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Over the past decades, adeno-associated viruses (AAV) have evolved to be one of the most promising viral vectors for gene therapy. This great interest mainly rises from its non-pathogenicity, its simple organization and the fact that all viral genes are dispensable. Although these properties equip AAV as very safe vector, it is of great importance to further study the biology of AAV infection, which in part is still poorly understood. During the past years, many studies focused on the viral transduction, revealing mechanisms that facilitate viral infection. Upon those mechanisms, a key step in the AAV infection process is the conformational change of the capsid within the endosome, which leads to exposition of VP1/2 N-terminus. Analysis of the VP1/2 N-terminus makes specific domains accessible that play important roles during infection. In contrast, other studies demonstrated that modifications of the viral capsid by the host cell, such as phosphorylation and ubiquitination, affect and partially inhibit viral infection. Therefore, the underlying study aimed to identify cellular proteins that specifically interact with the unique N-terminus of the VP1/2 capsid proteins. For that, tandem affinity purification was performed using the VP1/2 N-termini of the AAV serotypes 2, 6, 8, 9 and 10. Double-tagged VP1/2 fusion proteins were overexpressed in and purified from HEK293TT cells, following mass-spectrometry to identify co-purified proteins. In total, 51 putatively interacting proteins were isolated. From those, 6 were identified with all of the serotypes used in the screen. Among the identified proteins that are involved in ubiquitination processes, SPOP, CAND1 and Cullin 3 were isolated. These proteins are known to form an ubiquitination machinery complex; hence they were used for further characterization. Interaction was confirmed by co-immunoprecipitation for SPOP and Cullin 3, as well as by indirect immunofluorescence analyzing, whether the proteins co-localize in transfected cells. Interestingly, when co-expressed with SPOP, VP1 was found in a speckled pattern localized in the nucleus, overlapping with SPOP, suggesting recruitment by SPOP. Sequence analysis of the VP1/2 N-terminus of all AAV serotypes revealed a putative SPOP binding consensus motif, which allowed mutational analysis for further characterization. Different AAV mutants showed a reduced transduction, as well as reduced binding to SPOP and differences in the subcellular localization. Overexpression of SPOP following infection with AAV did not change the level of transduction, suggesting endogenous SPOP levels to be sufficient to act on the infection. Knockdown experiments of SPOP, showed a slight increase in the transduction efficiency for AAV. Taken together, this study identified SPOP to be involved in the AAV life-cycle. Moreover it revealed cellular proteins that might be involved in the AAV infection process. Whether these proteins are captured by the virus to facilitate its infection or serve as host defense mechanism needs to be determined.
|Supervisor:||Müller, Prof. Dr. Martin|
|Date of thesis defense:||3 June 2014|
|Date Deposited:||07 Jul 2014 09:27|
|Faculties / Institutes:||Service facilities > German Cancer Research Center (DKFZ)|
|Subjects:||570 Life sciences|