Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein
Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein
复制标题
埃博拉病毒糖蛋白假型流感病毒的表征
作者：
J. Xiao;P. Rijal;L. Schimanski;A. Tharkeshwar;E. Wright;W. Annaert;A. Townsend
我们已经产生了一种新的埃博拉病毒假型，E-S-FLU，可以在生物安全等级1/2的容器中处理，用于实验室分析。E-S-FLU病毒是一种包裹有埃博拉病毒糖蛋白的单循环流感病毒，它编码增强型绿色荧光蛋白作为报告蛋白，取代流感病毒血凝素。MDCK-SIAT1细胞被转导表达埃博拉病毒糖蛋白，作为E-S-FLU病毒生产的稳定跨膜蛋白。E-S-FLU病毒感染细胞依赖于Niemann-Pick C1蛋白，该蛋白是埃博拉病毒进入内核体/溶酶体晚期膜的典型受体。E-S-FLU病毒被一种抗埃博拉病毒糖蛋白抗体和多种已知可抑制野生型埃博拉病毒进入的小药物分子特异性中和。为了证明这种新的埃博拉病毒假型的应用，我们表明单个实验室批次足以筛选1,280个具有药理活性的化合物文库（LOPAC1280; Sigma）以抑制病毒进入。共有215种化合物抑制E-S-FLU病毒感染，而只有22种化合物抑制H5血凝素包被的对照H5- s - flu病毒。这些抑制性化合物的靶点和作用机制非常分散，如钙通道阻滞剂、雌激素受体拮抗剂、抗组胺药、血清素摄取抑制剂等，这与文献中其他假型或野生型埃博拉病毒的抑制剂筛选结果相关。E-S-FLU病毒是埃博拉病毒进入细胞研究的新工具，很容易应用于小分子抑制剂或抗体的高通量筛选试验。埃博拉病毒属于丝状病毒科，是生物安全4级病原体。目前还没有fda批准的治疗埃博拉病毒的药物。这些特点使得开发埃博拉病毒的替代品成为必要，这些替代品可以在更方便的实验室控制中处理，以研究病毒的生物学特性并筛选抑制剂。在这里，我们描述了一种新的替代品，命名为E-S-FLU病毒，它是基于一种被埃博拉病毒表面蛋白包裹的失活流感病毒核心，但不包含任何来自埃博拉病毒本身的遗传信息。我们发现E-S-FLU病毒使用与野生型埃博拉病毒相同的细胞进入途径。作为E-S-FLU病毒在1/2生物安全等级容器中易于使用的一个例子，我们表明，单个生产批次可以提供足够的替代病毒，以筛选包含1,280个候选病毒进入抑制剂的标准小分子文库。
ABSTRACT We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies. IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.
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影响因子：
3.7
作者：
Rossman JS;Lamb RA
通讯作者：
Lamb RA
影响因子：
5.3
作者：
van der Linden WA;Schulze CJ;Herbert AS;Krause TB;Wirchnianski AA;Dye JM;Chandran K;Bogyo M
通讯作者：
Bogyo M
影响因子：
20.3
作者：
Klein, U;Gloghini, A;Carbone, A
通讯作者：
Carbone, A
影响因子：
4.9
作者：
Funk RS;Krise JP
通讯作者：
Krise JP
DOI：
10.1126/science.1258758
发表时间：
2015-02-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Sakurai Y;Kolokoltsov AA;Chen CC;Tidwell MW;Bauta WE;Klugbauer N;Grimm C;Wahl-Schott C;Biel M;Davey RA
通讯作者：
Davey RA