To approach the goals of this project, we set up state-of-the-art high-throughput methods to study autophagy during viral infection. Thus, a high-throughput screening system for autophagy was established, including optimization of the method as well as the generation of the cell lines required. This preparatory work was summarized as a methods publication and submitted to the open-access journal Scientific Reports and is currently in revision (Koepke et al, 2020, Scientific Reports, submitted).
We have applied this method for our recent collaborative study on the antagonism of autophagy by the human immunodeficiency virus 1 (HIV-1) accessory protein Nef. Here we could show, that Nef recruits an inhibitory factor of autophagy, Bcl-2, to bind to the initiation complex and thus shuts down autophagy at an early step. The virus uses this factor to escape the anti-viral function of autophagy, and thus its replication is decreased if Nef is defective for Bcl-2 recruitment. This study was published in the journal Autophagy (Castro-Gonzalez et al, 2020, Autophagy)
During the current SARS-CoV-2 pandemic we have applied our method to study the SARS-CoV-2 protein Nsp1, resulting in the publication of a pre-print on BioRxiv (Thoms et al, 2020, BioRxiv, published; Science submitted). We could show that Nsp1 blocks the mRNA entry tunnel of the ribosome, shutting down translation in the process. Thus, innate immune responses depending on translation such as the type-I interferon system are blocked, whereas systems, such as autophagy, which do not depend on de novo protein production still function.
Finally, using our tools, a genome-wide CRISPR screen was performed to identify cellular proteins that inhibit autophagy. Depletion of these factors decreases replication of autophagy-sensitive viruses like HIV. We hypothesize that these proteins act as gatekeepers of autophagy, and their normal function is to prevent excessive induction of this catabolic process. However, during an HIV-1 infection they block the autophagic response, promoting HIV replication. Consequently, releasing the block on anti-viral autophagy by depletion of these factors drastically restricted replication of HIV.
Taken together, we defined the molecular mechanism of autophagy evasion strategies by HIV and discovered novel cellular key factors that may be therapeutically inhibited to boost anti-viral autophagy to restrict HIV and other autophagy-sensitive viruses.
