Genome-wide analysis of gene expression and nuclear/cytoplasmic distribution by compound 1 treatment [293T_NS1-2]

Analysis of cellular response to DHODH inhibition at gene expression and nuclear/cytoplasmic distribution level. The NS1 protein of influenza virus is a major virulence factor essential for virus replication as it re-directs the host cell to promote viral protein expression. NS1 inhibits cellular mRNA processing and export, down-regulating host gene expression and enhancing viral gene expression. We report here the identification of a non-toxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for *de novo* pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of VSV M protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. Five million 293T cells were non-transfected or transfected with 6ug of pCAGGS-NS1 for 16h. Then, cells were untreated or treated with compound 1(5uM) for 24h. RNA from total cell extracts or from nuclear or cytoplasmic fractions were obtained

本研究针对二氢乳清酸脱氢酶（dihydroorotate dehydrogenase, DHODH）抑制后的细胞应答，从基因表达与核质分布层面展开分析。
流感病毒NS1蛋白（NS1 protein）是病毒复制必需的主要毒力因子，其通过重编程宿主细胞以促进病毒蛋白表达。NS1蛋白可抑制宿主细胞mRNA的加工与核出转运，下调宿主基因表达并增强病毒基因的表达。
本研究报道了一种无毒的喹啉羧酸类化合物，可在病毒存在或缺失的情况下，逆转NS1蛋白介导的mRNA核出转运抑制作用。该喹啉羧酸类化合物可直接抑制二氢乳清酸脱氢酶（DHODH）——一种宿主细胞从头嘧啶生物合成所需的酶——并部分降低嘧啶类物质的水平。该效应可诱导核输出因子1（NXF1）的表达，进而在NS1蛋白存在的情况下促进mRNA的核出转运。
通过抑制DHODH解除NS1介导的mRNA出核转运阻滞的效应，同样在水泡性口炎病毒（Vesicular Stomatitis Virus, VSV）M蛋白（另一种病毒来源的mRNA出核转运抑制剂）存在的情况下发生。这种mRNA出核转运阻滞的逆转，使得抗病毒因子得以正常表达。由此可见，嘧啶类物质在毒力因子介导的mRNA核出转运抑制过程中发挥了必要作用。
实验处理如下：将5×10^6个293T细胞分为两组，一组不予转染，另一组用6μg的pCAGGS-NS1质粒转染并孵育16小时；随后将两组细胞分别不予处理或用5μM的化合物1处理，继续孵育24小时，最终收集总细胞提取物、细胞核组分或细胞质组分中的RNA。