Biochemical characterization of the Lassa virus L protein
Biochemical characterization of the Lassa virus L protein
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DOI：
10.1074/jbc.ra118.006973
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发表时间：
2019-03
期刊：
影响因子：
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通讯作者：
D. Vogel;M. Rosenthal;N. Gogrefe;S. Reindl;S. Günther
中科院分区：
文献类型：
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作者：
D. Vogel;M. Rosenthal;N. Gogrefe;S. Reindl;S. Günther
竞技场病毒和布尼亚病毒的L蛋白在结构和功能上与正粘病毒聚合酶复合体相关。它在病毒生命周期中起着核心作用，因为它复制病毒基因组并通过夺帽机制产生病毒mRNA。在这里，我们的目的是生物化学表征拉沙病毒，在西非流行的人类致病性沙粒病毒的L蛋白。使用杆状病毒表达系统表达全长250-kDa L蛋白。从小角X-射线散射数据计算的低分辨率结构揭示了类似于正粘病毒聚合酶复合物的晶体结构的构象。虽然L蛋白没有表现出夺帽核酸内切酶活性，但它在体外合成RNA。RNA聚合需要锰而不是镁离子，是独立的核苷酸引物，并抑制病毒Z蛋白。最大活性由最小长度为17个核苷酸的双链启动子序列介导，该序列含有非模板化的5′-G突出端（如在天然基因组环境中）以及互补启动子链之间天然存在的碱基错配。各种短引物的实验揭示了存在两个复制起始位点的模板链和引物易位的证据所提出的引物和重新对齐的假设。总的来说，我们的研究结果提供了一个详细的了解的机制差异和共同性的分段负链RNA病毒的聚合酶蛋白，并为寻找针对拉沙病毒的RNA聚合酶的抗病毒化合物的基础。
The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro. RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5′-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.