Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation

Abstract

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 mTORindependent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.ranslational control is critical for mammalian cells and the viruses that infect them. For example, picornaviruses and some flaviviruses have an internal ribosomal entry site (IRES) (1) that allows cap-independent translation of viral RNAs and provides preferential translation over most host cell mRNAs (2). Structural modifications at the 5′ ends of viral RNAs, such as differences in cap methylation or absence of a cap, often distinguish viral mRNAs from their cellular counterparts (3). Multiple cellular proteins recognize foreign RNAs or DNAs, which trigger specific signaling pathways that lead to general translational arrest and/or selective viral RNA degradation (4). Many viral RNAs trigger the IFN signaling pathway and translational inhibition, but viruses encode various proteins or RNAs that mute this response (5). Recognition of foreign nucleic acids by cellular surface or cytosolic receptors also leads to signaling events that provide an innate antiviral response (6).Retroviruses are positive-sense RNA viruses that have been used extensively for introduction of genes or small hairpin RNAs (shRNAs) into cells, both in culture and for gene therapy. Unlike most RNA viruses, retroviruses replicate through a DNA intermediate and use RNA polymerase II to produce mRNAs with structures that are very similar to those of host mRN...