Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.
Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.
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DOI：
10.1371/journal.ppat.1004879
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发表时间：
2015-05
期刊：
影响因子：
6.7
通讯作者：
Spiropoulou CF
中科院分区：
文献类型：
--
作者：
Bergeron É;Zivcec M;Chakrabarti AK;Nichol ST;Albariño CG;Spiropoulou CF
克里米亚刚果出血热病毒（CCHFV）是布尼亚病毒科（Nairovirus属）的负链RNA病毒。在人类中，CCHFV引起发热、出血、严重血小板减少和高死亡率。精确确定CCHFV高致病性的基础的主要障碍是缺乏生产重组CCHFV的方法。我们建立了一个基于质粒转染BSR-T7/5和Huh 7细胞的反向遗传学系统。在我们的系统中，噬菌体T7 RNA聚合酶产生互补的RNA拷贝的病毒的S，M，和L节段的支持下，在反式，CCHFV核蛋白和L聚合酶的双核苷酸。该系统进行了优化，系统地回收高产量的传染性CCHFV。此外，我们测试了该系统产生专门设计的CCHFV突变体的能力。M片段编码由宿主前蛋白转化酶（PC）加工的多蛋白，包括位点-1蛋白酶（S1 P）和弗林蛋白酶样PC。S1 P和弗林蛋白酶切割是产生非结构糖蛋白GP 38所必需的，而S1 P切割产生结构Gn。我们研究了弗林蛋白酶切割的作用，拯救重组CCHFV编码的病毒糖蛋白前体缺乏功能弗林蛋白酶切割基序（RSKR突变为ASKA）。ASKA突变阻断了糖蛋白前体向GP 38的成熟，并且Gn前体向Gn的成熟略有减少。弗林蛋白酶切割对于复制不是必需的，因为阻断弗林蛋白酶切割仅导致CCHFV滴度的瞬时降低，表明GP 38和/或Gn成熟降低导致病毒体产生减少。我们的数据表明，内罗病毒可以通过反向遗传学产生，并且我们的系统的实用性揭示了弗林蛋白酶切割的功能。该病毒拯救系统可进一步用于研究CCHFV复制周期，并促进有效疫苗的开发，以应对这种生物和公共卫生威胁。克里米亚刚果出血热（CCHF）是一种严重的病毒性疾病，其特征是快速发热、出血和高病死率。CCHF病毒（CCHFV）是布尼亚病毒科（Bunyaviridae）内罗病毒属（Nairovirus）的负链RNA病毒，是CCHF的病原体。目前还没有专门的治疗方法或有效的疫苗来对抗CCHF。为了研究内罗病毒发病机制和生物学的分子决定因素，我们开发了一种反向遗传学系统，能够产生CCHFV变体，其基因组序列由转染到细胞中用于病毒回收的质粒定义。我们的系统是第一个证明可以从质粒DNA的简单转染中有效地回收内罗病毒的系统，为特异性编辑CCHFV和其他内罗病毒的基因组铺平了道路。使用该系统，我们设计突变阻断CCHFV的非结构糖蛋白在宿主蛋白酶弗林蛋白酶识别的基序处的切割。使用这种弗林蛋白酶抗性CCHFV变体，我们证明了弗林蛋白酶直接裂解病毒糖蛋白导致病毒粒子产生滞后，揭示了这些糖蛋白在高效CCHFV复制中的功能。我们的实验突出了反向遗传学系统的效用，用于开发病毒变异体，用于调查CCHFV蛋白质功能和合理设计疫苗株。
Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat. Crimean Congo hemorrhagic fever (CCHF) is a severe viral disease characterized by rapid-onset fever, hemorrhage, and high case fatality rates. CCHF virus (CCHFV), the causative agent of CCHF, is a negative-strand RNA virus of the family Bunyaviridae (genus Nairovirus). No specific treatments or efficacious vaccines exist to combat CCHF. To investigate molecular determinants of nairovirus pathogenesis and biology, we developed a reverse genetics system capable of generating CCHFV variants with genome sequences defined by the plasmids transfected into cells for virus recovery. Our system is the first to demonstrate that a nairovirus can be efficiently recovered from the simple transfection of plasmid DNA, paving the way for specifically editing genomes of CCHFV and other nairoviruses. Using this system, we engineered mutations blocking the cleavage of CCHFV’s non-structural glycoproteins at a motif recognized by the host protease furin. Using this furin-resistant CCHFV variant, we demonstrate that direct cleavage of the viral glycoprotein by furin results in a lag in virion production, revealing a function of these glycoproteins in efficient CCHFV replication. Our experiments highlight the utility of a reverse genetics system for developing viral variants for investigating CCHFV protein function and for rationally designing vaccine strains.
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