Chang Pey-Jium, Hung Chien-Hui, Wang Shie-Shan, Tsai Ping-Hsin, Shih Ying-Ju, Chen Li-Yu, Huang Hsiao-Yun, Wei Ling-Huei, Yen Ju-Bei, Lin Chun-Liang, Chen Lee-Wen
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang-Gung University, Taoyuan, Taiwan Department of Medical Research, Chang-Gung Memorial Hospital, Chiayi, Taiwan
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang-Gung University, Taoyuan, Taiwan.
J Virol. 2014 Sep 1;88(17):10092-109. doi: 10.1128/JVI.01445-14. Epub 2014 Jun 25.
The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle.
Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.
已知卡波西肉瘤相关疱疹病毒（KSHV）的orf47-orf46-orf45基因簇依次编码糖蛋白L（gL）、尿嘧啶DNA糖基化酶和一种病毒被膜蛋白。在此，我们鉴定出两种新的mRNA变体，即orf47/45-A和orf47/45-B，它们是从一个三顺反子orf47-orf46-orf45 mRNA中选择性剪接而来，该mRNA在病毒重新激活早期阶段在orf47-orf46-orf45基因位点表达。剪接后的基因产物ORF47/45-A和ORF47/45-B仅由gL（ORF47）的部分区域、一个独特的7个氨基酸基序和完整的被膜蛋白ORF45组成。与ORF45蛋白一样，在细胞中表达的ORF47/45-A和ORF47/45-B足以激活p90核糖体S6激酶（RSK）和细胞外信号调节蛋白激酶（ERK）的磷酸化。然而，与ORF45不同，ORF47/45-A和ORF47/45-B都含有信号肽序列，并定位于内质网（ER）。此外，我们发现ORF47/45-A和ORF47/45-B具有一种额外功能，可介导内质网稳态的主要调节因子GRP78的上调。在病毒重新激活后的所有测试KSHV阳性细胞系中均可观察到GRP78上调这一重要事件，并且在细胞中敲低GRP78会显著损害病毒裂解周期进程，尤其是在裂解后期。与其他一些通过内质网合成的病毒糖蛋白相比，我们的结果有力地表明，ORF47/45蛋白可能是控制细胞中GRP7&表达和内质网稳态的关键效应因子。综上所述，我们的发现提供了证据，表明内质网稳态调节与KSHV裂解周期进程之间存在相互关联。
新出现的证据表明，几种病毒似乎采用不同策略来控制内质网稳态以支持其有效感染。本研究的两部分确定了内质网稳态调节与KSHV裂解周期进程之间关联的两个方面。第一部分描述了两种早期裂解周期蛋白ORF47/45-A和ORF47/45-B对主要内质网伴侣蛋白GRP78激活的功能。除了促进GRP78上调的能力外，ORF47/45蛋白还激活RSK和ERK的磷酸化。第二部分揭示，GRP78上调对于KSHV裂解周期进程至关重要，尤其是在后期。因此，我们提出病毒蛋白在裂解早期激活GRP78表达可能有助于保护宿主细胞免受严重内质网应激，并可能直接参与病毒粒子的组装或释放。
