Interactions between coronavirus non-structural proteins 7-10 and RNA

Coronaviruses (including SARS-CoV) are enveloped, positive-sense RNA viruses and posses the largest viral RNA genomes known, encoding several structural and auxiliary proteins as well as two large overlapping polyproteins, pp1a (replicase 1a, around 450 kDa) and pp1ab (replicase 1ab, around 750 kDa) necessary for viral RNA synthesis. These two polyproteins are cleaved extensively by the main protease (Mpro, also often called 3C-like protease) and the papain-like cysteine proteases (PL1pro and PL2pro), all of which are encoded by the viral genome, to yield a multi-subunit protein complex termed “viral replicase-transcriptase” (RTC). In the course of an infection cycle, the replicase-transcriptase complex amplifies the genomic RNA and synthesizes subgenomic mRNAs. Amplification of the genomic RNA involves full-length negative-strand templates, and the synthesis of subgenomic (sg) mRNA involves subgenome-length negative-strand templates. The cis-acting RNA elements involved in the different phases of coronavirus RNA synthesis have been studied extensively. It has been shown that 5’- and 3’-UTR of the genome are required for viral replication and transcription, as well as the so-called transcription-regulating sequence (TRS) element. In contrast, there is still very little known about the structure, functions and interactions of viral and cellular proteins in the RTC as it is engaged in different modes of RNA synthesis.

 

In order to address these questions, the specific binding affinities of various SARS-CoV nonstructural proteins (Nsps) with viral genome-derived RNA fragments will be tested by biochemical and biophysical methods. Building on preliminary data that we already have, computational biology will be employed to predict the secondary structures of the RNA fragments that have been or are yet to be identified as recognition elements for Nsps. Also, mutations will be designed by computational methods both in the RNA and in the interacting proteins in order to identify the key elements of the interactions. The binding processes will be characterized by Surface Plasmon Resonance, gel shift assays, isothermal titration calorimetry, and fluorescence methods (including single-molecule spectroscopy). Reverse genetics will be used to prove the function of each protein within the RTC. Well-defined complexes between Nsps and RNA will be subjected to crystallization and X-ray structure determination (a process that involves a large share of computation (>60%). The project is highly interdisciplinary; parts of the work will be carried out between the following institutes: Biochemistry, Chemistry, Molecular Medicine, Neuro- and Bioinformatics, Physics, and Virology & Cell Biology.

 

People in this Project

Yibei Xiao, M.Sc.Doctoral Candidate
Rolf Hilgenfeld, Prof. Dr. Dr. h.c.Investigator
Georg Sczakiel, Prof. Dr. rer. nat.Investigator

Publications

No publications so far.