Biology subjects

Gotte, M.

Publications and source records attributed to Gotte, M..

4 recordsLinked to original sources

bioRxiv2025

Single-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complex

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.

biophysics↗

bioRxiv2025

A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

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 nsps essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.

biophysics↗

bioRxiv2024

Mechanism and spectrum of inhibition of a 4'-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate (TP) metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

biochemistry↗

bioRxiv2022

Distinct genetic determinants and mechanisms of SARS-CoV-2 resistance to remdesivir

The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC50. Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a [~]10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings. One Sentence SummarySARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase

microbiology↗