EFFECT OF THE TRIPARTITE LEADER ON SYNTHESIS OF A NON-VIRAL PROTEIN IN AN ADENOVIRUS-5 RECOMBINANT
EFFECT OF THE TRIPARTITE LEADER ON SYNTHESIS OF A NON-VIRAL PROTEIN IN AN ADENOVIRUS-5 RECOMBINANT
复制标题
DOI：
10.1093/nar/13.3.841
复制
发表时间：
1985-01-01
影响因子：
14.9
通讯作者：
SHARP, PA
中科院分区：
文献类型：
--
作者：
BERKNER, KL;SHARP, PA
腺病毒5重组体的EIa区已被编码二氢叶酸还原酶（DHFR）的模块基因取代。在该重组体中，小鼠DHFR cDNA位于主要晚期启动子和完整的三方前导序列之后。前导序列终止于第三个前导序列的正常5′剪接位点（SS），因此RNA剪接将三联前导序列连接到DHFR cDNA上游的3′剪接位点。在感染的晚期，合成高水平的DHFR mRNA。在晚期的早期，这种mRNA被有效地翻译;然而，在后期，DHFR的翻译减少，可能是由于与其他晚期mRNA的竞争力差。平行研究了从仅含有第一个晚期前导序列的类似腺病毒5重组体合成DHFR蛋白。用这种重组病毒感染后表达了同等水平的DHFR mRNA;然而，DHFR翻译的效率比含有三方前导的DHFR mRNA低至少20倍。这表明三重前导序列在感染后期的翻译中是重要的。如前所述，仅含有第一前导序列的Ad 5重组体从一种新的mRNA中大量过量表达多肽IX，该新的mRNA是通过将模块化DHFR基因中的第一前导序列剪接到EIb区的3′剪接位点而形成的。被这种重组体感染的细胞从EIb区合成很少的正常mRNA。在这里，我们证明了用这种重组腺病毒和野生型腺病毒5共感染293细胞也导致EIb mRNA合成减少。我们建议，过量生产的多肽IX抑制mRNA的表达，从EIb和IX启动子位点，可能是通过一个自动调节环活性裂解生长过程中。
The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5′ splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3′ splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptlde IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3′ splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.