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Translational Control of MRNAs Transcribed from HIV-1 Provirus and HIV-1 Based Lentiviral Vectors

出版	Ohio State University, 2007
URL	http://books.google.com.hk/books?id=wKKjAQAACAAJ&hl=&source=gbs_api

註釋Abstract: Efficient translation of mRNA is an essential step in expression of all genes. Retroviruses encode obstacles to efficient viral mRNA translation. Study of retrovirus mRNA translational control is relevant to the processes of immunodeficiency, progression to neoplasia, and safe and efficacious gene transfer vector by this diverse class of RNA virus. This dissertation investigated fundamental mechanisms that control translation of retroviral mRNA and applied our findings to improve translation of transgene mRNA in lentiviral vectors. Chapter 2 unraveled a dynamic interface between HIV-1-induced cell cycle arrest and host translation suppression. Metabolic labeling experiments demonstrated that HIV-1-induced cell cycle arrest attributable to Vpr and Vif accessory proteins significantly limits the translation capacity of infected lymphocytes. Kinetic and ribosomal profile analysis determined that HIV-1 gag mRNA translation is resistant to this potential block to virus production. Cytosolic fractionation experiments demonstrated that gag RNA/ribosome complexes are translated on membrane-bound ribosomes and that amino-terminal myristate of Gag provides an ER partitioning signal. These results overturn the common notion that retrovirus translation is confined to soluble polyribosomes. HIV-1 mRNA partitioning to ER ribosomes represents a novel co-adaptation strategy to promote synthesis of viral structural protein. Chapter 3 developed a sucrose gradient fractionation method to interrogate the translation activity of the entire HIV transcriptome in lymphocytes. The assay verified that alternatively spliced transcripts are associated with polyribosomes. Chapter 4 applied for the first-time the post-transcriptional control element (PCE) of spleen necrosis virus (SNV) to facilitate translation of vector transgene mRNA. Coordinate enhancement of transgene transcription and translation in a lentiviral vector was achieved by combination of SNV PCE with a CMV transcriptional enhancer. Their combination increased protein yield up to 17-fold in transfected cells and transduced cells. Chapter 5 developed a novel retroviral vector to select PCE variants that increase output from the puromycin resistance gene by virtue of the error-prone reverse transcription process during consecutive passages of the viral vector. In aggregate, this dissertation produced fundamental research important for understanding infections by human and animal viruses, and applied research that for gene delivery in a broad array of clinical and research applications.