Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Abstract

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

“…This mechanistic model is similar to the mechanisms suggested previously for other SF1B helicases such as RecD2(28), and provides a framework for the understanding of the NSP13 translocation mechanism and possible sites of inhibition including possible allosteric sites that may differ between the two states and block structural transitions that occur as part of the catalytic cycle. We do not describe an active base separating mechanism for NSP13 consistent with biochemical and single molecule analysis of NSP13 which was found to be a predominantly passive helicase (advancing upon the spontaneous opening of base pairs), with a strong force dependent stimulation of activity that suggests mechanoragulation by the RNA polymerase NSP12(8).…”

“…NSP13 is a 67 kDa protein that belongs to the helicase superfamily 1B, it utilizes the energy of nucleotide triphosphate hydrolysis to catalyze the unwinding of double stranded DNA or RNA in a 5' to 3' direction (7). Although NSP13 is believed to act on RNA in vivo enzymatic characterization shows a significantly more robust activity on DNA in in vitro assays with relatively weak non processive helicase activity when compared to other superfamily 1B enzymes (8,9). NSP13 has been shown to interact with the viral RNA-dependent RNA polymerase NSP12 (10,11), and acts in concert with the replicationtranscription complex (NSP7/NSP8/NSP12) (12).…”

“…NSP13 has been shown to interact with the viral RNA-dependent RNA polymerase NSP12 (10,11), and acts in concert with the replicationtranscription complex (NSP7/NSP8/NSP12) (12). This interaction has been found to significantly stimulate the helicase activity of NSP13 possibly by means of mechanoregulation (11,13). In addition to its helicase activity NSP13 also possess RNA 5' triphosphatase activity within the same active site (14), suggesting a further essential role for NSP13 in the formation of the viral 5' mRNA cap.…”

Structural basis for backtracking by the SARS-CoV-2 replication–transcription complex

“…The overall architecture of the nsp13-RTC places the nucleic acid binding site of nsp13 directly in the path of the downstream template-strand RNA (t-RNA), and cryo-EM difference maps revealed the 5′-single-stranded t-RNA overhang engaged with nsp13 before entering the RdRp active site (14). The nsp13 helicase translocates on single-stranded nucleic acid in the 5′→3′ direction (15)(16)(17)(18)(19)(20)(21)(22). Thus, this structural arrangement presents a conundrum: The RdRp translocates in the 3′→5′ direction on the t-RNA strand, while nsp13 translocates on the same strand in the opposite direction.…”

Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

“… 6 Four nonstructural proteins (nsp) are thought to be involved in the capping process: nsp10, nsp13, nsp14, and nsp16. The primary function of nsp13 is the unwinding of viral RNA during replication, 7 but it also has 5′-RNA triphosphatase activity; 8 thereby it is responsible for cleaving monophosphate at the 5′-end of the polynucleotide. The protein responsible for initial cap creation, the guanylyltransferase, is not known.…”