Oncolytic adenoviruses are being examined as potential therapeutic agents for malignant gliomas and other solid tumors. We have reported that autophagy is a key component of the lysis process in adenovirus-infected cells, influencing the adenoviral spread. We hypothesize that understanding the regulation of autophagy should result in the generation of more potent oncolytic adenoviruses. Our group showed that the function of adenoviral early E1B-proteins include regulation of autophagy. Thus, E1B19k, a Bcl2-like protein, interacts with Beclin 1 and activates autophagy in adenovirus-infected cells. (Piya et al., PLOS One 2011). In this work, we sought to examine the capability of the E1B55K to modulate the autophagic process. Using mutant viruses deficient for the expression of E1B55k, E4orf6 or E1B55k/E4orf6, we observed that viruses lacking the expression of E1B55k were attenuated for the induction of autophagy. Upon examining the sequence of E1B55k, we discovered the presence of an LC3-interacting motif, termed LIR. Co-immunoprecipation assays revealed that E1B55k heterodimerized with LC3. As expected, mutations in the LIR sequence of E1B55k abrogated the physical interaction with LC3. Importantly, we showed that disruption of E1B55k/LC3 complexes had functional repercussions resulting in attenuation of the adenovirus-mediated autophagy. Because the complex of E1B55k and E4orf6 functions as a sumo ligase, we examined whether E1B55k/E4orf6 complexes were involved in LC3 sumoylation. Using co-immunoprecipitation studies in HeLa-6X-his-Sumo-1 infected with wild type and mutant adenoviruses, we showed that LC3 was sumoylated during adenoviral infection and that E1B55k/LC3 interactions were required for this posttranslational modification. Importantly, inhibition of the sumoylation process drastically reduced adenovirus replication. This is the first report showing that adenovirus induced sumoylation of cellular proteins to regulate autophagy. Preliminary studies of cytoplasmic and nuclear contents of LC3 and sumoylated LC3 suggest that adenovirus-mediated sumoylation of LC3 prolongs the presence of LC3 in the cytoplasm by modifying subcellular localization and decreasing protein degradation. We conclude that adenoviral E1B proteins cooperate in the induction and maintenance of autophagy in host cells. Our data provide new insights about the regulation of autophagy and the intracellular environment generated in cells infected with adenoviruses. It is hoped that these findings will propel the development of more potent and selective oncolytic adenoviruses.
Citation Format: Sujan Piya, Hong Jiang, Sarah Klein, W.K.Alfred Yung, Raymond Sawaya, Candelaria Gomez-Manzano, Juan Fueyo. Oncolytic adenoviruses modulate autophagy in cancer cells via sumoylation of LC3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2014-726