Bergeron Éric, Zivcec Marko, Chakrabarti Ayan K, Nichol Stuart T, Albariño César G, Spiropoulou Christina F
Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS Pathog. 2015 May 1;11(5):e1004879. doi: 10.1371/journal.ppat.1004879. eCollection 2015 May.
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.
克里米亚刚果出血热病毒（CCHFV）是布尼亚病毒科（属：内罗毕病毒属）的一种负链RNA病毒。在人类中，CCHFV会引发发热、出血、严重血小板减少症，并导致高死亡率。精确确定CCHFV高致病性基础的一个主要障碍是缺乏生产重组CCHFV的方法。我们开发了一种基于将质粒转染到BSR-T7/5和Huh7细胞中的反向遗传学系统。在我们的系统中，噬菌体T7 RNA聚合酶产生病毒S、M和L片段的互补RNA拷贝，这些拷贝在CCHFV核蛋白和L聚合酶的反式支持下被包装。该系统经过优化，以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特定设计的CCHFV突变体的能力。M片段编码一种多蛋白，该多蛋白由宿主前蛋白转化酶（PCs）加工，包括1型位点蛋白酶（S1P）和类弗林蛋白酶PCs。S1P和弗林蛋白酶切割对于产生非结构糖蛋白GP38是必需的，而S1P切割产生结构蛋白Gn。我们通过拯救一种编码缺乏功能性弗林蛋白酶切割基序（RSKR突变为ASKA）的病毒糖蛋白前体的重组CCHFV，研究了弗林蛋白酶切割的作用。ASKA突变阻止了糖蛋白前体成熟为GP38，并且Gn前体成熟为Gn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滴度的短暂降低，这表明要么是GP38和/或Gn成熟减少导致了病毒粒子产生减少。我们的数据表明，可以通过反向遗传学产生内罗毕病毒，并且我们系统的实用性揭示了弗林蛋白酶切割的一种功能。这种病毒拯救系统可进一步用于研究CCHFV复制周期，并促进开发有效的疫苗以应对这种生物和公共卫生威胁。
