Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins.
Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins.
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DOI：
10.1186/1743-422x-5-82
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发表时间：
2008-07-18
期刊：
影响因子：
4.8
通讯作者：
Roy P
中科院分区：
文献类型：
--
作者：
Liu L;Celma CC;Roy P
裂谷热病毒(RVFV)感染过程和形态发生的研究一直受到阻碍，因为处理这种病毒所需的生物安全条件，使替代系统，如重组病毒样颗粒，可能有助于了解这些过程是非常必要的。在这份报告中，我们介绍了RVFV结构蛋白N、Gn和GC的表达和特性，并证明了利用杆状病毒表达系统有效地产生RVFV病毒样颗粒(VLP)。构建了在多角体启动子控制下高效表达RVFV核衣壳蛋白的重组杆状病毒。凝胶过滤分析表明，表达的N蛋白可形成复杂的多聚体。此外，电子显微镜(EM)显示N蛋白复合体呈颗粒状、核衣壳样颗粒(NLP)。随后，利用双杆状病毒载体构建了表达RVFV糖蛋白(Gn/GC)和N蛋白的单一重组病毒。Gn和Gc糖蛋白不仅存在于细胞质中，而且存在于感染细胞的细胞表面。此外，在昆虫细胞中表达的Gn/GC能够在低pH漂移后诱导细胞-细胞融合，表明其功能特性保持不变。此外，通过酒石酸钾-甘油梯度离心法纯化细胞上清液，然后进行EM分析，鉴定了这三种结构蛋白组装成VLP。纯化后的颗粒呈现出与野生型RVFV病毒颗粒结构相似的包膜结构。同时，构建了只表达GC蛋白和N蛋白的重组病毒。这种双重重组病毒也产生了具有明显刺状结构的VLP，但似乎比同时含有两种糖蛋白的VLP具有更多的多形性，这表明GC和Gn可能与N蛋白复合体相互作用。我们的结果表明，杆状病毒表达系统具有产生大量VLP的巨大潜力，可用于RVFV的基础和应用研究。
Studies on Rift Valley Fever Virus (RVFV) infection process and morphogenesis have been hampered due to the biosafety conditions required to handle this virus, making alternative systems such as recombinant virus-like particles, that may facilitate understanding of these processes are highly desirable. In this report we present the expression and characterization of RVFV structural proteins N, Gn and Gc and demonstrate the efficient generation of RVFV virus-like particles (VLPs) using a baculovirus expression system. A recombinant baculovirus, expressing nucleocapsid (N) protein of RVFV at high level under the control of the polyhedrin promoter was generated. Gel filtration analysis indicated that expressed N protein could form complex multimers. Further, N protein complex when visualized by electron microscopy (EM) exhibited particulate, nucleocapsid like-particles (NLPs). Subsequently, a single recombinant virus was generated that expressed the RVFV glycoproteins (Gn/Gc) together with the N protein using a dual baculovirus vector. Both the Gn and Gc glycoproteins were detected not only in the cytoplasm but also on the cell surface of infected cells. Moreover, expression of the Gn/Gc in insect cells was able to induce cell-cell fusion after a low pH shift indicating the retention of their functional characteristics. In addition, assembly of these three structural proteins into VLPs was identified by purification of cells' supernatant through potassium tartrate-glycerol gradient centrifugation followed by EM analysis. The purified particles exhibited enveloped structures that were similar to the structures of the wild-type RVFV virion particle. In parallel, a second recombinant virus was constructed that expressed only Gc protein together with N protein. This dual recombinant virus also generated VLPs with clear spiky structures, but appeared to be more pleomorphic than the VLPs with both glycoproteins, suggesting that Gc and probably also Gn interacts with N protein complex independent of each other. Our results suggest that baculovirus expression system has enormous potential to produce large amount of VLPs that may be used both for fundamental and applied research of RVFV.