Co-translational genetic switching during protein synthesis: the HIV-1 Nef gene as a paradigm
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Lakmini Premadasa (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Ethan Taylor
Abstract: The old maxim of “one gene, one mRNA, one protein” no longer holds, especially with viral genes. It is possible for one mRNA to encode several proteins of unrelated functions in overlapping reading frames of a single oligonucleotide, or for an additional protein domain to be added on to a protein at the C-terminal by the readthrough of a stop codon. The question of how and when stop does not always mean stop, how slippage from one reading frame into another is controlled, and the factors that trigger those genetic switches, are the subjects of this research. The focus of the project is the HIV-1 nef gene, which has examples of both of these types of co-translational switching events (translational frameshifting and stop codon readthrough). Nef is a myristoylated protein expressed in the early stage of the HIV-1 life cycle, which functions as a fundamental factor for efficient viral replication and pathogenesis. One of the notable features of nef is the highly conserved 3’-UGA stop codon, and the potential for the protein to be extended by about 30 amino acids if readthrough of that stop codon can occur. We hypothesize that antisense tethering interactions (ATIs) between viral mRNA and host selenoprotein mRNA enables capture of the host selenocysteine insertion sequence (SECIS) element to enable the expression of virally encoded selenoprotein modules via translation of in frame UGA stop codons as selenocysteine (SeC). This mRNA hijack mechanism was predicted theoretically using computational analysis and was experimentally supported at the DNA level by gel shift assay. Readthrough of UGA was proved at the mRNA level by fluorescence microscopy image analysis and flow cytometry of transfected HEK 293 cells with engineered reporter gene plasmid vector constructs, in which the downstream reporter gene can only be expressed if the UGA is translated. siRNA knockdown of thioredoxin reductase 1 (TR1) mRNA in transfected cells resulted in decreased GFP expression, consistent with the hypothesis that host-virus mRNA tethering may enable selenocysteine incorporation for the stop codon readthrough. Furthermore quantitative analysis of TR1 mRNA knockdown demonstrated using RT-PCR confirmed that the siRNA treatment results in approximately 20% knockdown of TR1. The HIV-1 nef coding region features a potential -1 frameshift site with a potential overlapping gene region near the middle of the coding sequence. A sequence matching the pattern (XXXYYYZ) of a known -1 frameshifting “slippery sequence” signal is present in the nef sequence at this point, immediately upstream of a G-quadruplex (QPX) sequence that serves to regulate frameshifting. An in vitro frameshift assay using a dual reporter vector was constructed, in which the putative HIV-1 nef-fs sequence with QPX was cloned between two fluorescent reporter genes. Cells transfected with this construct showed orange fluorescence, which is only possible if the -1 frameshifting occurs. Treating the transfected cells with QPX stabilizing synthetic drug TMPYP4 increased the frameshifting efficiency by 27%, specifically confirming the role of the QPX as an enhancer of -1 frameshifting efficiency.
Co-translational genetic switching during protein synthesis: the HIV-1 Nef gene as a paradigm
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Created on 5/1/2016
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Additional Information
- Publication
- Dissertation
- Language: English
- Date: 2016
- Keywords
- Frameshift, HIV-1, Stop codon readthrough
- Subjects
- HIV (Viruses) $x Genetic aspects