A novel replicase-mediated self-amplifying RNA amplification mechanism of the SARS-CoV-2 replication-transcription systemLiu, Lin, Hung
et alBiochem Biophys Res Commun (2025) 758, 151654
Abstract: A novel self-amplifying mRNA (samRNA) amplification mechanism was first discovered in the SARS-CoV-2 replication-transcription system and named replicase cycling reaction (RCR). In principle, RCR is a replicase-mediated transcription reaction driven by the SARS-CoV-2 RNA-dependent RNA polymerases (RdRPs), which amplify a specific samRNA construct consisting of an RNA/mRNA sequence flanked by a 5'-end RdRP-reverse-promoter (5'-RdRP-RP) and a 3'-end RdRP-forward-promoter (3'-RdRP-FP) on both sides. Based on this samRNA composition, we had not only successfully established the first in-vitro RCR reaction for directly amplifying the SARS-CoV-2 genomic and subgenomic RNAs but also further used it in a combined in-vitro-transcription and RCR (IVT-RCR) protocol to identify new functions of the SARS-CoV-2 NSP7, NSP8, and NSP12 proteins, leading to a fast diagnostic assay for measuring the SARS-CoV-2 RdRP activity. These findings may shed a new light on the molecular mechanisms of SARS-CoV-2 replication and transcription. As a result, in addition to the previously found primer-dependent RNA synthesis activity of the coronaviral RdRP complexes, we herein reported another new 5'/3'-promoter-dependent, primer-independent samRNA synthesis mechanism mediated by the SARS-CoV-2 RdRP complex. Based on this novel RCR mechanism, the associated samRNA composition is conceivably useful for facilitating the design and development of next-generation RNA/mRNA medicines and vaccines.Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.
Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7Singh, Kushwaha, Kulandaisamy
et alMol Ther Nucleic Acids (2025) 36 (1), 102452
Abstract: RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication.© 2025 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.
A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competentKlein, Das, Bera
et albioRxiv (2025)
Abstract: Coronaviruses (CoV) encode sixteen non-structural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsp's essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.
Single-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complexLovell, Dewling, Li
et albioRxiv (2025)
Abstract: The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multi-subunit replication complex consisting of non-structural proteins (nsps) 12, 7 and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein-induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or co-expressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none) nsp8 and nsp12, in nsp8-nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, transient, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic, and the correct conformation must be achieved before stable RNA binding can occur. Notably, the co-expressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity, and protein activity, and underscores differences between co-expressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.
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