W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
mBio. 2020 Feb 11;11(1):e03253-19. doi: 10.1128/mBio.03253-19.
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 replication of SINV in neural cells and 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 and and determined the outcome from alphavirus encephalomyelitis in mice. 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.
宏结构域 (MD) 是一种高度保守的蛋白质折叠结构，存在于部分正链 RNA 病毒中，可结合并水解 ADP-核糖基 (ADPr) 从 ADP-核糖基化的蛋白质。甲病毒非结构蛋白 3 (nsP3) MD 对 ADPr 的结合和水解对于病毒在神经细胞中的复制起始是必需的，而水解酶活性则促进了复制复合物的扩增。为了确定这些活性对甲病毒脑炎发病机制的重要性，我们引入了辛德毕斯病毒 (SINV) nsP3 MD 中的突变，并评估了对 ADPr 结合和水解酶活性、病毒复制、免疫反应和疾病的影响。消除 ADPr 结合和水解酶活性 (G32E) 严重损害了 SINV 在神经细胞中的复制和 2 周龄小鼠中枢神经系统中的复制，在复制过程中恢复为野生型 (WT) (G) 或选择更不妥协的变化 (S)。与 WT 相比，结合和水解酶活性降低的 SINVs (G32S 和 G32A) 或水解酶缺陷与更好的 ADPr 结合 (Y114A) 的 SINVs 的毒力降低。与 WT 相比，G32S 病毒在大脑和脊髓中的复制情况较差，诱导相似的先天反应，并且幸存者完全恢复后疾病的严重程度较低，而 Y114A 病毒复制良好，诱导干扰素刺激和 NF-κB 诱导基因的表达更高，并且从脊髓中清除速度较慢，幸存者持续瘫痪。因此，MD 功能对于神经细胞复制 both 和 均很重要，并决定了小鼠中甲病毒脑炎的结局。病毒性脑炎是导致长期残疾和急性致命疾病的重要原因。确定病毒结局的决定因素有助于评估疾病严重程度并开发新的治疗方法。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 的高度保守宏结构域结合并可以从 ADP-核糖基化的蛋白质中去除 ADP-核糖基 (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，则病毒无法生长。如果结合和水解酶活性受损，病毒的生长不如野生型病毒好，诱导相似的先天反应，并且疾病的严重程度较低，大多数感染的小鼠都恢复了。如果结合得到改善，但水解酶活性降低，则病毒复制良好，诱导的先天反应大于 WT，但从神经系统中清除受损，小鼠仍瘫痪。因此，宏结构域功能决定了甲病毒脑炎的结局。
