A PhD position on « Packaging of the SARS-CoV-2 genomic RNA: molecular mechanism and consequences for RNA sensing» is available in our team « Viral ribonucleoproteins, incorporation of the genome and viral assembly » (https://ibmc.cnrs.fr/en/laboratoire/arn-en/equipes/viral-ribonucleoproteins-incorporation-of-the-genome-and-assembly/ ) and current Master 2 students who will graduate this summer are eligible to apply. The PhD thesis is funded by the NetRNA Labex research cluster and incorporated in the IMCBio international PhD program. Application for this project (Project PhD 2023-14) has to be done through the PhD program website (https://imcbio-phdprogram.unistra.fr ) before March 6th and the candidate have to register on this website before March 1st.
The ~30,000 nucleotide long SARS-CoV-2 RNA serves as mRNA for translation of the non-structural precursor proteins, as a template for synthesis of genomic and subgenomic RNAs, and as genomic RNA (gRNA) that must be specifically incorporated in the new viral particles. Incorporation of the SARS-CoV-2 gRNA is coupled with viral assembly, which takes place at the endoplasmic reticulum-Golgi intermediate complex (ERGIC). The N protein (nucleoprotein) plays a central role in this process by binding to yet unidentified packaging signals (PS) (1). In vitro, RNA binding to N causes liquid-liquid phase separation (LLPS), and this phenomenon might be involved in RNA packaging. Interestingly, in mouse hepatitis virus and closely related lineage A betacoronaviruses, which are the only coronaviruses for which the PS have been precisely identified, mutations in the PS not only affected RNA packaging and viral fitness, but mutant viruses also failed to overcome the innate immunity. Furthermore, targeting LLPS of SARS-CoV-2 N protein promotes innate antiviral immunity by elevating MAVS activity (2). Therefore, we hypothesize that binding of N to the PS might suppress the innate antiviral immune response.
We plan to (i) identify SARS-CoV-2 PS by analyzing RNA binding of wild-type N and of a collection of N mutants already available in the laboratory in vitro, (ii) analyze the effect of point mutations in PS on N binding in vitro and RNA packaging in virus-like particles (3), (iii) study the impact of these mutations on LLPS in vitro and in cells, and (iv) analyze the impact of the PS mutations on the MAVS activity.