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-κB-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.

中文翻译：

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Alphavirus nsP3宏域的ADP-核糖结合和水解酶活性都会影响小鼠的神经毒性。

巨域（MD）是存在于正链RNA病毒子集中的高度保守的蛋白质折叠，它结合并水解来自ADP核糖基化蛋白质的ADP核糖（ADPr）。甲型病毒非结构蛋白3（nsP3）MD的ADPr结合对于启动神经细胞中的病毒复制是必需的，而水解酶活性则有助于复制复合物的扩增。为了确定这些活性对于甲型病毒性脑脊髓炎发病机理的重要性，已将突变引入辛德比斯病毒（SINV）的nsP3 MD中，并评估了其对ADPr结合和水解酶活性，病毒复制，免疫应答和疾病的影响。消除ADPr结合和水解酶活性（G32E）严重损害了SINV在神经细胞中的体外复制以及在2周龄小鼠的中枢神经系统中的体内复制，并恢复了野生型（WT）（G）或选择复制期间影响较小的更改（S）。结合和水解酶活性降低的SINV（G32S和G32A）或水解酶缺乏与更好的ADPr结合（Y114A）结合的SINV的毒性低于野生型病毒。与WT相比，G32S病毒在脑和脊髓中的复制均较差，诱导了类似的先天反应，并且在幸存者完全恢复的情况下所引起的严重程度较低的疾病，而Y114A病毒则复制得很好，诱导了受干扰素刺激和抑制的高表达。 NF-κB诱导的基因 幸存者在持续麻痹的情况下从脊髓清除的速度更慢。因此，MD功能对于体内和体外神经细胞复制都很重要，并决定了小鼠甲型病毒性脑脊髓炎的预后。重要提示病毒性脑脊髓炎是长期致残以及急性致死性疾病的重要原因。鉴定结果的病毒决定因素有助于评估疾病的严重程度并开发新的治疗方法。蚊子传播的α病毒感染神经元并在小鼠中引起致命的疾病。高度保守的非结构蛋白3的巨域结合并可以从ADP-核糖基化的蛋白质中去除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。如果大域突变消除了ADPr结合或水解酶活性，病毒没有增长。如果结合和水解酶活性受损，病毒的生长速度将不及野生型病毒，引起相似的先天反应，并引起较轻的疾病，大多数感染的小鼠得以康复。如果结合力得到改善，但水解酶活性降低，则病毒可以很好地复制并诱导比野生型更大的先天应答，但会损害神经系统的清除能力，并使小鼠瘫痪。因此，宏结构域功能决定了α病毒性脑脊髓炎的预后。与WT相比，该病毒能够很好地复制并诱导更大的先天应答，但是从神经系统的清除受到损害，小鼠仍然处于瘫痪状态。因此，宏结构域功能决定了α病毒性脑脊髓炎的预后。与WT相比，该病毒能够很好地复制并引起更大的先天应答，但是从神经系统的清除受到损害，小鼠仍然处于瘫痪状态。因此，宏结构域功能决定了α病毒性脑脊髓炎的预后。