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Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection
报告基因试验法研究精液介导增强HIV-1感染   

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Abstract

Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear cells (PBMCs).

Keywords: HIV(艾滋病咨询门诊), Semen(精液), Amyloid(淀粉样蛋白), SEVI(SEVI), Sexual transmission(性传播)

Materials and Reagents

  1. Flat-bottom 96-well plates (Sarstedt AG, catalog number: 83.3924 )
  2. White 96-well polystyrene plates (Thermo Fisher Scientific, NuncTM, catalog number: 136101 )
  3. Round-bottom 96-well plates (Sarstedt AG, catalog number: 83.3925 )
  4. V-bottom 96-well plates (Sarstedt AG, catalog number: 83.3926 )
  5. Peripheral blood mononuclear cells (PBMCs)
    Note: These cells were isolated from healthy blood donor buffy coats by Ficoll (Biocoll) gradient centrifugation (see: http://www.bio-protocol.org/e323), and stimulated with 1 µg/ml phytohaemagglutinin (PHA) and 10 ng/ml IL-2 for 3 days.
  6. TZM-bl/JC53bl-13 (HeLa CD4+ CCR5+ LTR-luciferase and LTR-lacZ) cells (Wei et al., 2002) (National Institutes of Health AIDS Research and Reference Reagent Program, catalog number: 8129 )
  7. β-Galactosidase Reporter Gene Assay System for mammalian cells (Thermo Fisher Scientific, Gal-ScreenTM, catalog number: T1027 )
  8. Biocoll separating solution (Merck Millipore, Biochrom, catalog number: L6115 )
  9. Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific, catalog number: 41965039 )
  10. Luminescent Cell Viability Assay (Promega, CellTiter-Glo®, catalog number: G7571 )
  11. Fetal bovine/calf serum (FCS) (Thermo Fisher Scientific, catalog number: 10270106 ) (inactivated for 30 min at 56 °C in a water bath)
  12. Gentamicin (Thermo Fisher Scientific, catalog number: 15710049 )
  13. HIV-1 stock generated by HEK293T cells transfected with proviral DNA (e.g., pBRHIV-1 NL4-3 or pBRHIV-1 NL4-3 92TH014-2, see Münch et al., 2007 and Kim et al., 2010) or from infected cells)
  14. HIV-1_firefly-luciferase stock [e.g., NL4-3 HIV-1_Luciferase (e.g., similar to Hiebenthal-Millow and Kirchhoff, 2002)] generated by HEK293T cells transfected with a proviral DNA encoding firefly-luciferase
  15. Human Interleukin-2 (IL-2) (Miltenyi Biotec, catalog number: 130-097-745 )
  16. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
  17. Luciferase Assay System (Promega, catalog number: E1501 )
  18. Penicillin-Streptomycin (PenStrep) (Thermo Fisher Scientific, catalog number: 15140122 )
  19. Phosphate buffered saline (PBS), no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14190094 )
  20. Phytohaemagglutinin (PHA) (Thermo Fisher Scientific, RemelTM, catalog number: R30852801 )
  21. RPMI-1640 (Thermo Fisher Scientific, catalog number: 21875034 )
  22. Semen (fresh or frozen)
    Note: Ejaculates are collected from healthy individuals with informed consent. To minimize inter-donor variations, semen derived from > 10 individual donors are pooled and aliquoted. Before pooling (on ice) the ejaculates are allowed to liquefy for 20-30 min at room temperature. Pooled semen samples can be stored at -20 °C or -80 °C in 250 to 1,000 µl aliquots. In all experiments, semen aliquots need to be rapidly thawed, diluted, and mixed with virus. The remaining sample is discarded.
  23. Complete medium for adherent TZM-bl cells (see Recipes)
  24. Complete medium for suspension primary PBMCs (see Recipes) 

Equipment

  1. 96-well plate centrifuge (Eppendorf, model: 5804 R ) equipped with A-2-MTP rotor
  2. CO2 humidified incubator
  3. 5-50 µl 12-channel pipette (HTL, Discovery comfort, model: DV-12-50 )
  4. 20-200 µl 12-channel pipette (HTL, Discovery comfort, model: DV-12-200 )
  5. Orion II Microplate Luminometer (Titertek Berthold, catalog number: 11300010 )

Software

  1. Microsoft Excel (Microsoft)
  2. Simplicity 4.02 (Berthold Detection Systems)

Procedure

  1. Seed target cells
    1. TZM-bl cells: Seed 10,000 TZM-bl cells per well in 96-well flat-bottom plates containing 100 µl complete medium.
    2. PBMCs: Seed 200,000 stimulated PBMCs (1 µg/ml PHA + 10 ng/ml IL-2 for 3 days) per well in 96-well flat-bottom plates containing 100 µl complete medium.
    Note: Fill the outer wells with 200 µl PBS to avoid evaporation of the inner wells containing the cells (Figure 1: wells in grey).
  2. Culture 18-24 h at 37 °C in a 5% CO2 humidified incubator.
  3. The next day, without removing the medium, add 180 µl fresh complete medium containing 100 µg/ml gentamicin (and 10 ng/ml IL-2 for PBMCs).
    Note: Gentamicin prevents the outgrowth of bacteria that may have contaminated the semen collection container.
  4. Dilute HIV-1 stock in the complete medium to achieve a MOI < 1 (e.g., 0.1, 0.01, 0.001).
    Note: The HIV-1 enhancing activity of semen cannot be observed at high MOIs as all target cells have been already infected in the absence of semen.
  5. Thaw semen samples quickly in a 37 °C water bath and immediately dilute in PBS to achieve semen concentrations of 0, 0.8, 4, 20, and 100% (v/v) in a 96-well round-bottom plate.
    Note: Semen’s enhancing activity is lost over time. Avoid long room temperature exposure.
  6. Add 40 µl of respective HIV-1 dilutions to 40 µl semen dilutions (1:1) in a 96-well round-bottom plate to achieve semen concentrations of 0, 0.4, 2, 10, and 50% (v/v).
  7. Resuspend HIV-1/semen mixture; add 20 µl to 280 µl (1 to 15) cells in triplicates; the final cell culture concentrations of semen are 0, 0.027, 0.134, 0.67, 3.33% (v/v) (Figure 1).
    Note: The low semen concentrations on the cells reduce its cytotoxic effect.
  8. Incubate for 2-3 h at 37 °C in a 5% CO2 humidified incubator.
  9. Thereafter, remove the medium and add 200 µl fresh complete medium containing 100 µg/ml gentamicin.
    1. TZM-bl cells: Simple medium change is sufficient.
    2. PBMCs: Centrifuge for 5 min at 300 x g at room temperature, discard 100 µl (of 300 µl) supernatant (without cells), resuspend cells and transfer the entire sample (200 µl) into a 96-well V-bottom plate. Centrifuge for 5 min at 300 x g at room temperature, discard supernatant, resuspend cells in 200 µl of fresh complete medium with 100 µg/ml gentamicin and 10 ng/ml IL-2 and transfer into 96-well F-bottom plates.
    Note: Medium change prevents the toxic effects of semen.
  10. Incubate at 37 °C in a 5% CO2 humidified incubator.
  11. After 2 and 3 days, analyze all samples by light microscopy to detect the cytopathic effects (CPE) caused by HIV-1 infection, and the possible cytotoxic effects of semen.
    Notes:
    1. For TZM-bl cells: In this cell line, massive HIV-1 infection results in the formation of syncytia (see Figure 2 or supplementary Figure 1 in Kim et al., 2010). If syncytia are detectable at Day 2, determine the cellular β-galactosidase activities in all samples. Continued incubation of cultures that already show CPE for another 24 h may lead to over-infection and cell loss and should be avoided. If no CPE is detectable at Day 2, incubate cells for another 24 h, and determine reporter gene activities at Day 3 post infection. Final cell culture concentrations of semen of 3.33% are sometimes cytotoxic. Therefore, we greatly recommend running cytotoxicity assay in the absence of virus in parallel.
    2. For PBMC: In PBMC, the HIV-1 induced CPE is less pronounced. If syncytia are detectable, determine the luciferase activities in all samples at Day 2. If no CPE is detectable at Day 2, incubate cells for another 24 h, and determine the reporter gene activities at Day 3 post infection. Note that PBMC are more susceptible to the cytotoxic effects of semen (supplementary Figure 17 in Münch et al., 2007). Therefore, we greatly recommend running cytotoxicity assay in the absence of virus in parallel.
  12. Determine reporter gene activities:
    1. For TZM-bl cells (Figures 3 and 4) using the Gal-ScreenTM β-Galactosidase Reporter Gene Assay System
      1. Remove supernatant.
      2. Add 40 µl of lysis buffer together with substrate (mix according to the manufacturer’s instructions).
      3. Incubate for 30 min at room temperature.
      4. Transfer 35 µl of the lysed cells into a white 96-well luminometer plate.
      5. Read luminescence as relative light units/s in a luminometer.
    2. For PBMCs infected with an HIV-1 reporter virus encoding firefly luciferase using the Luciferase Assay System
      1. Resuspend PBMCs and transfer into a 96-well V-bottom plate.
      2. Centrifuge for 5 min, 550 x g at room temperature.
      3. Discard supernatant.
      4. Add 40 µl of 1x lysis buffer.
      5. Incubate for 10 min at room temperature.
      6. Resuspend and transfer 30 µl of the lysed sample into a white 96-well luminometer plate.
      7. Add 100 µl substrate.
      8. Immediately, read luminescence as relative light units/s in a luminometer.
  13. Evaluate raw data (Figures 3 and 4).
  14. Calculate average values obtained from non-infected cells (background) (Figure 3B).
  15. Subtract average background values from each sample (Figure 3B).
  16. Calculate average values and standard deviations of each triplicate measurement (Figures 3C and 4A).
  17. Calculate n-fold enhancement values by setting reporter gene activities observed in the absence of semen = 1 (Figures 3D and 4B). 

Representative data



Figure 1. Typical layout of a microtiter plate used in experiments to study the effect of semen on HIV-1 infection. Cells seeded in the inner 60 wells are inoculated with HIV-1 of indicated MOIs pretreated with 0, 0.4, 2, 10 and 50 % semen, or are left uninfected. grey: PBS; white: uninfected; colored: different MOIs of HIV-1.


Figure 2. Light microscopy analysis of infected TZM-bl cells in presence of semen. TZM-bl cells were inoculated with PBS (uninfected) or HIV-1 that has been preincubated with indicated concentrations of semen. Final semen concentrations on cells are given in brackets. Minor (0% semen), little (2% semen) and strong (10% semen) CPE. Scale bars are 100 µm.


Figure 3. Results and evaluation of a representative reporter gene assay. Different MOIs (red: 0.1, blue: 0.01, green: 0.001) of HIV-1 NL4-3 92Th014-2 (R5-tropic) were preincubated with indicated concentrations of semen before infection of TZM-bl cells. A. Raw data representing relative light units/second of β-galactosidase activity measured in each well. B. Average background (uninfected cells) is calculated and subtracted. C. Average and standard deviations of triplicate infections are calculated. D. Fold enhancement is calculated by setting 0% semen sample = 1.


Figure 4. Graphical presentation of data described above. A. Shown are average β-galactosidase activities (n = 3) measured 3 days after virus exposure. RLU/s: relative light units per sec. The numbers above the bars give n-fold enhancement of HIV infection by semen relative to that measured for the corresponding PBS control. B. n-fold enhancement values represented as bar graphs. Values represent average values obtained from triplicate infection ± standard deviation. Note that 50% semen during virion treatment (corresponding to final cell culture concentrations of semen of 3.3 %) are cytotoxic resulting in reduced infection rates.

Notes

The following issues need to be carefully considered:

  1. Do not add DEAE-Dextran, polybrene, or other additives that are often used in TZM-bl assays to increase infection rates. Addition of separate enhancers will mask the infection enhancing activity of semen.
  2. Use low MOIs that are either sub-infectious in the absence of semen or result in infection rates of less than 10%. Only then, an enhancing effect of semen on viral infection can be determined. Furthermore, a low MOI resembles physiological HIV-1 concentrations during sexual transmission.
  3. The HIV-1 enhancing activity of semen decreases over time. Thus, semen needs to be handled quickly.
  4. Pre-exposure of virus to semen allows binding of seminal amyloids to viral particles as it likely also occurs in vivo.
  5. Semen, after collection, is often not sterile thus gentamicin needs to be added to avoid bacterial outgrowth.
  6. Even low semen concentrations (starting from 1% on cells) are cytotoxic. Therefore, semen concentrations need to be reduced and the exposure time shortened (e.g., by media change).
  7. In parallel, it is recommended to run cytotoxicity assays. [e.g., MTT assay (see Kim et al., 2010) or Luminescent Cell Viability Assay]
  8. Assay does not need to be performed in flat 96-well plates, but can also be scaled into different formats.

If these points are considered, the experimental set up can be adapted and modified to allow measurement of infection of different target cells with any HIV strain and reporter virus (i.e., GFP followed by a flow cytometry readout), as well as other viruses such as HCMV and HSV (Tang et al., 2013; Torres et al., 2015).
Infection enhancement by in vitro generated amyloid fibrils can be examined using the same protocol, except that the toxicity avoiding steps, i.e., addition of gentamicin, high volumes of medium, removal of toxic semen after 2-3 h can be omitted.

Recipes

  1. Complete medium for adherent TZM-bl cells
    DMEM
    10% heat-inactivated FCS
    1% PenStrep
    1% L-glutamine
    (100 µg/ml gentamicin)
  2. Complete medium for suspension primary PBMCs
    RPMI
    10% heat-inactivated FCS
    1% PenStrep
    1% L-glutamine
    10 ng/ml IL-2
    (100 µg/ml gentamicin)

Acknowledgments

This assay was first published in (Münch et al., 2007) and the protocol described in detail in (Kim et al., 2010). Thanks to Annika Röcker, Edina Lump, and Onofrio Zirafi for carefully reading and revising the protocol. Janis A. Müller is part of the International Graduate School in Molecular Medicine Ulm.

References

  1. Arnold, F., Schnell, J., Zirafi, O., Sturzel, C., Meier, C., Weil, T., Standker, L., Forssmann, W. G., Roan, N. R., Greene, W. C., Kirchhoff, F. and Munch, J. (2012). Naturally occurring fragments from two distinct regions of the prostatic acid phosphatase form amyloidogenic enhancers of HIV infection. J Virol 86(2): 1244-1249.
  2. Hiebenthal-Millow, K. and Kirchhoff, F. (2002) The most frequent naturally occurring length polymorphism in the HIV-1 LTR has little effect on proviral transcription and viral replication. Virology 292 (1), 169-75.
  3. Kim, K. A., Yolamanova, M., Zirafi, O., Roan, N. R., Staendker, L., Forssmann, W. G., Burgener, A., Dejucq-Rainsford, N., Hahn, B. H., Shaw, G. M., Greene, W. C., Kirchhoff, F. and Munch, J. (2010). Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI. Retrovirology 7: 55.
  4. Munch, J., Rucker, E., Standker, L., Adermann, K., Goffinet, C., Schindler, M., Wildum, S., Chinnadurai, R., Rajan, D., Specht, A., Gimenez-Gallego, G., Sanchez, P. C., Fowler, D. M., Koulov, A., Kelly, J. W., Mothes, W., Grivel, J. C., Margolis, L., Keppler, O. T., Forssmann, W. G. and Kirchhoff, F. (2007). Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131(6): 1059-1071.
  5. Roan, N. R., Munch, J., Arhel, N., Mothes, W., Neidleman, J., Kobayashi, A., Smith-McCune, K., Kirchhoff, F. and Greene, W. C. (2009). The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection. J Virol 83(1): 73-80.
  6. Roan, N. R., Muller, J. A., Liu, H., Chu, S., Arnold, F., Sturzel, C. M., Walther, P., Dong, M., Witkowska, H. E., Kirchhoff, F., Munch, J. and Greene, W. C. (2011). Peptides released by physiological cleavage of semen coagulum proteins form amyloids that enhance HIV infection. Cell Host Microbe 10(6): 541-550.
  7. Roan, N. R., Liu, H., Usmani, S. M., Neidleman, J., Muller, J. A., Avila-Herrera, A., Gawanbacht, A., Zirafi, O., Chu, S., Dong, M., Kumar, S. T., Smith, J. F., Pollard, K. S., Fandrich, M., Kirchhoff, F., Munch, J., Witkowska, H. E. and Greene, W. C. (2014). Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection. J Virol 88(13): 7221-7234.
  8. Tang, Q., Roan, N. R. and Yamamura, Y. (2013). Seminal plasma and semen amyloids enhance cytomegalovirus infection in cell culture. J Virol 87(23): 12583-12591.
  9. Torres, L., Ortiz, T. and Tang, Q. (2015). Enhancement of herpes simplex virus (HSV) infection by seminal plasma and semen amyloids implicates a new target for the prevention of HSV infection. Viruses 7(4): 2057-2073.
  10. Usmani, S. M., Zirafi, O., Muller, J. A., Sandi-Monroy, N. L., Yadav, J. K., Meier, C., Weil, T., Roan, N. R., Greene, W. C., Walther, P., Nilsson, K. P., Hammarstrom, P., Wetzel, R., Pilcher, C. D., Gagsteiger, F., Fandrich, M., Kirchhoff, F. and Munch, J. (2014). Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen. Nat Commun 5: 3508.
  11. Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag, M. S., Wu, X., Shaw, G. M. and Kappes, J. C. (2002). Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46(6): 1896-1905.

简介

精液含有增强HIV-1感染的淀粉样蛋白原纤维(Münch等,2007; Kim等,2010; Roan等,2011; Arnold等,2012; Usmani等,2014; Roan et al。 ,2014)。 带正电的精液淀粉样蛋白捕获带负电的病毒颗粒并增加其对细胞表面的附着率,导致增强的融合和感染(Roan等人,2009)。 由于精液具有高度的细胞毒性,我们开发了一种能够定量精液感染增强活性同时最大限度降低其细胞损伤活性的测定方法。 在这里,我们描述允许使用报道细胞系(TZM-bl细胞)或外周血单核细胞(PBMC)定量精液的感染性增强活性的两种方案。

关键字:艾滋病咨询门诊, 精液, 淀粉样蛋白, SEVI, 性传播

材料和试剂

  1. 平底96孔板(Sarstedt AG,目录号:83.3924)
  2. 白色96孔聚苯乙烯板(Thermo Fisher Scientific,Nunc ,目录号:136101)
  3. 圆底96孔板(Sarstedt AG,目录号:83.3925)
  4. V-底96孔板(Sarstedt AG,目录号:83.3926)
  5. 外周血单核细胞(PBMC)
    注意:这些细胞通过Ficoll(Biocoll)梯度离心从健康血液供体白细胞层中分离(参见:
  6. TZM-b1/JC53b1-13(HeLa CD4 + + CCR5 + LTR-萤光素酶和LTR-lacZ)细胞(Wei等人,2002)(National Institutes of Health AIDS Research and Reference Reagent Program,目录号:8129)
  7. 用于哺乳动物细胞的β-半乳糖苷酶报告基因测定系统(Thermo Fisher Scientific,Gal-Screen TM ,目录号:T1027)
  8. Biocoll分离溶液(Merck Millipore,Biochrom,目录号:L6115)
  9. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,目录号:41965039)
  10. 发光细胞活力测定(Promega,CellTiter-Glo ,目录号:G7571)
  11. 胎牛/牛血清(FCS)(Thermo Fisher Scientific,目录号:10270106)(在56℃下在水浴中灭活30分钟)
  12. 庆大霉素(Thermo Fisher Scientific,目录号:15710049)
  13. 由原病毒DNA转染的HEK293T细胞产生的HIV-1原种(例如,pBRHIV-1 NL4-3或pBRHIV-1 NL4-3 92TH014-2,参见Münch等人 >,2007和Kim 等人,2010)或来自感染细胞)
  14. 由HEK293T细胞转染产生的HIV-1_萤火虫荧光素酶原液[例如],NL4-3HIV-1_荧光素酶(例如,类似于Hiebenthal-Millow和Kirchhoff,2002)与编码萤火虫荧光素酶的前病毒DNA
  15. 人白细胞介素-2(IL-2)(Miltenyi Biotec,目录号:130-097-745)
  16. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
  17. 萤光素酶测定系统(Promega,目录号:E1501)
  18. 青霉素 - 链霉素(PenStrep)(Thermo Fisher Scientific,目录号:15140122)
  19. 磷酸盐缓冲盐水(PBS),无钙,无镁(Thermo Fisher Scientific,目录号:14190094)
  20. 植物血细胞凝集素(PHA)(Thermo Fisher Scientific,Remel TM ,目录号:R30852801)
  21. RPMI-1640(Thermo Fisher Scientific,目录号:21875034)
  22. 精液(新鲜或冷冻)
    注意:在知情同意的情况下从健康个体收集射精。为了使供体间变化最小化,合并10个单独的供体并等分。在混合(在冰上)之前,使射精在室温下液化20-30分钟。合并的精液样品可以在-20℃或-80℃下以250至1,000μl等分试样储存。在所有实验中,精液等分试样需要快速解冻,稀释并与病毒混合。剩余的样品将被丢弃。
  23. 完全培养基用于贴壁TZM-bl细胞(参见配方)
  24. 用于悬浮主要PBMC的完全培养基(参见Recipes)

设备

  1. 配备有A-2-MTP转子的96孔板离心机(Eppendorf,型号:5804R)
  2. CO 2加湿培养箱
  3. 5-50μl12通道移液管(HTL,Discovery comfort,型号:DV-12-50)
  4. 20-200μl12通道移液管(HTL,Discovery comfort,型号:DV-12-200)
  5. (Orion II Microplate Luminometer)(Titertek Berthold,目录号:11300010)

软件

  1. Microsoft Excel(Microsoft)
  2. 简单4.02(Berthold检测系统)

程序

  1. 种子靶细胞
    1. TZM-bl细胞:在含有100μl完全培养基的96孔平底板中每孔接种10,000 TZM-bl细胞。
    2. PBMC:在含有100μl完全培养基的96孔平底板中每孔接种200,000个刺激的PBMC(1μg/ml PHA + 10ng/ml IL-2,持续3天)。
    注意:向外孔中加入200μlPBS,以避免含有细胞的内孔蒸发(图1:灰色孔)。
  2. 在37℃,5%CO 2湿润培养箱中培养18-24小时。
  3. 第二天,在不除去培养基的情况下,加入180μl含有100μg/ml庆大霉素(和PBNs的10ng/ml IL-2)的新鲜完全培养基。
    注意:庆大霉素可防止可能污染精液收集容器的细菌的生长。
  4. 在完全培养基中稀释HIV-1原液以达到MOI < 1(例如。,0.1,0.01,0.001)。
    注意:由于所有靶细胞在没有精液的情况下已经感染,因此在高MOI下不能观察到精液的HIV-1增强活性。
  5. 在37℃水浴中迅速解冻精液样品,立即在PBS中稀释,以在96孔圆底板中达到0,0.8,4,20和100%(v/v)的精液浓度。 > 注意:精液的增强活性随着时间的推移而消失。避免长时间暴露于室温。
  6. 在96孔圆底板中加入40μl各自的HIV-1稀释液至40μl精液稀释液(1:1),以达到0,0.4,2,10和50%(v/v)的精液浓度。
  7. 重悬HIV-1 /精液混合物;添加20微升到280微升(1到15)细胞一式三份;最终的细胞培养物浓度为0,0.027,0.134,0.67,3.33%(v/v)(图1)。
    注意:细胞上的精液浓度低会降低其细胞毒性作用。
  8. 在37℃,5%CO 2湿润培养箱中孵育2-3小时。
  9. 然后,取出培养基并加入200μl含有100μg/ml庆大霉素的新鲜完全培养基
    1. TZM-bl细胞:简单的培养基更换就足够了。
    2. PBMC:在室温下在300×g离心5分钟,弃去100μl(300μl)上清液(无细胞),重悬细胞并将整个样品(200μl)转移到96孔V底板。在室温下以300×g离心5分钟,弃去上清液,将细胞重悬于200μl具有100μg/ml庆大霉素和10ng/ml IL-2的新鲜完全培养基中,并转移至96孔F底板。
    注意:中度改变可以防止精液的毒性作用。
  10. 在37℃下在5%CO 2湿润培养箱中孵育
  11. 2和3天后,通过光学显微镜分析所有样品,以检测由HIV-1感染引起的细胞病变效应(CPE),以及精液可能的细胞毒性效应。
    注意:
    1. 对于TZM-bl细胞:在该细胞系中,大量HIV-1感染导致合胞体形成(参见图2或Kim等人,2010中的补充图1)。如果在第2天可检测合胞体,则确定所有样品中的细胞β-半乳糖苷酶活性。继续孵育已经显示CPE另一个24小时的文化可能导致过度感染和细胞损失,应该避免。如果在第2天没有检测到CPE,将细胞孵育另外24小时,并在感染后第3天确定报告基因活性。最终细胞培养物浓度为3.33%的精液有时是细胞毒性的。因此,我们极力建议在没有病毒的情况下并行运行细胞毒性测定。
    2. 对于PBMC:在PBMC中,HIV-1诱导的CPE较不明显。如果合胞体是可检测的,在第2天确定所有样品中的荧光素酶活性。如果在第2天没有可检测到CPE,将细胞孵育另外24小时,并在感染后第3天确定报告基因活性。注意,PBMC更易受精液的细胞毒性影响(补充图17在Münch等人,2007)。因此,我们极力建议在没有病毒的情况下并行运行细胞毒性测定。
  12. 确定报告基因活性:
    1. 对于使用Gal-Screen TMβ-半乳糖苷酶报告基因测定系统的TZM-bl细胞(图3和4)
      1. 除去上清液。
      2. 加入40μl裂解缓冲液与底物(根据制造商的说明书混合)。
      3. 在室温下孵育30分钟。
      4. 将35μl裂解的细胞转移到白色96孔光度计板。
      5. 在发光计中读取发光度作为相对光单位/s。
    2. 对于使用荧光素酶测定系统感染编码萤火虫荧光素酶的HIV-1报道病毒的PBMC
      1. 重悬PBMCs并转移到96孔V底板。
      2. 在室温下离心5分钟,550×g
      3. 弃去上清液。
      4. 加入40μl的1x裂解缓冲液
      5. 在室温下孵育10分钟。
      6. 重悬并将30μl裂解的样品转移到白色96孔光度计平板中
      7. 加入100μl底物
      8. 立即,在发光计中读取作为相对光单位/s的发光。
  13. 评估原始数据(图3和图4)。
  14. 计算从未感染细胞获得的平均值(背景)(图3B)。
  15. 减去每个样品的平均背景值(图3B)。
  16. 计算每个重复测量的平均值和标准偏差(图3C和4A)。
  17. 通过设置在没有精液= 1时观察到的报道基因活性来计算n倍增强值(图3D和4B)。

代表数据



图1.用于研究精液对HIV-1感染的影响的实验中使用的微量滴定板的典型布局接种在内部60孔中的细胞用预先用0预处理的指定MOI的HIV-1接种,0.4,2,10和50%的精液,或保留未感染。灰色:PBS;白色:未感染;有色:HIV-1的不同MOI

图2.在存在精液的情况下感染的TZM-bl细胞的光学显微镜分析。用已经用指定浓度的精子预孵育的PBS(未感染的)或HIV-1接种TZM-bl细胞。细胞上的最终精液浓度在括号中给出。次要(0%精液),少(2%精液)和强(10%精液)CPE。比例尺为100μm。


图3.代表性报道基因测定的结果和评价 HIV-1 NL4-3 92Th014-2(R5-嗜性)的不同MOI(红色:0.1,蓝色:0.01,绿色:0.001)在感染TZM-bl细胞之前用指定浓度的精液预培养。 A.表示在每个孔中测量的β-半乳糖苷酶活性的相对光单位/秒的原始数据。计算并减去平均背景(未感染细胞)。 C.计算三次感染的平均和标准偏差。 D.通过设置0%精液样品= 1.计算折叠增强。


图4.显示在病毒暴露后3天测量的平均β-半乳糖苷酶活性(n = 3)。 RLU/s:相对光单位/秒。条上方的数字相对于相应的PBS对照测量的,给出了通过精液的HIV感染的n倍增强。 B.表示为条形图的n倍增强值。值表示从一式三份感染获得的平均值±标准偏差。注意,在病毒粒子处理期间(对应于3.3%的精液的最终细胞培养物浓度)50%的精液是细胞毒性的,导致感染率降低。

笔记

以下问题需要仔细考虑:

  1. 不要添加DEAE葡聚糖,聚凝胺或其他常用于TZM-bl检测以增加感染率的添加剂。加入单独的增强剂将掩盖精液的感染增强活性
  2. 使用低MOIs,在感染率低于10%的情况下是亚传染性的或没有精液。只有这样,才能确定精液对病毒感染的增强作用。此外,低MOI类似于性传播过程中的生理HIV-1浓度
  3. 精液的HIV-1增强活性随时间降低。因此,精液需要快速处理。
  4. 病毒预先暴露于精液允许精液淀粉样蛋白与病毒颗粒结合,因为它可能也在体内发生。
  5. 精液,收集后,通常不是无菌的,因此需要加入庆大霉素以避免细菌生长。
  6. 甚至低的精液浓度(从细胞上的1%开始)是细胞毒性的。因此,需要减少精液浓度并缩短暴露时间(例如,通过培养基改变)。
  7. 同时,建议进行细胞毒性试验。 [例如。,MTT assay(见Kim et al 。,2010)或发光细胞活力测定]
  8. 测定不需要在平的96孔板中进行,而是也可以按比例改变成不同的形式

如果考虑这些点,则可以修改和修改实验装置以允许测量不同靶细胞与任何HIV毒株和报道病毒的感染(即GFP,接着流式细胞术读出) ,以及其他病毒如HCMV和HSV(Tang等人,2013; Torres等人,2015)。
可以使用相同的方案检查由体外产生的淀粉样蛋白原纤维的感染增强,除了避免步骤的毒性,即添加庆大霉素,大量培养基,去除的有毒精液2-3小时后可以省略。

食谱

  1. 完全培养基用于贴壁TZM-bl细胞 DMEM
    10%热灭活的FCS
    1%PenStrep
    1%L-谷氨酰胺 (100μg/ml庆大霉素)
  2. 用于悬浮主PBMC的完整培养基
    RPMI
    10%热灭活的FCS
    1%PenStrep
    1%L-谷氨酰胺 10 ng/ml IL-2 (100μg/ml庆大霉素)

致谢

该测定首次公开在(Münch等人,2007)和在(Kim等人,2010)中详细描述的方案中。感谢AnnikaR?cker,Edina Lump和Onofrio Zirafi仔细阅读和修订协议。 Janis A.Müller是国际分子医学研究生院Ulm的一部分。

参考文献

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Copyright: © 2017,  Müller et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Müller, J. A. and Münch, J. (2016). Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection . Bio-protocol 6(14): e1871. DOI: 10.21769/BioProtoc.1871.
  2. Lump, E., Castellano, L. M., Meier, C., Seeliger, J., Erwin, N., Sperlich, B., Stürzel, C. M., Usmani, S., Hammond, R. M., von Einem, J., Gerold, G. 6., Kreppel, F., Bravo-Rodriguez, K., Pietschmann, T., Holmes, V. M., Palesch, D., Zirafi, O., Weissman, D., Sowislok, A., Wettig, B., Heid, C., Kirchhoff, F., Weil, T., Klärner, F. G., Schrader, T., Bitan, G., Sanchez-Garcia, E., Winter, R., Shorter, J. and Münch, J. (2015). A molecular tweezer antagonizes seminal amyloids and HIV infection. eLife 4: 05397.
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