Biology subjects

Tersteeg, S.

Publications and source records attributed to Tersteeg, S..

3 recordsLinked to original sources

bioRxiv2025

Escape from SARS-CoV-2 Nsp1-mediated host shutoff by TIAR transcript reveals general features of Nsp1 resistance

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape strategies include general inhibition of host gene expression referred to as host shutoff. Viral non-structural protein 1 (Nsp1) is the main host shutoff factor that blocks protein translation and induces messenger RNA (mRNA) cleavage and degradation. Viral mRNAs are resistant to the translation shutoff and cleavage induced by Nsp1, and the 5 leader sequence present in all viral mRNAs has been shown to confer resistance. However, the exact molecular mechanism for escape from Nsp1 host shutoff has not been demonstrated. In our previous work, we analyzed the effects of Nsp1 on the expression and function of cellular proteins important for stress granule formation. We discovered that the host transcript for the TIA1 cytotoxic granule-associated RNA binding protein like 1 (TIAL1, commonly referred to as TIAR) is resistant to SARS-CoV-2 Nsp1 host shutoff. In this work, using reporter shutoff assays, we examined sequence and structural features of the TIAR 5 untranslated region (UTR) and discovered that the first 23 nucleotides of the TIAR transcript are both necessary and sufficient to confer resistance to the Nsp1. Furthermore, our work revealed that the lack of guanosines within a window of 10 to 18 nucleotides downstream from the 5 end is a defining feature of Nsp1-resistant transcripts shared between the SARS-CoV-2 leader sequence and the TIAR 5 UTR. Our findings are consistent with the model in which sequence features of 5 UTRs, rather than their secondary structure, confer resistance to Nsp1 host shutoff to both viral and cellular mRNAs.

molecular biology↗

bioRxiv2025

A Guanine-quadruplex located on the negative strand of the hepatitis C virus facilitates efficient genomic RNA synthesis

Guanine-rich nucleic acids can form a unique secondary structure called Guanine-quadruplexes (G4s). These G4s are conserved across all domains of life and in viruses, where they can play important regulatory roles in the viral lifecycle. In flaviviruses, the (-) strand 3 untranslated region (UTR) is essential for initiating genomic RNA synthesis. Within the (-) strand 3 UTR of hepatitis C virus (HCV), a highly conserved G4 is located within stem-loop IIy (SLIIy), spanning nucleotides 110-131--a region that is essential for efficient replication. Using bioinformatics, we demonstrate that nucleotides 110-131 are highly conserved across HCV genotypes 1-7, and this region within SLIIy can likely adopt a more energetically favorable G4 conformation rather than the predicted hairpin. From biophysical and cell biology experiments, we show that stabilizing the predicted hairpin structure in SLIIy disrupts RNA synthesis, while successive guanine-to-adenine mutations within the G4 sequence impair G4 formation and hinder replication. Combining our findings with prior studies, we propose that the SLIIy G4 plays a crucial role in recruiting NS3 helicase, which is necessary for unwinding RNA structures and facilitating access for NS5B polymerase. The G4 may serve as a regulatory switch that modulates helicase binding and replication efficiency. We propose that in the absence of a stable G4, NS3 recruitment or unwinding is impaired, leading to inefficient RNA unwinding and reduced polymerase activity, ultimately hindering viral replication. These results reveal a previously unrecognized role for G4 structures in the HCV replication cycle, highlighting their importance in regulating (+) strand RNA synthesis. ImportanceHepatitis C virus is a positive-sense single-stranded RNA virus that relies on an intermediate negative strand to generate new genomic RNA. While the last 157 nucleotides of the negative-strand 3 untranslated region are known to be essential for replication, the underlying regulatory mechanisms have remained poorly understood. Our study demonstrates that a highly conserved guanine-rich sequence (107-131 nt) located on the negative strand SLIIy forms a guanine-quadruplex secondary structure that plays a pivotal role in orchestrating viral RNA synthesis. This G4 not only promotes efficient replication but likely acts as a molecular target to recruit the NS3 helicase, thereby allowing access for the NS5B polymerase. Disruption of the G4 structure and stabilization of the stem-loop severely impairs viral replication. These findings reveal an unrecognized layer of post-transcriptional regulation in the HCV lifecycle and establish G4 RNA structures as a critical element in HCV genome replication.

biophysics↗

bioRxiv2022

Purification and Characterization of Inorganic Pyrophosphatase for in vitro RNA Transcription

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and of high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering (SEC-MALS-DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for labs involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production. Statement of SignificanceWe show an easy two-step purification procedure to efficiently produce large quantities of iPPase, an expensive component of IVT reactions. Laboratory-produced iPPase can significantly reduce the financial strain on research labs that rely on large-scale RNA production for experiments. Furthermore, we show for the first time, using a combination of orthogonal biophysical techniques, that yeast iPPase assembles into higher-order oligomers similar to bacterial iPPase.

biochemistry↗