologies,Invitrogen TM,目录号:31985-070)
  23. 50ml离心管(Dot Scientific,目录号:451-PG)
  24. 完成DMEM(cDMEM)(参见配方)

设备

  1. 96孔BioCoat胶原包被的组织培养板(Corning,目录号:356698)
  2. BioCoat胶原包被的T75cm 2烧瓶(Corning,目录号:356485)
  3. 倒置显微镜
  4. 血细胞计数器
  5. 37℃,5%CO 2细胞培养箱中培养
  6. 台式离心机(例如Sorvall,型号:T6000D或Eppendorf,型号:5810R)

程序

  1. 非生长Huh7细胞培养物的制备
    1. 种子6,000 Huh7细胞/孔在BioCoat胶原包被的96孔板中在200μl/孔10%FBS cDMEM中。
    2. 将板在37℃下在5%CO 2细胞培养箱中孵育过夜或直到细胞达到95%-99%汇合。
    3. 吸出或倾析电镀培养基,并更换为200μl/孔   含有1%DMSO的10%FBS cDMEM,并继续在37℃下培养细胞 在5%CO 2细胞培养箱中孵育10天,改变1%DMSO, 10%FBS cDMEM。 (介质更换应缓慢进行   其中移液管尖端朝向孔的壁倾斜)。 在此期间   孵育期细胞仍然代谢活跃,但停止 (Sainz和Chisari,2006)。 到第10天,每个孔含有 约65,000个细胞。

  2. 感染开始
    1. 在2%FBS cDMEM中稀释HCVcc至50-100个病灶形成单位(ffu)/100μl。
    2. 向每个孔中加入100μl病毒接种物并在37℃下孵育 在5%CO 2细胞培养箱中过夜(约16小时)以允许初始 病毒输入。

  3. 阻止后续的"无细胞"细胞外病毒进入
    该步骤应在接种后不晚于18小时进行,以防止随后从第一轮感染细胞释放到培养基中的后代病毒感染细胞。
    1. 感染后16小时移除病毒接种物,并用1×PBS轻轻冲洗
    2. 加入含有10μg/ml HCV E2的10%FBS,1%DMSO cDMEM 抗体MAb AR3A加入到每个孔中,总体积为150μl/孔 中和HCVcc的无细胞扩散(Barretto等人,2014; Timpe等人 al。,2008)。 (可以加入另外的因子特异性抑制剂 平行样本在这一点,以评估其对HCV的影响 细胞间传播; 见下文F部分。)
    3. 继续孵化 在37℃在5%CO 2细胞培养箱中培养以允许时间 (例如感染后48-72小时)发生HCV的细胞间传播。
    4. 在进入D部分之前,从中收集所有培养基 将样品孔转移至新的96孔板中进行分析 中和,如下文E节所述。 (这可以测定 立即或储存于-80℃,直到可以进行测定)。

  4. 通过免疫染色可视化HCV细胞与细胞的传播
    在这些条件下,多细胞HCV病灶只能通过细胞间传播形成。 因此,单个HCV病灶中的HCV阳性细胞的数量可以用作细胞 - 细胞传输的读出。 在这里,免疫染色被描述为可视化HCV感染的细胞,但是可以使用其他方法(例如补充方案A)。
    1. 通过向每个孔中加入等体积的4%PFA来固定细胞,最终浓度为2%,并在室温下孵育25分钟。
    2. 滗析并用1x PBS冲洗细胞三次
    3. 通过与100μl/孔冰冷孵育阻断内源性过氧化物酶   1×含有0.3%(v/v)过氧化氢的PBS 5分钟 温度(RT)。
    4. 用1×PBS滗出和冲洗3次
    5. 渗透和封闭细胞1小时用100μl/孔1×PBS含有 0.5%(v/v)Triton X-100,3%(w/v)BSA和10%(v/v)FBS。
    6. 从封闭溶液中吸出。
    7. 立即加入50μl/well适当的HCV特异性原发性 抗体稀释在含有0.5%(v/v)Triton X-100和3% (w/v)BSA在室温下1小时。 (例如人类抗HCV E2 MAb AR3A可以 使用2.3μg/ml或小鼠多克隆血清抗HCV NS5A 9E10可以 以1:500稀释使用。)
    8. 倾析或吸出一抗,并用1x PBS冲洗细胞三次。
    9. 用适当的HRP共轭二级培养物孵育细胞 抗体(50μl/孔)在室温下孵育1小时(例如,我们检测结合的MAb AR3A 与HRP缀合的抗人抗体和9E10的1:1000稀释液接触 用HRP缀合的抗小鼠的1:500稀释液)
    10. 用1x PBS冲洗细胞3次。
    11. 孵育细胞与30微升/孔AEC检测基板在室温下30分钟
    12. 用dH 2 O 3洗涤细胞3次,加入150μl/孔的溶液 dH 2 O:甘油(1:1)。 板可以在4℃下储存
    13. 使用适当的显微镜量化和拍摄焦点。 的 每个病灶的HCV阳性细胞数是HCV细胞对细胞的读出 (见图1)。

  5. 中和控制测定
    为了确保在测定期间培养基中的无细胞病毒被中和,应当测定在实验培养基的培养基中感染性无细胞病毒的存在。 这些样品在扩展测定结束时收获,如C4)部分所述
    1. Huh7细胞培养物的制备。
      1. 种子4,000 Huh7细胞/孔在96孔细胞培养板中在200μl/孔10%FBS cDMEM中。
      2. 将板在37℃下在5%CO 2细胞培养箱中孵育过夜。
    2. 吸出电镀培养基,并将培养基样品(〜200微升)转移到初始Huh7细胞单层。
    3. 在24小时,吸出病毒接种物,并加入200μl/孔新鲜的10%FBS cDMEM
    4. 在接种后72小时,固定细胞并进行免疫染色以检测HCV感染的细胞(如上)。
      如果在上清液中检测到感染性病毒,则不能从测定中准确地测定细胞与细胞的扩散。

  6. 评估不同因素对HCV细胞与细胞传播的影响
    除了监测HCV细胞到细胞扩散的动力学之外,该测定可用于鉴定抑制病毒生命周期的这一步骤的细胞因子或抗病毒剂。
    1. 抑制剂的使用。
      抑制剂对特定因素或抗病毒药物 具有未知的作用机制可以在无细胞病毒时加入 在步骤C2阻塞条目。 在这种情况下,适当的控制应该 也包括在内:
      1. "模拟"处理阴性对照样品   (例如稀释剂或IgG对照)以控制 非特异性抑制。
      2. 作为抑制的阳性对照   细胞至细胞扩散,可以用抑制剂处理平行培养物   其阻断已知为HCV细胞至细胞扩散所需的因素 (例如,20μg/ml抗紧密连接蛋白抗体,30μg/ml抗NPC1L1抗体或 30μM的NPC1L1抑制剂Ezetimibe)。 在没有细胞的情况下 在这些HCV扩散抑制剂的存在下形成的病灶 限于1-2个细胞。
        注意:在中进行此测定时 主动分裂细胞(补充协议B),这些控制是 特别有助于确定细胞分裂的数目 ,即在这些对照中的病灶的大小 样品提供细胞分裂可能程度的指示 有助于增加灶灶大小。
    2. siRNA敲除。
      RNAi是评估细胞参与的替代方法   HCV细胞到细胞扩散的基因。 在这种情况下,进行测定 在沉默感兴趣的基因后,如上所述 补充方案C)。 在这种情况下,应该有以下控件   包括:
      1. 监测和控制转染的非特异性效应。
        1. 未处理和模拟转染的对照样品。
        2. 非特异性siRNA转染的对照样品(例如siScramble或siGFP)。
      2. 作为平行抑制细胞与细胞扩散的阳性对照   可以用靶向已知的因子的siRNA转染培养物 (例如,紧密连接蛋白1或NPC1L1)所必需的。
      3. 在扩展测定期间,siRNA靶向的表达水平 基因应当在平行样品中监测(例如,RT-qPCR和/或) 免疫印迹,和/或功能测定(如果可获得的话) 虽然不是 这种沉默方法需要进行初步实验 确定特定基因沉默的效率和动力学 确保测定在最佳沉默条件下进行。 也, 因为感兴趣的因子可能涉及无细胞的细胞外 病毒进入,病毒接种应在沉默前进行 使得第一轮感染可以发生。
        注意: 如果沉默在感染开始之前开始部分抑制 但是,感染可能导致形成的灶的数目减少 细胞到细胞扩散的读数是焦点尺寸而不是焦点数 这个技术问题不应阻止细胞与细胞扩散的分析 除非初始感染完全阻止。

  7. 示例结果和解释
    当细胞 - 细胞扩散被阻断时,观察到仅有1-2个细胞大小的病灶。因此,在非分裂细胞培养中,由1-2个细胞组成的病灶被分类为没有扩散(图1,右图),而具有多于2个细胞的病灶被解释为由细胞到细胞扩散产生1,中间图)。通过计算不显示细胞间传播(即,1-2细胞灶)的每个孔中感染事件的百分比相对于感染事件的百分比,可以半定量地表示数据每个孔显示出细胞到细胞的扩散(即> 2个细胞灶)。或者,为了更定量地测量扩散(和不同抑制剂/细胞因子的效应),可以计数孔内每个个体焦点中的细胞数目。


    图1. HCV细胞到细胞扩散测定。左图:感染后72小时的Huh7细胞单层对HCV染色。中:当细胞 - 细胞扩散允许进行时形成的典型的HCV病灶。右:当传播阻塞时观察到HCV病灶。

食谱

  1. 完成DMEM(cDMEM)
    100单位/ml青霉素
    100mg/ml链霉素 2mM L-谷氨酰胺

第二部分。补充协议

补充方案A:可视化HCV阳性细胞的替代策略
如上所述,免疫染色是检测HCV阳性细胞的方便的方法;然而可以使用其他方法。例如,如果需要HCV细胞至细胞扩散的实时活细胞成像,则Huh7.5-RFP-NLS-IPS报道细胞系(Jones等人,2010)可以使用。在未感染的细胞中,RFP-IPS-NLS蛋白定位于细胞质中的线粒体。感染后,HCV NS3/4A蛋白酶切割RFP-IPS-NLS离开线粒体,允许蛋白质定位于细胞核。因此,可以基于RFP从细胞质到细胞核的重新定位来实时监测细胞的感染。

材料和试剂

注意:与主协议中的相同,添加以下内容。

  1. Huh7-5.1TRIP-RFP-NLS-IPS(来自Charles Rice,Rockefeller University,New York,NY)(Jones等人,2010)

设备

注意:与主协议中的相同,添加以下内容。

  1. 倒置荧光显微镜(例如Nikon Corporation,型号:TE2000U)

程序

按照主协议中所述进行实验,除了:

  1. 在主协议部分A(制备非生长Huh7细胞培养物)中利用Huh7-5.1TRIP-RFP-NLS-IPS细胞代替亲代Huh7细胞。
  2. 在基于RFP信号的核定位的显微镜下监测细胞与细胞的铺展(代替主要方案D节中描述的免疫染色)(参见图2的实施例结果)。由于敏感性增加,在该测定中细胞 - 细胞扩散的证据可早在感染后12小时检测。


    图2.由被HCV感染的Huh7.5-RFP-NLS-IPS细胞( 即 核RFP)包围的Foci未感染的销售( 细胞质RFP)

补充方案B:正在生长的细胞培养物中的HCV细胞至细胞扩散测定
由于技术原因,有时优选在生长Huh7细胞中进行测定(例如,当使用较高浓度的一些商业抗体制剂时,限制抑制剂浓度,叠氮化钠的毒性作用,或者更灵活地 siRNA敲低的时间)。 在这种情况下,可以如上所述进行测定,具有以下修改:

材料和试剂

注意:与主协议相同。

设备

注意:与主协议相同。

程序

  1. 生长的Huh7细胞培养物的制备
    1. 在测定前一天,种子12,000 Huh7细胞/孔的96孔组织 培养板在10%FBS cDMEM中。 (包括用于计数细胞的额外孔 如下所示。)
    2. 在37℃下在5%CO 2细胞中孵育烧瓶 培养箱中培养过夜,然后进行如上所述的测定 主协议步骤B.
  2.  细胞分裂的会计。 因为细胞分裂也可以有助于增加每个焦点的细胞数目,所以在测定期间监测细胞分裂的程度是关键的。 这可以通过 计数在感染时和在测定结束时存在于一式三份孔中的细胞数目。在测定结束时每孔中的细胞数目与感染时每个孔中细胞数目的比率提供了在测定期间发生的细胞分裂数目的估计。结合主协议步骤F2中描述的阳性对照,直接监测细胞扩增可以建立可能纯粹来自细胞分裂的病灶的大小,从而有助于控制细胞生长的效果。

补充方案C:非生长Huh7细胞的siRNA转染反转

材料和试剂

注意:与主协议相同。

设备

注意:与主协议相同。

程序

  1. 非生长Huh7细胞培养物的制备
    1. 种子〜2×10 6 Huh7细胞在BioCoat T75cm 2烧瓶中的15ml 10%FBS cDMEM中。
    2. 将烧瓶在37℃下在5%CO 2细胞培养箱中孵育过夜或直到细胞达到95%-99%汇合。
    3. 用20ml含有1%DMSO的10%FBS cDMEM替换培养基 并在37℃下在5%CO 2细胞培养箱中继续孵育细胞   每隔一天更换1%DMSO,10%FBS cDMEM。
  2. RNAiMAX siRNA转染。
    1. 在DMSO处理后14-20天,用5ml胰蛋白酶胰蛋白酶处理烧瓶   在37℃下10分钟。 用7ml含有DMEM的血清中和 抗生素。
    2. 将细胞转移到50ml离心管中,并在台式离心机中以1,000×g旋转5分钟。
    3. 冲洗细胞在35毫升OptiMEM两次,收集细胞沉淀 通过其间的离心。 将细胞重悬于7.8×10 5个细胞/ml OptiMEM。
    4. 转染混合物的制备(作为主混合物或 单独转染在Eppendorf管或板格式,as 适当)。
      对于待转染的96孔板的每个孔:
      1. 将0.84μl10μMsiRNA原液与30μlOptiMEM混合。
      2. 添加1.2微升RNAiMax到siRNA-OptiMEM并轻轻混合。
      3. 在室温下孵育20分钟。
      4. 在孵育过程中,将30μl转染混合物转移至等待的96孔转染板的合适孔中。
    5. 孵育20分钟后,加入90μl的细胞 悬浮液到含有转染混合物和移液管的每个孔中 轻轻地混合一次。 (目标是重建非繁殖细胞 单层〜65,000个细胞。 基于上述,每口井应接受   〜70,000个细胞,最终体积为120μl,含有最终siRNA 浓度为70nM,允许一些细胞损失。)
    6. 在37℃下在5%CO 2细胞培养箱中孵育过夜。
    7. 继续感染,如主协议步骤B2中所述。 (如果沉默基因可能是无细胞胞外病毒所需的 进入,建议感染文化20-24 h 在靶基因有效沉默之前的转染后)。

笔记

在每个使用者的手中,重要的是确定感染单位的数量和孵育的时间,其将导致形成足够数量的分离良好的焦点,其足够大以清楚地观察扩散。 这可以通过用不同体积的病毒接种物进行中试测定并孵育不同时间段来实现。

致谢

这项工作由NIH授权R01-AI078881和R21-AI097809支持。

参考文献

  1. Barretto,N.,Sainz,B.,Jr.,Hussain,S。和Uprichard,S.L。(2014)。 确定宿主细胞因子在丙型肝炎病毒细胞至细胞传播中的参与和治疗影响。 J Virol 88(9):5050-5061。
  2. Jones,C.T.,Catanese,M.T.,Law,L.M.,Khetani,S.R.,Syder,A.J.,Ploss,A.,Oh,T.S.,Schoggins,J.W.,MacDonald,M.R.,Bhatia,S.N.and Rice,C.M。 使用基于荧光细胞的报道系统对丙型肝炎病毒感染的实时成像。 Nat Biotechnol 28(2):167-171
  3. Knipe,D。和Howley,P.M。(2007)。病毒学原理。 In:Williams,L.and Wilkins(eds)。第5版
  4. Law,M.,Maruyama,T.,Lewis,J.,Giang,E.,Tarr,AW,Stamataki,Z.,Gastaminza,P.,Chisari,FV,Jones,IM,Fox, McKeating,JA,Kneteman,NM和Burton,DR(2008)。 广泛中和抗体可防止丙型肝炎病毒准种挑战。

    /em> 14(1):25-27。
  5. Lindenbach,B.D.,Evans,M.J.,Syder,A.J.,Wolk,B.,Tellinghuisen,T.L.,Liu,C.C。,Maruyama,T.,Hynes,R.O.,Burton,D.R.,McKeating,J.A.and Rice,C.M。 在细胞培养中完全复制丙型肝炎病毒 科学 309(5734):623-626。
  6. Sainz,B.,Jr.and Chisari,F.V。(2006)。 通过分化良好,生长停滞的人类肝癌细胞产生感染性丙型肝炎病毒。/a> J Virol 80(20):10253-10257。
  7. Timpe,JM,Stamataki,Z.,Jennings,A.,Hu,K.,Farquhar,MJ,Harris,HJ,Schwarz,A.,Desombere,I.,Roels,GL,Balfe,P.and McKeating,JA 2008)。 在存在中和抗体的情况下,肝癌细胞中的丙型肝炎病毒细胞和细胞传播。 Hepatology 47(1):17-24。
  8. Yu,X。和Uprichard,S.L。(2010)。 基于细胞的丙型肝炎病毒感染荧光共振能量转移(FRET)测定用于抗病毒化合物筛选。/a> Curr Protoc Microbiol Chapter 17:Unit 17 15.
  9. Zhong,J.,Gastaminza,P.,Cheng,G.,Kapadia,S.,Kato,T.,Burton,D.R.,Wieland,S.F.,Uprichard,S.L.,Wakita,T.and Chisari,F.V。 体外耐药性丙型肝炎病毒感染 > Proc Natl Acad Sci USA 102(26):9294-9299
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barretto, N. and Uprichard, S. L. (2014). Hepatitis C virus Cell-to-cell Spread Assay. Bio-protocol 4(24): e1365. DOI: 10.21769/BioProtoc.1365.
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