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标题：Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation
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作者：Yongqiang Gou ; Hyewon Byun ; Adam E. Zook 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：3505-3510
DOI：10.1073/pnas.1420477112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceTranslation is a key process that is regulated by cellular health and responses to the environment, including virus infection. We show here that introduction of lentivirus and gammaretroviral vectors into cells by transfection increased translation (superinduction) of cotransfected genes but not most endogenous proteins. Superinduction was independent of the unfolded protein, stress, and interferon responses and did not require retroviral vector transcription. Retroviral vectors elevated cap-dependent translation initiation without increased mammalian target of rapamycin (mTOR) kinase activity. Thus, DNA sequences from HIV-1 and other retroviruses increase translation of cotransfected genes in trans by mTOR complex 1-independent signaling. Our results suggest that retroviral DNA manipulates translation, which has practical implications for protein expression and design of vectors for transfection assays, DNA vaccines, and shRNA knockdown experiments. Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.
关键词：superinduction ; retroviruses ; translation ; transfection