Biochemical and biophysical analysis of the adenovirus early region 4, open reading frame 3 protein

The adenovirus Early region 4 Open Reading Frame 3 (E4 ORF3) protein has been the subject of much scrutiny given its importance in the viral life cycle. To date, several functions have been attributed to E4 ORF3 including disruption of the subnuclear Promyelocytic leukemia Oncogenic Domain (POD), inhibition of host cell double-strand break repair machinery, and enhancement of viral DNA replication. While mutational analysis is able to separate these capabilities, it falls short of ascribing specific functional domains to the protein. In addition, the method of ORF3 POD disruption and its post-translational modification status are currently unknown. In order to address these shortcomings, biochemical and biophysical approaches were implemented. The E4 ORF3 protein was recombinantly produced and purified in large quantities for structural studies. While crystallization screening proved unsuccessful, secondary structure information was ascertained through Circular Dichroism (CD) spectroscopic analysis. The protein was found to be primarily alpha helical and remained so in spite of temperature and ionic strength fluctuations. In addition, evidence suggesting E4 ORF3 oligomerization was found. The mechanism of E4 ORF3 POD disruption was explored through affinity chromatography experiments. Two novel ORF3 binding partners, Transcriptional Intermediary Factors (TIF) 1alpha and 1gamma, were identified and validated through these experiments. While the functional relevancy of TIF1alpha binding remains unknown, ORF3 appears responsible for the proteasome-dependent degradation of TIF1gamma. However, the importance of the E4 ORF3:TIF1alpha interaction is underscored by its evolutionary conservation among other adenovirus serotypes. E4 ORF3 was found to interact with TIF1alpha by co-immunoprecipitation and a recombinant protein pull-down assay excluding other mammalian proteins. These data suggest a direct interaction between the proteins, while immunofluoresence analyses suggest that the N-terminal region of TIF1alpha is sufficient for ORF3 binding. The possibility of ORF3 post-translational modification by the small ubiquitin-like modifier (SUMO) was also explored. While mutation of a SUMO consensus sequence within ORF3 generates a nonfunctional protein, sumolation of the ORF3 protein was not identified. In addition to generating the first E4 ORF3 structural information, these data mark novel ORF3 functions in the TIF family member interactions.