Strategies of viral multi-functional regulator proteins
: Adeno-associated virus rep

Student thesis: Doctoral ThesisDoctor of Philosophy


Adeno-­‐associated virus (AAV) is a small non-­‐pathogenic human DNA parvovirus. The AAV life cycle, which includes transcriptional regulation, DNA replication, assembly and site-­‐specific integration, is orchestrated by AAV’s four Rep proteins. Structurally, these proteins share a AAA+ domain characteristic of the SF3 family of helicases, with the larger Rep68 and Rep78 additionally containing an N-­‐terminal origin-­‐binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains is the basis for the remarkable multi-­‐functionality displayed by Rep68 and Rep78. To date, structural studies of Rep68 and Rep78 have been limited by the tendency of these proteins to aggregate when purified. Here, we describe a fully functional Rep mutant that does not aggregate even at high concentrations and use this mutant to investigate the structural requirements for Rep functions. We demonstrate that one of the determinants regulating the oligomerisation of the Rep proteins lies in the linker connecting the helicase domain and OBD. We also identify a series of key residues at the interface between Rep monomers and show that mutating them has drastic effects both on the oligomerisation and functionality of the Rep proteins. Importantly, these oligomerisation-­‐deficient mutants do not support the AAV life-­‐cycle and fail to bind DNA efficiently, an important Rep function necessary for DNA nicking, transcriptional regulation, viral DNA replication and site-­‐specific integration.
Finally, understanding the molecular details of Rep and its functions will contribute to the development of new AAV-­‐based vectors that exploit the Rep-­‐ mediated integration mechanism and potentially have a lower risk of insertional mutagenesis than retroviral vectors. In the last chapter, we describe an AAV vector for testing the safety and feasibility of AAV-­‐mediated targeted gene addition in induced pluripotent stem (iPS) cells within the therapeutic context of SCID-­‐X1, an immunodeficiency caused by mutations in the common gamma chain gene.
Date of Award2016
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMichael Linden (Supervisor) & Els Henckaerts (Supervisor)

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