RNA G-quadruplex in TMPRSS2 reduces SARS-CoV-2 infection

The COVID-19 pandemic, caused by SARS-CoV-2, presents a significant global health crisis. Understanding SARS-CoV-2 pathogenesis is crucial for developing effective treatments. While ACE2 and TMPRSS2 are known host factors essential for SARS-CoV-2 entry, the regulatory mechanisms of these and other host molecules remain unclear. The SARS-CoV-2 genome, as a positive-sense RNA, functions as a template for both replication and translation, likely containing functional RNA elements that modulate gene regulation. RNA G-quadruplexes (RG4s), non-canonical secondary structures formed by guanine-rich sequences, are present in various viruses and have been implicated in pathogenesis. RG4s are also found in the human transcriptome, suggesting their role in both physiological and pathological processes. Given the known functions of RG4s in viral infections and their abundance in both viral and human genomes, this study explores the potential role of RG4s in SARS-CoV-2 infection.

Previous research has identified ACE2 and TMPRSS2 as critical host factors for SARS-CoV-2 infection. TMPRSS2, a transmembrane serine protease, facilitates the entry of the virus into host cells by activating the viral spike protein. Inhibitors of TMPRSS2, such as camostat mesylate, have shown promise as potential antiviral drugs. However, the regulatory mechanisms governing the expression of these host factors remained elusive. Studies have also shown the presence of RG4 structures in various viruses, impacting viral pathogenesis. However, the role of RG4 in SARS-CoV-2 infection specifically was yet to be determined. Existing literature on RG4 structures in other viruses and their influence on gene expression provides a foundation for investigating a similar role in SARS-CoV-2.

This research employed a multi-faceted approach combining bioinformatics, biochemical, and biophysical techniques, in vitro and in vivo experiments, and analysis of clinical samples. Bioinformatics analysis was used to predict potential G4-forming sequences (PQSs) within the SARS-CoV-2 genome and host factors (ACE2 and TMPRSS2). Selected PQSs were experimentally validated using circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy with G4-specific probes (NMM and ThT), and fluorescence resonance energy transfer (FRET) assays to confirm RG4 formation and assess their stability. Gel mobility shift assays were performed to analyze structure compaction. Bio-layer interferometry (BLI) was employed to measure the binding affinity of G4-stabilizing ligands (PDS) to the target sequences. In vitro experiments involved the use of cell lines (H1299, HBE, hACE2-293T) to investigate the effects of RG4 stabilization on TMPRSS2 expression using plasmids encoding wild-type and RG4-mutant TMPRSS2. Polysome profiling was used to assess the effects on TMPRSS2 translation. A pseudovirus system (VSV pseudotyped with SARS-CoV-2 S glycoprotein) was employed to analyze the effects of RG4 stabilization on SARS-CoV-2 entry. In vivo studies were conducted using C57BL/6J mice expressing human ACE2 to evaluate the impact of RG4 stabilization (using PDS) on SARS-CoV-2 pseudovirus infection. Immunohistochemistry (IHC), ELISA, and Western blot were used to assess TMPRSS2 levels in mouse lungs. Finally, IHC staining was performed on lung tissues from healthy individuals and COVID-19 patients to assess TMPRSS2 expression levels.

Bioinformatics predicted multiple PQSs in the SARS-CoV-2 genome and host factors (ACE2 and TMPRSS2). Experimental validation confirmed RG4 formation for PQS-13385 in the SARS-CoV-2 NSP10 and PQS-675 within TMPRSS2. The RG4 structure within TMPRSS2 (GQS-675) inhibited TMPRSS2 translation, as demonstrated by reduced protein levels but unchanged mRNA levels upon RG4 stabilization. G4-specific stabilizers (PDS, cPDS, TMPyP4) reduced TMPRSS2 protein levels in a GQS-675-dependent manner in several cell lines. Polysome profiling showed a shift of TMPRSS2 mRNA towards lighter polysome fractions upon PDS treatment, indicating translational inhibition. In pseudovirus infection assays, RG4 stabilizers significantly reduced SARS-CoV-2 entry efficiency in human lung cells, with PDS exhibiting comparable efficacy to camostat mesylate. In vivo experiments demonstrated that PDS treatment reduced SARS-CoV-2 pseudovirus infection in hACE2-expressing mice, accompanied by a decrease in lung TMPRSS2 levels. Furthermore, IHC analysis revealed increased TMPRSS2 expression in the lungs of COVID-19 patients compared to healthy controls.

This study provides compelling evidence for a novel regulatory mechanism involving RG4 in SARS-CoV-2 infection. The identification of RG4s in both the SARS-CoV-2 genome and host factors, particularly the functional RG4 in TMPRSS2, highlights a previously unknown aspect of viral pathogenesis. The ability of G4 stabilizers to reduce viral entry both in vitro and in vivo suggests RG4-targeting strategies as promising avenues for COVID-19 prevention and treatment. The observed increase in TMPRSS2 protein in the lungs of COVID-19 patients corroborates the findings and suggests a potential clinical relevance. The findings suggest that manipulating RG4 formation could be an effective antiviral strategy. The research also expands our understanding of the complex interplay between viral and host factors in SARS-CoV-2 infection.

This research demonstrates the involvement of RG4s in SARS-CoV-2 infection, specifically highlighting the role of a TMPRSS2 RG4 in regulating viral entry. The effectiveness of G4 stabilizers in reducing infection both in vitro and in vivo suggests a novel therapeutic target for COVID-19. Future research should focus on developing specific RG4-targeting agents, validating these findings using authentic SARS-CoV-2, and conducting larger clinical studies to establish the clinical relevance of RG4 regulation in COVID-19 pathogenesis. Investigating the mechanisms regulating TMPRSS2 RG4 and the roles of other potentially relevant host factors containing RG4s warrants further study.

This study used a pseudovirus system, which may not fully recapitulate the complexity of authentic SARS-CoV-2 infection. The in vivo studies utilized a pseudovirus and AAV9, which has both pulmonic and hepatic tropism leading to observation in both lung and liver. The clinical correlation was based on a limited number of COVID-19 patient samples. Future research should address these limitations by employing authentic SARS-CoV-2 infection assays, using lung-specific AAV vectors and conducting larger clinical studies.