The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.
The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.
复制标题
DOI：
10.3390/v8050144
复制
发表时间：
2016-05-21
期刊：
影响因子：
--
通讯作者：
Ogino T
中科院分区：
文献类型：
--
作者：
Ogino M;Ito N;Sugiyama M;Ogino T
狂犬病病毒大蛋白（L蛋白）在病毒RNA的合成和加工过程中起着多种酶促作用。然而，没有一个假定的酶活性已在体外直接证明。在这项研究中，我们表达并纯化了重组形式的RABV L蛋白，并验证了其鸟苷5′-三磷酸酶和GDP多核糖核苷酸转移酶（PRNTase）的活性，这是通过非常规机制形成病毒mRNA帽所必需的。RABV L蛋白用GDP将RABV mRNA起始序列5′-AACA（C/U）的5′-三磷酸化而非5′-二磷酸化帽状结构G（5′）ppp（5′）A加帽。发现底物RNA中的5′-AAC序列对于RABV L蛋白的RNA加帽是严格必需的。此外，定点诱变显示RABV L蛋白的PRNTase基序A至E中的一些保守氨基酸残基（G1112、T1170、W1201、H1241、R1242、F1285和Q1286）是帽形成所需的。这些发现表明，RABV L蛋白中推定的PRNTase结构域催化弹状病毒特异性加帽反应，该反应涉及pRNA转移至GDP的共价催化，从而提供该结构域作为开发抗病毒剂的靶标。
The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U), with GDP to generate the 5′-terminal cap structure G(5′)ppp(5′)A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.