Molecular role of the viral nucleoprotein in the SARS-Cov-2 replication cycle – SARS2NUCLEOPROTEIN

In order to develop rational strategies for the development of inhibitors of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) it is essential to understand the molecular basis of different aspects of the viral cycle. Coronaviruses are enveloped positive-sense single strand RNA viruses that express their own replication machinery. Replication of SARS-CoV-2 is carried out by the RNA-dependent RNA polymerase complex, comprising nsp12, bound to nsp7 and nsp8. The processes of genome replication and transcription represent important targets for viral inhibition.
The nucleoprotein of SARS-CoV-2 (N) is an important cofactor of the viral replication complex, encapsidating the viral genome in the nucleocapsid, thereby protecting it from the host cell environment, and regulating gene transcription in the infected cell. N colocalizes to the replicase-transcriptase complex, although the molecular basis of its role in regulation of these processes remains unknown. It has recently been shown in SARS-CoV-1 that this colocalization depends on an essential interaction with the N-terminal component of another viral protein Nsp3 (Nsp3a).
Coronaviral N is a highly dynamic protein, comprising five domains, three of which are predicted to be intrinsically disordered (ID), intercalated by two folded domains. The structural features of coronaviral N are strongly conserved, with 90% sequence identity between SARS-CoV-1 and SARS-CoV-2. Both folded domains have been shown to bind RNA, and the C-terminal folded domain is responsible for oligomerization and the formation of higher-order assemblies that form the nucleocapsid. The central linker domain comprises a serine-arginine (SR) rich domain that is phosphorylated, a modification that impacts gene regulation and replication efficiency.
N has recently been shown to undergo liquid-liquid phase separation (LLPS), forming biomolecular condensates upon mixing with RNA. Phosphorylation of the central linker domain modulates the biophysical properties of the droplets. The formation of such membraneless organelles has been shown to occur for negative sense RNA viruses, such as measles, rabies and nipah viruses, where nucleo- and phosphoproteins form liquid droplets in solution. The formation of such viral factories has been shown to enhance processes such as encapsidation of the viral genome as shown recently by the coordinating group. The molecular basis of the stabilization of this phenomenon remains unknown in the case of SARS-CoV-2.
Nsp3a is also a highly dynamic protein, with a ubiquitin-like folded domain and a long, acidic, ID domain at the C-terminus. The role of the ID domain is not known but is predicted to be 23 amino acids longer in the case of SARS-CoV-2 compared to SARS-CoV-1.
In this project we aim to characterize the conformational behavior of N from SARS-CoV-2 at atomic resolution using NMR spectroscopy, small angle X-ray scattering (SAXS) and fluorescence microscopy and spectroscopy as well as its interaction with the viral cofactor Nsp3a. We will characterize the impact of phosphorylation on the conformational dynamics and on the interaction with Nsp3a. We will investigate the impact of both phosphorylation and Nsp3a interaction on the formation and nature of SARS-CoV-2 liquid droplets at atomic resolution. We will also investigate the possibility of observing the kinetics and the structural basis of nucleocapsid assembly upon RNA in vitro, as recently demonstrated for measles virus
This study will characterize the key interactions involving N, thereby laying the basis of the conception of possible development of inhibitors of viral replication (for example peptides that mimic interaction sites in the ID domains) and therefore falls into the category ‘Physiopathogenesis of the disease’. The coordinating group is a member of the COVID19-NMR consortium (covid19-nmr.de) whose aim is to acquire and rapidly disseminate NMR-based information on Sars-CoV-2 proteins and RNA.