Rapid Hybrid Structure Determination of Coronavirus Protein-RNA Complexes as a Basis for Drug Screening for the Treatment of COVID-19

The ongoing pandemic caused by SARS-CoV-2 calls for world-wide, concerted efforts to generate new insights into the biology of betacoronaviruses. Because coronaviruses are single-stranded RNA viruses with a large (approximately 30 kilobases) RNA, the interactions between viral proteins and the viral RNA are of central importance to essential functions of the virus, for example replication in the host cell. Surprisingly, very little is known about the details of these interactions at a molecular level as high-resolution structures of protein-RNA complexes of SARS-CoV-1 and 2 or MERS-CoV are completely absent. Insights into the three-dimensional organization of the interplay between protein and RNA which are highly conserved in this virus family would however enable the development of new therapeutic approaches, also in preparation for the likely re-emergence of a related virus.To fill this gap, we propose to perform structural studies on two very important RNA-binding proteins of SARS-CoV, the nucleocapsid N protein and the non-structural protein 3 (Nsp3), by characterizing their interactions with RNA which will be the basis for performing rational drug screens. To achieve this, we will combine the expertise of the two applicant groups in the structural analysis of ribonucleoproteins: Nuclear magnetic resonance (NMR) spectroscopy and protein-RNA cross-linking coupled to mass spectrometry (MS). In a stepwise fashion, we plan to apply these techniques individually, starting with individual domains of the target proteins from both SARS-CoV-1 and 2. Results from these complementary methods will then be combined in an integrative approach to obtain structural insights of their mode of binding to RNA. Also complexes between full-length proteins and RNA will be investigated. With the availability of structural data of two viruses, commonalities and differences between them will allow the generation of new hypotheses related to their pathogenesis and provide the basis for new therapeutic approaches. We will take advantage of these information to perform drug screening, adding an element of immediate translational impact to our structural biology efforts. The combination of NMR- and MS-based approaches is known to accelerate structure determination of challenging targets, which is of particular urgency in the context of the current and future global health threat by this group of RNA viruses.