Pettit Kneller Elizabeth L, Connor John H, Lyles Douglas S
Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
J Virol. 2009 Jan;83(2):770-80. doi: 10.1128/JVI.01279-08. Epub 2008 Nov 12.
Vesicular stomatitis virus (VSV) matrix protein inhibits nuclear-cytoplasmic mRNA transport. The goal of this work is to determine whether VSV inhibits the nuclear-cytoplasmic transport of heterogeneous ribonucleoproteins (hnRNPs), which are thought to serve as mRNA export factors. Confocal microscopy experiments showed that hnRNPA1, hnRNPK, and hnRNPC1/C2, but not hnRNPB1 or lamin A/C, are relocalized to the cytoplasm during VSV infection. We determined whether protein import is inhibited by VSV by transfecting cells with a plasmid encoding enhanced green fluorescent protein (EGFP) tagged with either the M9 nuclear localization sequence (NLS) or the classical NLS. These experiments revealed that both the M9 NLS and the classical NLS are functional during VSV infection. These data suggest that the inhibition of protein import is not responsible for hnRNP relocalization during VSV infection but that hnRNP export is enhanced. We found that hnRNPA1 relocalization was significantly reduced following the silencing of the mRNA export factor Rae1, indicating that Rae1 is necessary for hnRNP export. In order to determine the role of hnRNPA1 in VSV infection, we silenced hnRNPA1 in HeLa cells and assayed three aspects of the viral life cycle: host protein synthesis shutoff concurrent with the onset of viral protein synthesis, replication by plaque assay, and cell killing. We observed that host shutoff and replication are unaffected by the reduction in hnRNPA1 but that the rate of VSV-induced apoptosis is slower in cells that have reduced hnRNPA1. These data suggest that VSV promotes hnRNPA1 relocalization in a Rae1-dependent manner for apoptotic signaling.
水泡性口炎病毒（VSV）基质蛋白抑制核质mRNA转运。本研究的目的是确定VSV是否抑制异质性核糖核蛋白（hnRNPs）的核质转运，hnRNPs被认为是mRNA输出因子。共聚焦显微镜实验表明，在VSV感染期间，hnRNPA1、hnRNPK和hnRNPC1/C2会重新定位到细胞质中，但hnRNPB1或核纤层蛋白A/C不会。我们通过用编码带有M9核定位序列（NLS）或经典NLS的增强型绿色荧光蛋白（EGFP）的质粒转染细胞，来确定VSV是否抑制蛋白质输入。这些实验表明，在VSV感染期间，M9 NLS和经典NLS均具有功能。这些数据表明，蛋白质输入的抑制并非VSV感染期间hnRNP重新定位的原因，而是hnRNP输出增强。我们发现，在mRNA输出因子Rae1沉默后，hnRNPA1的重新定位显著减少，这表明Rae1是hnRNP输出所必需的。为了确定hnRNPA1在VSV感染中的作用，我们在HeLa细胞中沉默了hnRNPA1，并检测了病毒生命周期的三个方面：与病毒蛋白合成开始同时发生的宿主蛋白合成关闭、通过空斑试验检测的复制以及细胞杀伤。我们观察到，宿主关闭和复制不受hnRNPA1减少的影响，但在hnRNPA1减少的细胞中，VSV诱导的凋亡速率较慢。这些数据表明，VSV以Rae1依赖的方式促进hnRNPA1重新定位以进行凋亡信号传导。
