Host innate immune responses to viral RNA

"Host innate immune responses to viral RNA"Background: Coronaviruses (CoVs) are RNA viruses that have long been known to cause severe disease in livestock and companion animals. In humans, severe and fatal respiratory diseases have been observed through the emergence of zoonotic CoVs, such as SARS-CoV and MERS-CoV. CoVs are well known to efficiently evade early innate immune responses and enzymatic functions within - and virus-host interactions at - the CoV replication/transcription complex (RTC) are key to efficiently evade early innate immune responses. Working hypothesis and aims: We hypothesize that induction of early innate immune responses to CoV heavily rely on the ability of host cell innate immune sensors to access and recognize viral RNA and that CoVs have evolved efficacious mechanisms to prevent early detection of viral RNA. We further hypothesize that these early virus-host interactions predominantly take place at the CoV RTC. In order to mechanistically understand these innate immune evasion strategies, we will "illuminate" the cellular environment of the CoV RTC to identify host cell factors that are required for CoV replication, and host cell factors that are targeting the CoV RTC to restrict CoV replication. The reverse genetic systems for the mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) and well characterized recombinant mutant viruses will be used in combination with murine and human models of infection to dissect key steps and key molecules involved in early innate immune responses on the molecular level. We will furthermore employ state-of-the-art technologies involving biotin ligase-mediated proximity labeling and proteomics, CRISPR/Cas9-based functional screens, and transcriptomics in combination with ribosomal profiling to obtain a detailed mechanistic view on (i) key interactions involved in early innate immune responses, (ii) the kinetics of these interactions and (iii) the kinetics of the global host cell response under well-defined conditions. These studies will provide spatial and temporal view of basic principles of viral RNA recognition and antiviral innate immune mechanisms in different primary cell types following virus infection. Expected significance: Our proposed studies will reveal basic principles of viral RNA sensing and antiviral innate immune effector mechanisms that are highly relevant also beyond CoV infections. We expect to identify key molecules, mechanisms and pathways that promote or restrict viral replication at the site of viral RNA synthesis. This information will further our understanding on fundamental aspects of viral RNA synthesis and innate immune responses to RNA virus infection, and will facilitate the development of novel strategies to interfere with viral RNA replication during the early phase of infections. Keywords viral RNA synthesis; innate immunity; type I interferon; RNA decay; reverse genetics; viral RNA sensing; RNA virus; Coronavirus; virus replication complex Hauptdisziplin Molekularbiologie