Host innate immune responses to viral RNA

"Host innate immune responses to viral RNA"1. Project summaryBackground: Coronaviruses are RNA viruses of both veterinary and medical importance. The SARS-CoV epidemic 2002/2003, and the recent emergence of the novel human coronavirus EMC, exemplified the zoonotic potential of coronaviruses and their ability to seriously impact on human health. Notably, in most cell types neither high nor low pathogenic coronaviruses elicit pronounced innate immune responses, such as type-I interferons (IFNs), during the early phase of viral infection, suggesting that coronaviruses counteract sensing of viral nucleic acid. During our previous SNF-funded projects we have uncovered a link between Mda5-mediated sensing of coronaviral RNA and ribose 2'O-methylation, suggesting that RNA modifications such as 2'O-methylation provide a molecular signature for discrimination of self and non-self mRNA. More recently, by assessing a coronaviral inhibitor targeting membrane-bound RNA synthesis, we found that inhibitor-resistant viruses can efficiently replicate without inducing double-membrane vesicles (DMVs). These DMVs are a hallmark of coronavirus replication and harbor dsRNA. Therefore, these inhibitor-resistant viruses represent an excellent tool to study sensing of viral RNA and antiviral effector mechanisms under conditions where dsRNA is not shielded by DMVs but accessible to binding by host cell cytosolic RNA sensors.Working hypothesis and aims: We hypothesize that induction of innate immune responses to viral RNA and restriction of RNA virus replication is dependent on (i) modification of viral mRNA, such as 2'O-methylation, and (ii) localization of viral RNA in the host cell. Furthermore, innate immune responses and restriction of RNA virus replication appears to be very different in particular cell types, depending on which particular innate immune pathways and antiviral mechanisms dominate. The reverse genetic systems for the mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) in combination with murine and human models of infection that are amenable to genetic modification will allow us to dissect key steps and key molecules involved in host innate immune responses on the molecular level. It will be important to analyze the molecular interactions within infected primary target cells with a particular focus on interactions between pathogen recognition receptors, antiviral effector proteins, dsRNA, and the viral replicase-transcriptase complex. To do this we will generate a number of recombinant murine and human coronaviruses that are deficient in 2'O-methylation, contain inhibitor-resistant mutations and combinations thereof. Using these novel tools we will then assess innate immune responses in primary human and murine cells in order to obtain a detailed view on kinetics and quality of innate immune responses under well-defined conditions. These studies will provide spatial and temporal view of basic principles of RNA recognition and antiviral innate immune mechanisms in different primary cell types following virus infection. Expected significance: The proposed studies will provide general insights into the biology of viral RNA sensing and antiviral effector mechanisms that are highly relevant also beyond coronavirus infections. We expect to identify key mediators of viral RNA sensing and the "antiviral state" that are apparently counteracted by many RNA viruses through methylation of viral mRNA and shielding of dsRNA by host cell membranes. Therefore, a better understanding of these fundamental aspects of innate immune responses to RNA virus infection will facilitate the development of novel strategies to interfere with viral RNA replication during the early phase of infections. Keywords localization of viral RNA; innate immunity; type I interferon; non-self RNA sensing; reverse genetics; RNA virus; viral RNA synthesis; Coronavirus; RNA modification Hauptdisziplin Molekularbiologie