NIH Research Festival
SARS-CoV-2, a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. We biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA, demonstrating strand-specific interactions. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 to disrupt a high-affinity nucleic acid-protein interaction, in a uni-directional manner and with a preferential displacement of streptavidin complex from biotinylated ssDNA versus ssRNA. Our studies demonstrate that this displacement is dependent on its intrinsic ATP hydrolysis function, building upon structural studies of the RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. We propose that this novel biochemical activity implicates a catalytic role of Nsp13 in protein-RNA remodeling during genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into coronavirus proofreading. In further studies, we have examined the ability of Nsp13 to resolve SARS-CoV-2 derived RNA secondary structures to enable the polymerase complex (Nsp12/7/8) during synthesis. Our results show that wild-type Nsp13 inhibited polymerase extension whereas the ATPase-dead mutant stimulated activity, implicating an ATP-dependent catalytic function or protein interaction of Nsp13 that modulates RNA synthesis by Nsp12/7/8 during replication.
Scientific Focus Area: Molecular Biology and Biochemistry
This page was last updated on Monday, September 25, 2023