Alternative role of motif B in template dependent polymerase inhibition

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on the central molecular machine RNA-dependent RNA polymerase (RdRp) for the viral replication and transcription. Remdesivir at the template strand has been shown to effectively inhibit the RNA synthesis in SARS-CoV-2 RdRp by deactivating not only the complementary UTP incorporation but also the next nucleotide addition. However, the underlying molecular mechanism of the second inhibitory point remains unclear. In this work, we have performed molecular dynamics simulations and demonstrated that such inhibition has not directly acted on the nucleotide addition at the active site. Instead, the translocation of Remdesivir from +1 to-1 site is hindered thermodynamically as the post-Translocation state is less stable than the pre-Translocation state due to the motif B residue G683. Moreover, another conserved residue S682 on motif B further hinders the dynamic translocation of Remdesivir due to the steric clash with the 1′-cyano substitution. Overall, our study has unveiled an alternative role of motif B in mediating the translocation when Remdesivir is present in the template strand and complemented our understanding about the inhibitory mechanisms exerted by Remdesivir on the RNA synthesis in SARS-CoV-2 RdRp. © 2022 Chinese Physical Society.

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15.

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.

SARS-CoV-2 viral RNA levels are not 'viral load'.

Ct values are commonly used as proxies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 'viral load'. Since coronaviruses are positive single-stranded RNA [(+)ssRNA] viruses, current reverse transcription (RT)-qPCR target amplification does not distinguish replicative from transcriptional RNA. Although analyses of Ct values remain informative, equating them with viral load may lead to flawed conclusions as it is presently unknown whether (and to what extent) variation in Ct reflects variation in viral load or in gene expression.

Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

Novel RT-ddPCR assays for simultaneous quantification of multiple noncoding and coding regions of SARS-CoV-2 RNA.

A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.