Characterization of the Adenovirus E4orf6/E1B55K E3 Ubiquitin Ligase.

摘要

Human adenovirus type 5 (Ad5) has been engineered as therapeutic oncolytic viruses to selectively kill cancer cells. The earliest and most widely used is the Ad5 ONYX-015 virus which lacks the viral protein E1B55K. The adenoviral proteins E1B55K and E4orf6 were shown previously to be important for multiple functions during viral infection. Most of these functions are dependent on their formation of the E3 ligase complex with cellular proteins Cul5, Elongins B and C, and Rbx1. Therefore, in order to improve the current oncolytic adenovirus therapy, a better understanding on this E3 ligase complex is required. As the E4orf6/E1B55K E3 ligase contains components similar to other cullin E3 ligase complexes, the mechanism of the E3 ligase assembly was investigated. I showed that the E4orf6/E1B55K E3 ligase complex is formed in an unconventional way: E4orf6 uniquely contains three BC box motifs for its interaction with Elongin C unlike other cellular proteins, which contain only one BC box. In addition, the complex utilises neither of the two known mechanisms for recruiting Cul5. Ad5 is by far the best characterized of the more than fifty different adenovirus serotypes; however, it is unclear how representative its properties are with respect to all adenoviruses. Thus, the conservation of the E4orf6/E1B55K E3 ligase was studied systematically in members of other adenovirus subgroups. I demonstrated that the E4orf6 and E1B55K proteins from all serotypes can form an E3 ligase complex but with different cullin specificities: Ad4, Ad5, Ad9 and Ad34 recruit primarily Cul5, Ad12 and Ad40 recruit primarily Cul2, and Ad16 can recruit both. As for function, I showed that different serotypes degrade different ranges of substrates with the only common substrate to all being DNA ligase IV. I found clear evidence that E1B55K is the substrate binding component of the complex; however, I demonstrated that there is no correlation between binding and the capability to degrade specific substrates. These studies have shown clearly that considerable heterogeneity exists in the formation and function of the adenovirus E4orf6/E1B55K E3 ligase.