Liu Ya-Fen, Tsai Pei-Yun, Chulakasian Songkhla, Lin Fong-Yuan, Hsu Wei-Li
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Department of Beauty Science, MeiHo University, Neipu, Taiwan.
FEBS J. 2016 Mar;283(5):899-911. doi: 10.1111/febs.13642. Epub 2016 Feb 3.
The virion host shutoff protein (vhs), encoded by the gene UL41, has RNase activity and is the key regulator of the early host shutoff response induced by type 1 herpes simplex virus. Despite low amino acid similarity, the vhs protein of the swine herpesvirus, pseudorabies virus (PrV), also exhibits RNase activity. However, the mechanism underlying the action of vhs remains undefined. Here, we report that the RNA degradation profile of PrV vhs is similar, but not identical, to that of type 1 herpes simplex virus vhs. Notably, the presence of a cap structure enhances both the degradation rate and the preferential targeting of the vhs protein towards the 3'-end of the encephalomyocarditis virus internal ribosome entry site (IRES). Furthermore, type 1 herpes simplex virus vhs produces a simple degradation pattern, but PrV vhs gives rise to multiple intermediates. The results of northern blotting using probes recognizing various regions of the RNA substrate found that PrV vhs also cleaves downstream of the IRES region and this vhs protein overall shows 5' to 3' RNase activity. Moreover, addition of the translation initiation factors eIF4H and eIF4B significantly increased the RNase activity of recombinant PrV vhs against capped RNA. Nonetheless, these proteins did not fully reconstitute the IRES-directed targeting pattern observed for vhs translated in a rabbit reticular lysate system. The interaction between PrV vhs and eIF4H/eIF4B implies that the translation initiation machinery within the cell is able to stimulate the nuclease activity of PrV vhs. However, this process remains inefficient in terms of the IRES-targeting pattern.
由UL41基因编码的病毒体宿主关闭蛋白（vhs）具有核糖核酸酶活性，是1型单纯疱疹病毒诱导的早期宿主关闭反应的关键调节因子。尽管猪疱疹病毒伪狂犬病病毒（PrV）的vhs蛋白氨基酸相似性较低，但也表现出核糖核酸酶活性。然而，vhs作用的潜在机制仍不明确。在此，我们报告PrV vhs的RNA降解谱与1型单纯疱疹病毒vhs的相似，但并不完全相同。值得注意的是，帽结构的存在提高了vhs蛋白对脑心肌炎病毒内部核糖体进入位点（IRES）3'端的降解速率和优先靶向性。此外，1型单纯疱疹病毒vhs产生简单的降解模式，但PrV vhs会产生多种中间体。使用识别RNA底物不同区域的探针进行Northern印迹分析的结果表明，PrV vhs也在IRES区域下游切割，并且这种vhs蛋白总体上表现出5'到3'的核糖核酸酶活性。此外，添加翻译起始因子eIF4H和eIF4B显著增加了重组PrV vhs对带帽RNA的核糖核酸酶活性。尽管如此，这些蛋白质并未完全重建在兔网织红细胞裂解物系统中翻译的vhs所观察到的IRES导向的靶向模式。PrV vhs与eIF4H/eIF4B之间的相互作用表明细胞内的翻译起始机制能够刺激PrV vhs的核酸酶活性。然而，就IRES靶向模式而言，这个过程仍然效率低下。
