Protein processing strategies by adeno-associated virus type 5 (AAV5) and the effects of the adenovirus E4orf6/E1b-55k/Cullin 5 E3 ubiquitin ligase complex on AAV protein stability

We report the initial identification of the ubiquitination of a parvovirus nonstructural protein by the adenovirus E4orf6/E1b-55k/Cullin 5 E3 ubiquitin ligase complex. The small Rep and capsid proteins of adeno-associated virus type 5 (AAV5) were found to be specifically targeted for degradation in a proteasome-dependent manner by the adenovirus E3 ubiquitin ligase complex. This effect was at least partially dependent upon the levels of substrate protein within the cell, as significant overexpression of Rep during transient transfection prevented the degradation of the protein in 293 cells in the presence of E4orf6. This finding is in agreement with the observation that during adenovirus infection or transient co-transfection, adenovirus VA RNA is capable of overcoming this degradative effect by enhancing the overall levels of translated AAV protein. AAV5 small Rep proteins were found to associate with adenovirus E1b-55k in the absence or presence of E4orf6; however, Rep only co-immunoprecipitated with Cullin 5, the largest anchor protein portion of the complex, when E4orf6 was present. We demonstrate that the adenovirus E3 ligase functions to add ubiquitin moieties in a specific manner to AAV proteins and results in the targeting of these AAV proteins for destruction by the