Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site
Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site
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DOI：
10.1128/jvi.75.4.1864-1869.2001
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Talbot, SJ
中科院分区：
文献类型：
--
作者：
Bieleski, L;Talbot, SJ
我们之前检查了原发性渗出性淋巴瘤细胞系 BCP-1（潜伏感染 KSHV）中卡波西肉瘤相关疱疹病毒 (KSHV) 的开放阅读框 (ORFS) 71 (K13)、72 和 73 的转录和剪接 (45)。这些 ORF 编码的三个基因（vFLIP、vCyclin 和潜伏期相关核抗原 [LANA]）在潜伏期和抒情周期期间从 BCP-1 细胞中的共同转录起始位点转录。将所得转录物拼接以产生编码 LANA、vCyclin 和 vFLIP 的 5.32 kb 消息以及编码 vCyclin 和 vFLIP 的 1.7 kb 双顺反子消息。为了研究 vFLIP 蛋白是否可以从该 vCyclin/vFLIP 信息中表达，我们利用了双顺反子荧光素酶报告系统。海肾和萤火虫荧光素酶（利用不同底物）的基因在 T7 RNA 聚合酶启动子下游串联克隆。紧邻 vFLIP 起始密码子上游的 DNA 片段被克隆到两个荧光素酶基因之间。两种荧光素酶的相对表达（一种由假定的内部核糖体进入位点 (IRES) 序列指导，另一种由帽子依赖性核糖体扫描指导）用于比较不同 DNA 片段的活性。发现 vCyclin 编码区内的最小 233 bp 片段可指导下游顺反子（萤火虫荧光素酶）的有效表达。该 IRES 的活性是方向依赖性的，并且不受用于抑制帽依赖性翻译的方法的影响。这是 DNA 病毒编码的 IRES 元件的首次演示，可能代表了 KSHV 控制蛋白质表达的一种新机制。
We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lyric cycles. The resulting transcript is spliced to yield a 5.32 kb, message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.