Periodic Reporting for period 1 - VIAR (Virus-induced autophagy restriction)
Reporting period: 2018-04-01 to 2020-03-31
Cellular innate immunity employs multiple ways to fight against invading pathogens, among them autophagy. During autophagy cytoplasmic cargoes, such as viral components, are targeted for destruction. This process has been previously recognized as an anti-viral mechanism against e.g. human immunodeficiency virus (HIV). However, HIV has evolved strategies to counteract this anti-viral defense system. While the basic core machinery of autophagy is well-described, the virus-autophagy interplay is little understood. The overall goal of this project was to (I) define cellular key factors restricting anti-viral autophagy that may be promising targets to therapeutically inhibit and boost autophagy and (II) to examine HIV autophagy evasion strategies. Thus, we intend to ultimately advance our understanding of the intricate interplay between cellular autophagy and the virus, which may be the basis for future innovative anti-viral therapeutic strategies.
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.
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.
This project advanced our insights on the fundamental regulation of autophagy during viral infections and has uncovered previously unknown strategies how viruses evade anti-viral autophagy.
As a direct result of this project, four publications were already published or are in revision, on two of them the Marie-Curie fellow is the corresponding author (Koepke et al, 2020, Scientific Reports, in revision, Thoms et al, 2020, BioRxiv; Science, in review) on two a co-author (Castro-Gonzalez et al, 2020, Autophagy, Braun et al, 2019, Cell Reports). Furthermore, the publication of the main part of the project is currently in the preparation phase and submission is planned to a prestigious international journal supporting open access. This project has allowed the Marie-Curie-Fellow to generate data to apply for funding to set up his own lab. He successfully was awarded with grants by the Deutsche Forschungsgemeinschaft (German Society for Research, DFG) and intramural funding. The fellow further received two awards for his work, one by the CRC1279 and one by the Schering-Stiftung, which were announced to the public on webpages and social media. Thus, this project has successfully launched an academic career of the Marie-Curie-Fellow at Ulm University.
Finally, we hope that targeting autophagy to combat viral infections will be a future therapeutic option available to treat patients suffering from infectious diseases.
As a direct result of this project, four publications were already published or are in revision, on two of them the Marie-Curie fellow is the corresponding author (Koepke et al, 2020, Scientific Reports, in revision, Thoms et al, 2020, BioRxiv; Science, in review) on two a co-author (Castro-Gonzalez et al, 2020, Autophagy, Braun et al, 2019, Cell Reports). Furthermore, the publication of the main part of the project is currently in the preparation phase and submission is planned to a prestigious international journal supporting open access. This project has allowed the Marie-Curie-Fellow to generate data to apply for funding to set up his own lab. He successfully was awarded with grants by the Deutsche Forschungsgemeinschaft (German Society for Research, DFG) and intramural funding. The fellow further received two awards for his work, one by the CRC1279 and one by the Schering-Stiftung, which were announced to the public on webpages and social media. Thus, this project has successfully launched an academic career of the Marie-Curie-Fellow at Ulm University.
Finally, we hope that targeting autophagy to combat viral infections will be a future therapeutic option available to treat patients suffering from infectious diseases.