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HIV-1 Single Cycle Infection
HIV-1单循环感染   

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Abstract

The role of a viral or cellular protein on HIV-1 infection can sometimes be difficult to assess in a system where the virus is able to replicate for several cycles. Indeed, some effects are only observable at early time points, and can be masked (or on the contrary, artificially increased) after several rounds of viral replication. Therefore, to clearly know at which step of HIV-1 replication cycle one protein acts, it is important to be able to study one cycle of infection only. This protocol allows rapid and robust quantification of HIV-1 single cycle infection, and can be used to compare mutant viruses, or treatments with different drugs.

Materials and Reagents

  1. HeLa CD4+ CCR5+ LTR lacZ (also known as P4C5) cells (Verrier et al., 1997)
  2. Dulbecco Eagle’s Modified Medium (DMEM) (Life Technologies, Gibco®)
  3. HIV-1 Δenv viral particles, produced by transfection of HEK-293T cells
  4. Nonidet P40 (NP40) (Sigma-Aldrich)
    No longer commercially available, but replaced by Igepal CA-630 (Sigma-Aldrich, catalog number: I8896 )
  5. Phosphate buffer saline (PBS) (Life Technologies, Gibco®)
  6. Chlorophenol Red-β-D-Galactopyranoside (CPRG) (F. Hoffmann-La Roche, catalog number: 10884308001 )
  7. Fetal bovine serum (FBS) (Thermo Fisher Scientific)
  8. Penicillin/Streptomycin (PS) (Life Technologies, Gibco®)

Equipment

  1. 96-well spectrophotometer
  2. Flat-bottom 96-well plates (Corning, Costar®)

Procedure

  1. P4C5 cells are HeLa cells, stably expressing CD4 and CCR5. Additionally, they express the lacZ gene under the control of HIV-1 Long Terminal Repeat (LTR) promoter. They are grown like normal HeLa cells, in DMEM, 10% heat-inactivated FBS, 10% PS, and kept at 5% CO2 and at 37 °C.
  2. 8 x 104 P4C5 cells were plated in flat-bottom 96-well plates in a final volume of 100 μl of complete medium for 24 h.
  3. Cells were infected in triplicate with HIV-1 Δenv, using 1 or 5 ng of Gag p24 per well. Specifically, 100 μl of virus-containing medium were added to the 100 μl of complete medium already in the well. Viral titers have to be previously determined using a Gag p24 ELISA assay (either home-made or commercial). The use of two different viral inputs is required to make sure that the saturation levels are not reached.
  4. At 36 h post-infection, cells were lysed in 80 μl of lysis buffer, then incubated for 1 min at room temperature with 80 μl of lysis buffer, containing CPRG at a final concentration of 3.65 mg/ml. CPRG, the substrate of the β-galactosidase enzyme, allows to monitor the levels of infection. OD (570 nm) was measured every 15 min and normalized against background levels of OD (690 nm).
  5. Infection levels - as monitored by percentage of Gag+ cells by flow cytometry, or quantification of Gag p24 in the culture supernatants by ELISA - are proportionnal to the normalized OD (570 nm). The assay is linear from 0.1 to 3 units of OD (570 nm). Representative results are shown in Figure 3B of Roesch et al. 2012.

Recipes

  1. Complete medium
    DMEM
    10% heat-inactivated FBS
    10% PS
  2. Lysis buffer
    PBS
    0.1% NP40
    5 mM MgCl2

Acknowledgments

This protocol is adapted from Roesch et al. (2012).

References

  1. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.
  2. Verrier, F. C., Charneau, P., Altmeyer, R., Laurent, S., Borman, A. M. and Girard, M. (1997). Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolateProc Natl Acad Sci U S A 94(17): 9326-9331.

简介

病毒或细胞蛋白质对HIV-1感染的作用有时可能难以在其中病毒能够复制几个周期的系统中评估。 事实上,一些作用仅在早期时间点可观察到,并且可以在几轮病毒复制后被掩蔽(或相反,人为增加)。 因此,为了清楚知道一种蛋白质作用于HIV-1复制循环的哪个步骤,重要的是能够仅研究一个周期的感染。 该协议允许快速和强大的量化HIV-1单周期感染,并可用于比较突变病毒或不同药物的治疗。

材料和试剂

  1. (也称为P4C5)细胞(Verrier等人,1997),其中所述癌细胞包括人类癌细胞和人类癌细胞(HeLa CD4 +
  2. Dulbecco Eagle's Modified Medium(DMEM)(Life Technologies,Gibco )
  3. 通过转染HEK-293T细胞产生的HIV-1ΔEvenv病毒颗粒
  4. Nonidet P40(NP40)(Sigma-Aldrich)
    不再可商购,但用Igepal CA-630(Sigma-Aldrich,目录号:I8896)代替
  5. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco )
  6. 氯酚红-β-D-吡喃半乳糖苷(CPRG)(F.Hoffmann-La Roche,目录号:10884308001)
  7. 胎牛血清(FBS)(Thermo Fisher Scientific)
  8. 青霉素/链霉素(PS)(Life Technologies,Gibco )

设备

  1. 96孔分光光度计
  2. 平底96孔板(Corning,Costar )

程序

  1. P4C5细胞是稳定表达CD4和CCR5的HeLa细胞。此外,它们表达在HIV-1长末端重复(LTR)启动子控制下的lacZ基因。它们在正常HeLa细胞中,在DMEM,10%热灭活的FBS,10%PS中生长,并保持在5%CO 2和37℃。
  2. 将8×10 4个P4C5细胞接种在平底96孔板中,最终体积为100μl的完全培养基24小时。
  3. 使用1或5ng的Gag p24 /孔,用HIV-1Δenv感染细胞一式三份。具体地,将100μl的含病毒培养基加入到已经在孔中的100μl完全培养基中。病毒滴度必须使用Gag p24 ELISA测定(自制或商业化)预先测定。需要使用两种不同的病毒输入,以确保未达到饱和度水平。
  4. 在感染后36小时,将细胞在80μl裂解缓冲液中裂解,然后在室温下用80μl含有终浓度为3.65mg/ml的CPRG的裂解缓冲液温育1分钟。 CPRG,β-半乳糖苷酶的底物,允许监测感染的水平。 每15分钟测量OD(570nm),并相对于OD的背景水平(690nm)进行标准化。
  5. 通过流式细胞术或通过ELISA对培养物上清液中Gag p24的定量来监测感染水平(通过Gag 细胞百分比监测)与标准化OD(570nm)成比例。 该测定为0.1至3个OD单位(570nm)的线性。 代表性的结果如Roesch等人2012年的图3B所示。

食谱

  1. 完成媒介
    DMEM
    10%热灭活的FBS 10%PS
  2. 裂解缓冲液
    PBS
    0.1%NP40
    5mM MgCl 2/

致谢

该协议改编自Roesch等人(2012)。

参考文献

  1. Roesch,F.,Meziane,O.,Kula,A.,Nisole,S.,Porrot,F.,Anderson,I.,Mammano,F.,Fassati,A.,Marcello,A.,Benkirane, Schwartz,O。(2012)。 热疗刺激HIV-1复制。 PLoS Pathog 8(7):e1002792。
  2. Verrier,F.C.,Charneau,P.,Altmeyer,R.,Laurent,S.,Borman,A.M.and Girard,M。(1997)。 针对gp120/gp41的几种构象依赖性表位的抗体抑制CCR-5依赖性细胞 - 由原代巨噬细胞向性HIV-1分离物的天然包膜糖蛋白介导的细胞间融合。 17):9326-9331。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Roesch, F. and Schwartz, O. (2013). HIV-1 Single Cycle Infection. Bio-protocol 3(7): e416. DOI: 10.21769/BioProtoc.416.
  2. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.
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