Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles.
Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles.
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DOI：
10.3390/v12111296
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发表时间：
2020-11-12
期刊：
影响因子：
--
通讯作者：
Guzzo C
中科院分区：
文献类型：
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作者：
Burnie J;Tang VA;Welsh JA;Persaud AT;Thaya L;Jones JC;Guzzo C
HIV-1 糖蛋白刺突 (gp120) 通常是细胞在启动免疫反应之前遇到的第一个病毒抗原，并且通常是疫苗设计中的唯一目标。因此，表征 HIV 颗粒表面细胞抗原的存在可能有助于识别新的抗病毒靶点或影响 gp120 的靶向。尽管表征病毒颗粒表面的蛋白质很重要，但目前用于此目的的技术不支持病毒的高通量分析，并且通常仅提供病毒相关蛋白质的半定量评估。传统的批量技术通常评估病毒制剂的平均值，这可能掩盖病毒亚群中微妙但重要的差异。另一方面，提供单个病毒体细节的显微镜技术难以以高通量方式使用，并且抗原检测的灵敏度较低。流式细胞术是一种传统上用于快速、高灵敏度表征单细胞的技术，但在检测病毒方面的用途有限，因为病毒颗粒的小尺寸阻碍了其检测。在此，我们报告了使用流式细胞术通过光散射和荧光对 HIV-1 假病毒颗粒进行检测和表面抗原表征，由于其特定于病毒的应用，称为流式病毒测定法。我们通过直接染色含有病毒粒子的细胞上清液来定量病毒包膜中的三种细胞蛋白（整合素α4β7、CD14和CD162/PSGL-1），无需额外的处理步骤来区分病毒颗粒或特定的病毒纯化技术。我们还表明，除了 α4β7、CD14 和 CD162/PSGL-1 之外，还可以在单个 HIV 病毒体表面同时检测到两种抗原，探测四跨膜蛋白标记 CD81。这项研究证明了校准流式病毒测定法作为一种工具的新进展，以比以前报道的技术更有效、定量的方式提供病毒包膜的灵敏、高通量表征。
The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.
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影响因子：
16
作者：
Crescitelli R;Lässer C;Szabó TG;Kittel A;Eldh M;Dianzani I;Buzás EI;Lötvall J
通讯作者：
Lötvall J
影响因子：
3.7
作者：
Bonar, Michal M.;Tilton, John C.
通讯作者：
Tilton, John C.
影响因子：
4.6
作者：
Arakelyan, Anush;Petersen, Jennifer D.;Margolis, Leonid
通讯作者：
Margolis, Leonid
影响因子：
15.9
作者：
Arakelyan, Anush;Fitzgerald, Wendy;Grivel, Jean-Charles
通讯作者：
Grivel, Jean-Charles
靶向α4β7整联蛋白可减少猿猴免疫缺陷病毒的粘膜传播，并保护与肠道相关的淋巴组织免受感染。
影响因子：
82.9
作者：
通讯作者：
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