Impact of RNA modification on coronavirus-induced innate immune responses

"Impact of RNA modification on coronavirus-induced innate immune responses"1. Project summaryBackground: Coronaviruses are RNA viruses of both veterinary and medical importance. In humans, coronavirus infections manifest usually as mild upper respiratory tract disease (common cold), however, the emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV), exemplified the zoonotic potential of coronaviruses and their ability to seriously impact also on human health. Therefore, we have to extend our knowledge on critical coronavirus-host interactions and basic mechanisms of viral RNA synthesis in order to develop novel and efficacious coronavirus vaccines and inhibitors.During our previous SNF-funded projects we have identified a number of coronaviral domains and functions that critically impact on pathogenicity. The identification of the replicase-encoded non-structural protein (nsp) 1 as a major pathogenicity factor led to the development of a novel type of live attenuated vaccines based on a modified coronavirus replicase gene. Furthermore, we discovered that the inactivation of replicase gene-encoded RNA-modifying enzymes by reverse genetics resulted in highly attenuated virus mutants in vivo, whereas virus replication in cell culture was only marginally affected. These functions include the nsp14-encoded N7-methyltransferase activity, the nsp15-encoded endonuclease activity, and the nsp16-encoded 2'O-methyltransferase activity. Our preliminary data suggest that these RNA-modifying enzymes are involved in host innate immune responses and therefore represent attractive targets for antiviral intervention and for the generation of rationally designed coronavirus vaccines.Working hypothesis: We hypothesize that modification of viral mRNA, such as N7- and 2'O-methylation, impact on two fundamental cellular processes, namely (mRNA 5'-cap-dependent) protein synthesis and the detection of molecular signatures for non-self RNA recognition. The reverse genetic system for the mouse hepatitis virus (MHV) in combination with a murine model for coronavirus infection allows for the use of genetically modified virus and host strains to assess the biological consequences of RNA methylation. Furthermore, it will be important to analyze the spatial-temporal molecular interactions within infected primary target cells with a particular focus on imaging interactions between pathogen recognition receptors, antiviral effector proteins, viral RNA, and the viral replicase-transcriptase complex. These studies will increase our understanding on foreign RNA recognition by the innate arm of the immune system and control of mRNA translation under cellular stress conditions, such as virus infection.Specific aims: The first aim of the project is to perform detailed phenotypic analyses of recombinant MHV mutants that are deficient in particular RNA-modifying enzymes. These studies will be complemented by high resolution imaging analyses (aim 2) of infected cells using confocal laser and electron microscopy.Expected significance: The proposed studies on the function of viral RNA-modifying enzymes and the nature of viral RNA modifications will provide general insights into the biology of RNA sensing. Furthermore, viruses rely on the host cell's translational machinery for protein synthesis, and thus control of mRNA translation represents an important focus for virus-host interactions. Therefore, a better understanding of viral RNA-modifying enzymes will facilitate the development of novel antiviral drugs and as well as the rational design of live attenuated viral vaccines. Keywords innate immunity; RNA methylation; non-self RNA sensing; type I interferon; pathogenicity factors; reverse genetics; RNA virus; Coronavirus; mRNA; cap-structure; mRNA methylation; innate immune responses; interferon Hauptdisziplin Molekularbiologie