在体外对HIV - 1核心脱壳
Summary
脱壳是在HIV – 1生命周期的早期阶段的重要一步，并定义为衣壳外壳的拆卸和释放病毒核蛋白复合物（vRNP）。在这里，我们展示了隔离HIV – 1病毒颗粒的完整的核心和量化其脱壳技术<em>在体外</em>。
Abstract
的逆转录病毒的基因组是由脂质包膜包围一个衣壳，包裹。慢病毒，如HIV – 1，锥形衣壳外壳组成的六角晶格排列的CA蛋白。衣壳是关闭的7 pentamers广泛年底，在窄的一端的锥1，2 5。在这衣壳外壳外壳是病毒的核蛋白复杂，和他们一起组成的核心。
HIV – 1病毒膜与靶细胞膜的融合，是释放到细胞质。衣壳，然后拆卸 3中的可溶性形式释放游离Ca简称为脱壳的过程。细胞内的位置和HIV – 1脱壳的时间是知之甚少。在CA的单一氨基酸替换改变衣壳的稳定性也削弱的HIV – 1感染细胞的能力4。这表明衣壳的稳定性是HIV – 1感染的关键。 </p>
HIV – 1脱壳已经很难研究由于缺乏这个过程中的敏感和可靠的检测。在这里，我们描述了一个定量方法研究使用体外传染性的HIV – 1颗粒分离的核心脱壳。该方法涉及了一层洗涤剂，并通过集中的病毒颗粒的沉淀核心的隔离成线性蔗糖梯度，在寒冷的。要量化脱壳，孤立的核心是在37 ° C的各种时间间隔，随后离心沉淀。脱壳程度上清液中的CA的一小部分通过量化分析。已采用这种方法分析HIV – 1衣壳的稳定性4，5，6的病毒突变的影响。研究HIV – 1脱壳的细胞因子的作用，还应该有用。
Protocol
1。生产的HIV – 1颗粒在继续之前，必须获得许可，并按照您的机构的生物安全办公室的工作在艾滋病毒传染性批准了一项生物安全设施的指引。您必须确保适当的照顾，并在生物安全实验室工作期间的安全防范措施。 HIV – 1颗粒的生产通常是由瞬时转染293T细胞与HIV – 1前病毒DNA使用一个磷酸钙转染方法7，8 。也适用于高滴度病毒感染T细胞的文化成?…
Discussion
这里描述的方法来净化HIV – 1核心，研究在体外脱壳研究这种HIV – 1生命周期的阶段，特别是由于没有一个有效的方法来分析靶细胞的HIV – 1脱壳。虽然这种方法使用平衡离心净化核心，其他人使用简要裂解后直接制粒，1％的Triton – X – 100 12 13或通过造粒蔗糖垫覆盖0.1％的Triton X – 100 的14，15 。
通过这种方法获得的内核的复苏是受实验误差。一个变量是在这个协议…
Disclosures
The authors have nothing to disclose.
Acknowledgements
HIV – 1脱壳在艾肯实验室的研究是由NIH的拨款AI40364，AI50423和AI076121支持。几个关键材料，包括在P24 ELISA试剂，由美国国立卫生研究院艾滋病研究和参考程序，艾滋病司，NIAID的，国立卫生研究院提供。
Materials
| Name of the reagent | Company | Catalog number | Comments (optional) |
| DMEM | Cellgro | 10-013-CV | |
| PBS | Cellgro | 21-031-CV | |
| Triton-X-100 | Mallinckrodt Baker Inc | 9002-93-1 | |
| Tween 20 | Acros | 9005-64-5 | |
| 0.25% Trypsin-EDTA | Cellgro | 25-053-CI | |
| 2XBBS | Composition: 50mM BES [pH 6.95], 280 mM NaCl and 1.5 mM Na2HPO4. Adjust pH to 6.95 at room temperature. Filter sterilize & store in aliquots at -20°C. | ||
| Coating antibody: Monoclonal antibody to p24 (183-H12-5C) | NIH AIDS Research and Reference Reagent Program | 3537 | |
| Primary antibody: HIV-Ig (hyperimmune human patient serum) |
NIH AIDS Research and Reference Reagent Program |
3957 | |
|
Secondary antibody: Goat anti-human IgG, peroxidase conjugated |
Pierce | 31130 | |
| HRP substrate | KPL Inc | 50-76-11 | |
| Immulon 2HB 96 well plates | Thermo Scientific | 3455 | |
| SW32Ti and SW32.1 Ti rotors and compatible ultracentrifuge | Beckman Coulter | ||
| TLA-55 rotor and tabletop ultracentrifuge | Beckman Coulter | ||
| High speed microfuge tubes | Beckman Coulter | 326823, 358123, 357448 | |
| Auto-Densi Flow density gradient fractionator | Labconco Corp. | 4517000 | |
| Gradient Former | CBS Scientific Co. Inc. | GM-20 |
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Tags
In Vitro UncoatingHIV-1 CoresRetrovirusesCapsidLipid EnvelopeLentivirusesCA ProteinHexagon LatticePentamersCone StructureViral Ribonucleoprotein ComplexFusionTarget Cell MembraneCytoplasmDisassemblySoluble CAUncoating ProcessIntracellular LocationTimingAmino-acid SubstitutionsStability Of The CapsidHIV-1 InfectionAssaysQuantitative MethodIsolation Of CoresSedimentationConcentrated VirionsDetergent LayerSucrose Gradient
Cite This Article
Shah, V. B., Aiken, C. In vitro Uncoating of HIV-1 Cores. J. Vis. Exp. (57), e3384, doi:10.3791/3384 (2011).