Saeed Mohammad F, Kolokoltsov Andrey A, Davey Robert A
Department of Microbiology and Immunology, and Western Regional Center for Excellence in Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1019, USA.
J Virol Methods. 2006 Aug;135(2):143-50. doi: 10.1016/j.jviromet.2006.02.011. Epub 2006 Apr 3.
Entry is the first and essential step in virus replication and is a target for therapeutic intervention. However, current knowledge on entry mechanism for the majority of viruses is poor, partly due to lack of a simple, sensitive and accurate entry assay that can be applied to diverse viruses. To overcome this obstacle, a novel contents-mixing-based virus entry assay is described that can be broadly applied to many enveloped viruses. By fusing firefly luciferase to the HIV Nef protein, luciferase was directly packaged into HIV particles pseudotyped with envelope proteins of diverse viruses including HIV, rabies and others. Upon cell entry, the luciferase-fusion protein was released into the cell cytoplasm, reacted with its substrates and was detected by light emission. The assay was validated by demonstrating its versatility in measuring virus entry. Entry was detected much more rapidly (in real-time) with higher sensitivity (a multiplicity of infection <0.1 gives a robust signal) and lower background (signal/noise ration >1000) than other comparable assays. In addition to its utility in studying virus entry mechanisms, the assay will aid in screening potential entry/fusion inhibitors and in diagnosis of virus infections.
病毒进入是病毒复制的首要且关键步骤，也是治疗干预的靶点。然而，目前对于大多数病毒进入机制的了解尚浅，部分原因是缺乏一种可广泛应用于多种病毒的简单、灵敏且准确的进入检测方法。为克服这一障碍，本文描述了一种基于内容物混合的新型病毒进入检测方法，该方法可广泛应用于多种包膜病毒。通过将萤火虫荧光素酶与HIV Nef蛋白融合，荧光素酶被直接包装到以包括HIV、狂犬病病毒等多种病毒包膜蛋白假型化的HIV颗粒中。病毒进入细胞后，荧光素酶融合蛋白被释放到细胞质中，与底物发生反应并通过发光进行检测。通过证明其在测量病毒进入方面的通用性，验证了该检测方法。与其他类似检测方法相比，该方法能更快速（实时）地检测到病毒进入，灵敏度更高（感染复数<0.1时能产生强烈信号），背景更低（信噪比>1000）。除了在研究病毒进入机制方面的应用，该检测方法还将有助于筛选潜在的进入/融合抑制剂以及诊断病毒感染。
