Identification and Characterization of Two Novel Spliced Genes Located in the orf47-orf46-orf45 Gene Locus of Kaposi's Sarcoma-Associated Herpesvirus
Identification and Characterization of Two Novel Spliced Genes Located in the orf47-orf46-orf45 Gene Locus of Kaposi's Sarcoma-Associated Herpesvirus
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DOI：
10.1128/jvi.01445-14
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发表时间：
2014-09-01
影响因子：
5.4
通讯作者：
Chen, Lee-Wen
中科院分区：
文献类型：
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作者：
Chang, Pey-Jium;Hung, Chien-Hui;Chen, Lee-Wen
已知卡波西肉瘤相关疱疹病毒 (KSHV) 的 orf47-orf46-orf45 基因簇连续编码糖蛋白 L (gL)、尿嘧啶 DNA 糖基化酶和病毒外皮蛋白。在这里，我们鉴定了两种新的 mRNA 变体，orf47/45-A 和 orf47/45-B，它们是由三顺反子 orf47-orf46-orf45 mRNA 选择性剪接而成，该 mRNA 在病毒重新激活的早期阶段在 orf47-orf46-orf45 基因位点中表达。剪接基因产物 ORF47/45-A 和 ORF47/45-B 仅由 gL (ORF47) 的部分区域、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，细胞中表达的ORF47/45-A和ORF47/45-B充分激活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不同的是，ORF47/45-A和ORF47/45-B都含有信号肽序列并且定位于内质网(ER)。此外，我们发现 ORF47/45-A 和 ORF47/45-B 具有介导 GRP78 上调的额外功能，GRP78 是 ER 稳态的主要调节因子。病毒重新激活后，在所有测试的 KSHV 阳性细胞系中都可以观察到有关 GRP78 上调的重要事件，细胞中 GRP78 的敲低会显着损害病毒裂解周期的进展，尤其是在裂解后期。与通过 ER 合成的一些其他病毒糖蛋白相比，我们的结果强烈暗示 ORF47/45 蛋白可能作为控制细胞中 GRP78 表达和 ER 稳态的关键效应子。总而言之，我们的研究结果提供了证据，表明 ER 稳态的调节与 KSHV 裂解周期的进展之间存在相互关联。 重要性 新出现的证据表明，几种病毒似乎使用不同的策略来控制 ER 稳态以支持其生产性感染。本研究的两个部分确定了 ER 稳态调节与 KSHV 裂解周期进展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 在主要 ER 伴侣蛋白 GRP78 激活中的功能。除了能够促进 GRP78 上调外，ORF47/45 蛋白还激活 RSK 和 ERK 的磷酸化。第二部分揭示了 GRP78 的上调对于 KSHV 裂解周期的进展至关重要，尤其是在后期。因此，我们认为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的 ER 应激，并可能直接涉及病毒粒子的组装或释放。
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.IMPORTANCEEmerging 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.