Huang W, Flint S J
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
J Virol. 1998 Jan;72(1):225-35. doi: 10.1128/JVI.72.1.225-235.1998.
The subgroup C human adenoviruses induce selective export of newly synthesized viral mRNA from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNA species. Such posttranscriptional regulation of viral and cellular gene expression in infected cells requires viral E1B and E4 proteins. To facilitate the investigation of parameters that govern selective export in adenovirus-infected cells, we constructed a marked human beta-actin minigene under the control of the glucocorticoid-inducible enhancer-promoter of mouse mammary tumor virus and introduced it into the left end of the adenovirus type 5 (Ad5) genome. Transcription of this reporter gene (designated MA) as well as of a sibling, which differed only in the inclusion of a cDNA copy of the Ad2 major late tripartite leader sequence upstream of beta-actin sequences (termed MtplA), in recombinant virus-infected cells was strictly dependent on the addition of dexamethasone to the medium. When transcription of the MA gene was induced during the late phase of infection, newly synthesized MA RNA entered the cytoplasm. These transcripts, which contain no viral sequences, therefore reproduce the behavior of exceptional cellular mRNA species observed when transcription of their genes is activated during the late phase of infection (U.-C. Yang, W. Huang, and S. J. Flint, J. Virol. 70:4071-4080, 1996). Unexpectedly, however, higher concentrations of newly synthesized RNA accumulated in the cytoplasm when the tripartite leader sequence was present in the reporter RNA, despite equal rates of transcription of the two reporter genes. Examination of the partitioning of both newly synthesized and steady-state populations of MA and MtplA RNAs between nuclear and cytoplasmic compartments indicated that the tripartite leader sequence did not increase RNA stability in the cytoplasm. Comparison of nuclear and cytoplasmic reporter RNA species by Northern blotting, primer extension, and reverse transcription-PCR provided no evidence for altered processing induced by the tripartite leader sequence. We therefore conclude that the tripartite leader sequence, long known to facilitate the translation of mRNAs during the late phase of adenovirus infection, can also modulate mRNA export from the nucleus.
C亚组人腺病毒可诱导新合成的病毒mRNA从细胞核选择性输出到细胞质，同时抑制大多数细胞mRNA种类的输出。感染细胞中病毒和细胞基因表达的这种转录后调控需要病毒E1B和E4蛋白。为便于研究腺病毒感染细胞中选择性输出的调控参数，我们构建了一个受小鼠乳腺肿瘤病毒糖皮质激素诱导型增强子-启动子控制的标记人β-肌动蛋白小基因，并将其引入5型腺病毒（Ad5）基因组的左端。在重组病毒感染的细胞中，该报告基因（命名为MA）以及一个仅在β-肌动蛋白序列上游包含Ad2主要晚期三联前导序列的cDNA拷贝（称为MtplA）的姊妹报告基因的转录严格依赖于向培养基中添加地塞米松。当在感染后期诱导MA基因转录时，新合成的MA RNA进入细胞质。因此，这些不含病毒序列的转录本再现了在感染后期其基因转录被激活时观察到的特殊细胞mRNA种类的行为（U.-C.杨、W.黄和S.J.弗林特，《病毒学杂志》70:4071-4080，1996）。然而，出乎意料的是，尽管两个报告基因的转录速率相同，但当报告RNA中存在三联前导序列时，细胞质中积累了更高浓度的新合成RNA。对MA和MtplA RNA新合成群体和稳态群体在核和细胞质区室之间的分配进行检查表明，三联前导序列不会增加细胞质中的RNA稳定性。通过Northern印迹、引物延伸和逆转录PCR对核和细胞质报告RNA种类进行比较，没有提供三联前导序列诱导加工改变的证据。因此，我们得出结论，长期以来已知在腺病毒感染后期促进mRNA翻译的三联前导序列也可以调节mRNA从细胞核的输出。
