hnRNPs Relocalize to the Cytoplasm following Infection with Vesicular Stomatitis Virus
hnRNPs Relocalize to the Cytoplasm following Infection with Vesicular Stomatitis Virus
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DOI：
10.1128/jvi.01279-08
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发表时间：
2009-01-15
影响因子：
5.4
通讯作者：
Lyles, Douglas S.
中科院分区：
文献类型：
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作者：
Kneller, Elizabeth L. Pettit;Connor, John H.;Lyles, Douglas S.
水泡性口炎病毒（VSV）基质蛋白抑制核质mRNA转运。这项工作的目标是确定VSV是否抑制异质性核糖核蛋白（hnRNP）的核质转运，这被认为是作为mRNA的输出因子。共聚焦显微镜实验表明，在VSV感染期间，hnRNPA 1、hnRNPK和hnRNPC 1/C2重新定位于细胞质，而hnRNPB 1或核纤层蛋白A/C则不然。我们确定蛋白质输入是否被VSV抑制，通过用编码增强型绿色荧光蛋白（EGFP）的质粒转染细胞，所述增强型EGFP用M9核定位序列（NLS）或经典NLS标记。这些实验表明，M9 NLS和经典NLS在VSV感染期间都是功能性的。这些数据表明，蛋白质进口的抑制不负责VSV感染过程中的hnRNP重新定位，但hnRNP出口增强。我们发现，hnRNPA 1的再定位显着减少后沉默的mRNA输出因子Rae 1，表明Rae 1是必要的hnRNP出口。为了确定hnRNPA 1在VSV感染中的作用，我们在HeLa细胞中沉默了hnRNPA 1，并测定了病毒生命周期的三个方面：宿主蛋白质合成关闭，同时病毒蛋白质合成的开始，通过空斑试验复制，和细胞杀伤。我们观察到宿主关闭和复制不受hnRNPA 1减少的影响，但在hnRNPA 1减少的细胞中，VSV诱导的细胞凋亡速率较慢。这些数据表明，VSV促进hnRNPA 1重新定位在Rae 1依赖的方式凋亡信号。
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.