Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1
Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1
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DOI：
10.1128/aac.00309-13
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发表时间：
2013-10-01
影响因子：
4.9
通讯作者：
Chaiken, Irwin
中科院分区：
文献类型：
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作者：
Contarino, Mark;Bastian, Arangassery R.;Chaiken, Irwin
人类免疫缺陷病毒(HIV)是导致艾滋病大流行的主要病原体。在这项工作中，我们使用嵌合重组蛋白策略来测试使用同时结合Env蛋白和病毒膜的试剂不可逆地破坏HIV-1病毒粒子的可能性。我们构建了凝集素氰基韦林-N(CVN)和gp41膜-近端外区(MPER)肽的融合，并在前者的C端和后者的N端之间添加了可变长度的(Gly(4)Ser)(X)连接子(其中x为4或8)。His标记的重组蛋白在BL21(DE3)pLysS细胞中表达，并通过固定化金属亲和层析和凝胶过滤纯化，发现在阻断HOS.T4.R5细胞Bal假型HIV-1感染方面显示出纳摩尔效应。这种抗病毒活性是HIV-1特异性的，因为它不能抑制水泡性口炎病毒(VSV)或两性小鼠白血病病毒对细胞的感染。重要的是，嵌合蛋白被发现在没有宿主细胞的情况下，以剂量依赖的方式从bal假型HIV-1和完全感染的bal HIV-1中释放病毒内的p24蛋白。MPER或CVN的添加被发现超过了这种病毒裂解作用，表明嵌合体的两个成分都是病毒裂解所必需的。使用嵌合配体与Env蛋白尖峰和膜结合可以破坏病毒的稳定并导致灭活，这一发现为研究病毒颗粒的亚传性和评估这种方法在暴露于病毒的最早阶段和宿主细胞遭遇之前的灭活提供了一种手段。
Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly(4)Ser)(x) linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.