The role of terminal protein in adenovirus nuclear delivery

The future of gene therapy is dependent on the development of vectors that can selectively and efficiently deliver therapeutic DNA to target cells in vivo with minimal toxicity. Virus vectors are the most widely used transducing systems. However, there are many ethical and safety concerns associated with the use of viruses in humans. Non-viral vectors are more suitable because of the lack of a specific immune response; but these are currently inefficient due to the intracellular barriers between nucleus and extracellular milieu preventing sufficient therapeutic DNA delivery to the cell nucleus. Non-viral vectors must be tailored to overcome these barriers for sufficient delivery. One of the most substantial barriers for DNA delivery is the nuclear envelope which is a double membrane surrounded the nucleus. The vector must pass the nuclear envelope to enter the nucleus. Research on viral tropism suggests that some viruses have evolved means of interaction with nuclear transport systems, and there is a good chance that active transport can be utilized for delivery of plasmid DNA. There is a widespread interest in the use of adenoviruses primarily because of their ability to efficiently deliver double-stranded DNA to the nucleus. In addition, their large genome allows for extensive modification and incorporation of therapeutic genes. The basic understanding of how adenovirus nuclear delivery systems work is fundamental to ensure further development and enhancement of the non-viral gene therapy. This PhD project investigated into the mechanisms of adenovirus nuclear delivery by examining the role of the adenovirus terminal protein, a protein that is covalently coupled to the 5’ ends of the adenoviral DNA. To identify the encoded NLS on terminal protein (Chapter 4), PCR-based approaches were used to amplify several truncated terminal protein derivatives and to create site-directed terminal protein mutants. The products were inserted into mammalian expression plasmids pcDNA6.2/C-EmGFP (fused in frame to the C-terminus of the EmGFP) and pcDNA6.2/N-YFP (fused in frame to the N-terminus of the YFP) using the Gateway recombination system. Transfection of mammalian cells (HeLa and COS-7 cells) allowed evaluation of the extent of nuclear delivery of each fusion protein by determining the nuclear/cytoplasmic fluorescence ratio F(n/c). Two NLSs were identified; the bipartite sequences (MRRRR370 and PVRRRRRRV390) and monopartite sequence (PGARPRGRF671). The arginine residues were found to be critical part of the bipartite NLS. The PGARPRGRF671 sequence was also shown to have a nucleolus delivery property when not masked by the flanking regions. To identify the nuclear import pathway of terminal protein (Chapter 5), ALPHAScreen® assay was carried out for the terminal protein and terminal protein derivatives encoding NLSs. It was found that the nuclear delivery of terminal protein is independent of the functional microtubules and the two NLSs employed different pathways. This suggested that TP is not involved in the intracellular trafficking of Adv. The nuclear delivery of the bipartite NLS (MRRRR370 and PVRRRRRRV390) is dependent on the IMPα/β mediated pathway, whereas the monopartite NLS (PGARPRGRF671) employs alternative nuclear trafficking pathway, most likely IMPβ mediated pathway. It was identified that the negatively rich domain, proximal to the bipartite NLS, reduces the binding specificity between NLS and its IMP binding partner. It also influences the binding affinity of NLS with IMP receptors by either masking or enhancing NLS-IMP interaction.