Berkner K L, Sharp P A
Nucleic Acids Res. 1985 Feb 11;13(3):841-57. doi: 10.1093/nar/13.3.841.
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 polypeptide 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.
腺病毒5型重组体的EIa区域已被编码二氢叶酸还原酶（DHFR）的模块化基因所取代。在这个重组体中，小鼠DHFR cDNA位于主要晚期启动子和完整的三联前导序列之后。前导序列在第三个前导序列的正常5'剪接位点（SS）处结束，这样RNA剪接就将三联前导序列与DHFR cDNA上游紧邻的一个3'剪接位点连接起来。在感染后期，合成了高水平的DHFR mRNA。在后期的早期阶段，这种mRNA能有效地被翻译；然而，在后期，DHFR的翻译减少，可能是因为与其他晚期mRNA的竞争较差。同时研究了来自仅包含第一个晚期前导序列的类似腺病毒5型重组体的DHFR蛋白的合成。用这种重组病毒感染后，表达了相当水平的DHFR mRNA；然而，DHFR的翻译效率比包含三联前导序列的DHFR mRNA至少低20倍。这表明三联前导序列对于感染后期的翻译很重要。如先前报道，仅包含第一个前导序列的Ad5重组体从一种新的mRNA中大量过表达多肽IX，这种新的mRNA是由模块化DHFR基因中的第一个前导序列与EIb区域中的3'剪接位点剪接形成的。用这种重组体感染的细胞从EIb区域合成的正常mRNA很少。在这里，我们证明用这种重组体和野生型腺病毒5共同感染293细胞也会导致EIb mRNA合成减少。我们提出多肽IX的过量产生可能通过在裂解生长过程中活跃的自动调节环抑制了EIb和IX启动子位点的mRNA表达。
