Oligomeric state of β-coronavirus non-structural protein 10 stimulators studied by OmniSEC and Small Angle X-ray Scattering (preprint)

Members of the beta-coronavirus family such as SARS-CoV-2, SARS, and MERS have caused pandemics over the last 20 years. Future pandemics are likely and studying the coronavirus family members is necessary for their understanding and treatment. Coronaviruses possess 16 non-structural proteins, many of which are involved in viral replication and other vital functions. Non-structural protein 10 (nsp10) is an essential stimulator of nsp14 and nsp16, modulating RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to understanding its multifunctional role. We report the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. Proteins were subjected to a combination of OmniSEC and SEC-MALS to characterise their oligomeric state. Full-length nsp10s were predominantly monomeric in solution, while truncated versions of nsp10 have a higher tendency to oligomerise. Small angle X-ray scattering (SAXS) experiments reveal a globular shape of nsp10 which is conserved in all three coronaviruses, including MERS nsp10, which diverges most from SARS and SARS-CoV-2 nsp10s. In conclusion, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 are globular and predominantly monomeric in solution. Additionally, we describe for the first time a functional role of the C-terminus of nsp10 for tight binding to nsp14.

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16 (preprint)

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5' end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2'-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the host's innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays, and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets.

Two ligand-binding sites on SARS-CoV-2 non-structural protein 1 revealed by fragment-based x-ray screening (preprint)

The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 through suppressing its replication and or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.95 A). Binding affinities of hits against nsp1 were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal sift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medially relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.