Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation.
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.
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Yongqiang
Y
Gou
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Hyewon
H
Byun
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Adam E
AE
Zook
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Gurvani B
GB
Singh
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Andrea K
AK
Nash
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191.
Mary M
MM
Lozano
Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191 jdudley@austin.utexas.edu.
Jaquelin P
JP
Dudley
D004247:DNA / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E / Q000096:biosynthesis; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism*; D007372:Interferons / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism*; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D013312:Stress, Physiological; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D039561:Eukaryotic Initiation Factor-4E; D012315:RNA Caps; D034741:RNA, Small Interfering; D004247:DNA; D007372:Interferons; D058570:TOR Serine-Threonine Kinases
retroviruses; superinduction; transfection; translation