Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice
Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice
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DOI：
10.1128/mbio.03253-19
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发表时间：
2020-01-01
期刊：
影响因子：
6.4
通讯作者：
Griffin, Diane E.
中科院分区：
文献类型：
--
作者：
Abraham, Rachy;McPherson, Robert L.;Griffin, Diane E.
宏结构域（Macrodomain，MD）是一种存在于正链RNA病毒亚群中的高度保守的蛋白质折叠结构，它与ADP核糖基化蛋白结合并水解ADP核糖。甲病毒非结构蛋白3（nsP 3）MD的ADPr结合是神经细胞中病毒复制起始所必需的，而水解酶活性促进复制复合物扩增。为了确定这些活动的甲病毒脑脊髓炎的发病机制的重要性，突变引入辛德毕斯病毒（SINV）的nsP 3 MD，并对ADPr结合和水解酶活性，病毒复制，免疫反应和疾病的影响进行了评估。ADPr结合和水解酶活性（G32 E）的消除严重损害了2周龄小鼠神经细胞中SINV的体外复制和中枢神经系统中的体内复制，并在复制过程中恢复为野生型（WT）（G）或选择损害较小的变化（S）。具有降低的结合和水解酶活性（G32 S和G32 A）或具有水解酶缺陷结合更好的ADPr结合（Y114 A）的SINV的毒性低于WT病毒。与WT相比，G32 S病毒在脑和脊髓中的复制较差，诱导了相似的先天性反应，并且引起的疾病不太严重，幸存者完全恢复，而Y114 A病毒复制良好，诱导了干扰素刺激和NF-κ B诱导的基因的更高表达，并且从脊髓中清除更慢，幸存者持续瘫痪。因此，MD功能是重要的神经细胞复制在体外和体内，并确定从甲病毒脑脊髓炎在mice.IMPORTANCE的结果病毒性脑脊髓炎是一个重要的原因，长期残疾，以及急性致命性疾病。确定结果的病毒决定因素有助于评估疾病的严重程度和开发新的治疗方法。蚊媒甲病毒感染神经元并导致小鼠致命疾病。非结构蛋白3的高度保守的宏结构域结合并可以从ADP-核糖基化蛋白质中去除ADP-核糖（ADPr）。为了确定这些功能对毒力的重要性，产生了重组突变病毒。如果宏结构域突变消除了ADPr结合或水解酶活性，则病毒不会生长。如果结合和水解酶活性受损，病毒的生长不如野生型病毒，诱导类似的先天反应，引起的疾病不太严重，大多数感染的小鼠恢复。如果结合得到改善，但水解酶活性降低，则病毒复制良好，并诱导比WT更大的先天反应，但神经系统的清除受损，小鼠仍然瘫痪。因此，宏结构域功能决定了甲病毒性脑脊髓炎的结局。
Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-kappa B-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice.IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.