Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein
Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein
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DOI：
10.1128/jvi.02730-06
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发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Doms, Robert W.
中科院分区：
文献类型：
--
作者：
Altamura, Louis A.;Bertolotti-Ciarlet, Andrea;Doms, Robert W.
克里米亚-刚果出血热病毒（CCHFV; Nairovirus属，布尼亚病毒科）的结构糖蛋白是通过内切蛋白水解切割1，684个氨基酸的M RNA片段编码的多蛋白而衍生的。该多聚蛋白被共价切割成PreG（N）和PreG（C）前体，然后它们被SKI-1和SKI-1样蛋白酶切割以分别产生G（N）和G（C）的N末端。然而，预测由G（N）和G（C）的N末端限定的所得多肽（58 kDa）大于成熟GN（37 kDa）。通过类比正布尼亚病毒属中病毒的拓扑相似的M片段编码的多蛋白，含有四个预测的跨膜结构域的PreG（N）的C末端区域也可能含有非结构蛋白NSM。为了表征潜在的PreG（N）C-末端切割事件，开发了一组表位标记的PreG（N）截短和内部缺失突变体。这些构建体允许在G（N）胞外域之后的第二个预测跨膜结构域内或非常接近第二个预测跨膜结构域的C-末端内切蛋白水解切割的鉴定和C-末端片段的后续产生。脉冲追踪实验表明，PreG（N）C-末端裂解发生在前体合成后不久，G（N）糖蛋白产生之前。由此产生的片段被运送到高尔基体，即病毒组装的场所。特异于PreG（N）的第二胞质环的抗血清的开发允许检测源自完整CCHFV M片段的瞬时表达以及病毒感染的情况下的细胞相关NSM蛋白。
The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreG(N) and PreG(C) precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of G(N) and G(C), respectively. However, the resulting polypeptide defined by the N termini of G(N) and G(C) is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreG(N) that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreG(N) C-terminal cleavage events, a panel of epitope-tagged PreG(N) truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the G(N) ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreG(N) C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the G(N) glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreG(N) allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.