Functional Characterisation of SARS-CoV-2 Helicase NSP13

Abstract

SARS-CoV-2 is a pathological nidovirus responsible for the COVID-19 pandemic, killing about 7 million people. Belonging to the Betacoronavirus genus in the Nidovirales order, its 30 kb ssRNA+ genome codes for many structural and accessory proteins, as well as 16 non-structural proteins (NSPs) which are involved in the virus’ replication as well as pathological activity within the host cells. Of these, NSP7, NSP8, and NSP12 together for the holo-RdRp complex. The complex further interacts with other NSPs like the helicase NSP13, and accessory proteins like NSP10 and NSP14 to facilitate viral replication by transcription. This replication-transcription complex (RTC) is not unique to SARS-CoV-2 and has high sequence homology to other pathological betacoronaviruses. This combined with the complex’s essential function makes it a suitable target for antiviral drug research. NSP13 is a 67 kDa ATP-dependent helicase that plays an important part of the RTC, interacting with it as two monomers. It helps the RTC to backtrack and repair the addition of mismatched nucleotides during replication. To understand and characterise the nucleic acid binding pocket of NSP13, we selected candidate residues that might interact with the nucleotide backbone. We also selected residues that bound exclusively to the 2’ – OH groups of the RNA backbone in the computational models to decouple RNA and DNA binding and activity. Selected residues were then alanine-substituted and purified successfully, and were characterised with respect to their ATPase and unwinding activity in DNA and RNA substrates. Mutant R178/N179A, present in the 1B domain, showed decreased ATPase and unwinding activity in both substrates, while N361A shown decreased activity in the ATPase assay only. We were unable to decouple the DNA vs RNA unwinding activity of the helicase, as the mutations did not affect the activity of the protein much.