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

The study investigates whether RNA G-quadruplex (RG4) structures in the SARS-CoV-2 genome and host mRNAs regulate infection and pathogenesis. SARS-CoV-2 infection relies on host factors ACE2 and TMPRSS2 for viral entry, yet regulatory mechanisms controlling these host molecules are unclear. RG4s are guanine-rich RNA secondary structures implicated in gene regulation and viral pathogenesis. The authors hypothesize that RG4s within the virus and host, particularly within TMPRSS2, modulate translation and thereby influence SARS-CoV-2 entry. They aim to identify RG4s bioinformatically and validate them biochemically/biophysically, determine effects on TMPRSS2 translation, and test whether pharmacological stabilization of RG4s reduces SARS-CoV-2 pseudovirus infection in cells and mice.

Prior work shows RG4s are prevalent in many viruses (ebola, HCV, HIV, SARS-CoV) and throughout the human transcriptome, regulating processes such as splicing, localization, and translation, with roles in diseases including cancer, neurological and metabolic disorders, and viral infections. SARS-CoV-related proteins (e.g., NSP3) can bind host RG4s, potentially affecting antiviral responses. Emerging reports identified putative RG4s in the SARS-CoV-2 genome and suggested they may affect viral gene expression. TMPRSS2 inhibition (e.g., camostat mesylate) has been proposed to block SARS-CoV-2 entry, but upstream regulatory mechanisms of TMPRSS2 remain poorly defined.

• Bioinformatic prediction of putative G-quadruplex-forming sequences (PQSs) using QGRS-mapper in SARS-CoV-2 genome and host Ace2/Tmprss2 mRNAs; conservation assessed via UCSC Genome Browser.
• Biophysical/biochemical validation of RG4 formation: circular dichroism (CD) spectroscopy, fluorescence emission with G4 probes N-methyl mesoporphyrin IX (NMM) and Thioflavin T (ThT), fluorescence resonance energy transfer (FRET) spectra and melting, CD melting to determine Tm, gel mobility shift assays (native PAGE) using FAM-labeled RNAs, and bio-layer interferometry (BLI) to quantify binding between pyridostatin (PDS) and PQS RNAs.
• In-cell RG4 visualization: transfection of FAM-labeled PQS-675 RNAs (WT and G4-mutant) into H1299 cells followed by immunofluorescence with BG4 G4-specific antibody; modulation with RG4 stabilizer PDS and antisense oligonucleotides (ASOs) targeting PQS-675.
• Construction of expression plasmids: full-length human and mouse Tmprss2 ORFs cloned into pcDNA3.1; RG4-disrupting synonymous mutations introduced into central G-tracts (human hG4mut1/2/3; mouse mG4mut). Transfections performed in H1299, HBE, hACE2-293T, and LLC cells.
• Pharmacological modulation: treatment with RG4 stabilizers PDS, carboxypyridostatin (cPDS), and TMPyP4; comparison with TMPRSS2 inhibitor camostat in entry assays.
• Assessment of TMPRSS2 expression: western blotting (HA-tagged and endogenous), ELISA (mouse lungs), immunohistochemistry (IHC) in mouse lungs and human lung tissues; qRT-PCR for mRNA levels.
• Polysome profiling to evaluate translation efficiency and ribosome association of endogenous and exogenous Tmprss2 mRNAs, with/without PDS.
• Pseudovirus entry assays: vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike (VSV-SARS-2-S-luc) carrying Renilla luciferase; luciferase readout in cell lysates post-infection.
• Mouse in vivo model: C57BL/6J mice transduced intrathoracically with AAV9-hACE2, then challenged with VSV-SARS-2-S-luc; daily PDS (6 mg/kg) versus saline; in vivo bioluminescence imaging (IVIS), lung Renilla mRNA quantification, TMPRSS2 protein by ELISA, western blot, and IHC; toxicity assessment by survival, behavior, histology, and serum biochemistry.
• Cell viability by MTS assay; statistics with two-tailed unpaired Student’s t-test; p<0.05 significant.

• SARS-CoV-2 genome harbors multiple PQSs; PQS-13385 within NSP10 forms a K+-dependent RG4 (CD signature with positive ~264 nm, negative ~238 nm; increased NMM/ThT fluorescence; higher Tm in KCl vs LiCl), indicating presence of viral RG4s.
• Host mRNAs Ace2 and Tmprss2 contain PQSs; Tmprss2 PQS-675 (GQS-675) in the ORF is highly conserved and forms a canonical 3-quartet RG4 in vitro: CD and fluorescence signatures in KCl, increased FRET, higher Tm in KCl, retarded mobility in native PAGE; PDS enhances CD/FRET and Tm; BLI shows high-affinity binding of PDS to PQS-675-WT (reported KD=604.9±2.84 (M), Ka=1356±5.12 (1/Ms), Kd=8.21±0.02E-04 (1/s)), with much weaker binding to mutant (KD=3327±0.07 (M)).
• In cells, FAM-labeled PQS-675 WT RNA colocalizes with BG4 foci; PDS increases and ASOs decrease colocalization, while G4-mutant shows minimal colocalization, supporting in-cell RG4 formation.
• RG4 stabilizers (PDS, cPDS, TMPyP4) reduce endogenous TMPRSS2 protein levels in H1299 without affecting Tmprss2 mRNA, indicating post-transcriptional repression (e.g., significant reductions with PDS p=0.00012; cPDS p=0.0082).
• Exogenous expression: hG4mut1 (RG4-disrupted) yields higher TMPRSS2 protein than hG4WT at similar mRNA levels in H1299 and hACE2-293T, demonstrating RG4-dependent translational repression; RG4 stabilizers suppress TMPRSS2 from hG4WT but not from hG4mut plasmids. Additional mutants (hG4mut2/3) corroborate RG4-specific effects and exclude codon bias.
• Polysome profiling: PDS shifts endogenous Tmprss2 mRNA toward lighter polysomes, indicating reduced translation; exogenous hG4WT shows decreased ribosome association with PDS, while hG4mut1 translation profile is largely unchanged despite global translation attenuation.
• Pseudovirus entry: RG4 stabilizers significantly decrease VSV-SARS-2-S-luc entry in H1299 and HBE, with PDS efficacy comparable to camostat; PDS reduces entry in hG4WT-expressing cells but not in hG4mut1-expressing cells, linking inhibition to GQS-675.
• Mouse model: Daily PDS (6 mg/kg) in AAV9-hACE2 mice reduces pseudovirus bioluminescence signal and Renilla mRNA in lungs (e.g., significant reductions at day 8), and lowers lung TMPRSS2 protein by IHC, ELISA, and western blot; similar reduction observed in liver TMPRSS2. PDS shows low apparent toxicity at tested doses.
• Clinical relevance: TMPRSS2 protein is increased in lungs of COVID-19 patients versus healthy controls, consistent with a role in pathogenesis and supporting therapeutic targeting of RG4-mediated regulation.

The study addresses whether RG4 structures regulate SARS-CoV-2 infection by modulating host and viral RNAs. Findings demonstrate that a conserved 3-quartet RG4 within the TMPRSS2 ORF represses translation, thereby limiting SARS-CoV-2 spike-mediated entry. Stabilizing RG4s with ligands like PDS inhibits pseudovirus infection in cells and reduces infection markers and TMPRSS2 expression in vivo, suggesting RG4s as actionable regulatory elements. The presence of viral RG4s (e.g., NSP10) indicates broader roles in the viral life cycle. The results position RG4-mediated translational control as a rapid post-transcriptional mechanism influencing host susceptibility. Given that other entry-associated host factors (AXL, furin, cathepsin L, neuropilin-1) also harbor putative RG4s, RG4 targeting may have wider antiviral applicability. However, PDS likely binds multiple G4s, raising specificity concerns; future work should develop gene- or site-specific RG4-targeted strategies and assess activity across SARS-CoV-2 variants and authentic viral infection models. Potential cellular regulators (e.g., RG4 helicases DHX9, DHX36, DDX3X, DDX5, DDX21) and viral proteins (NSP3, NSP13) may modulate RG4 dynamics during infection.

This work uncovers a previously unappreciated RG4-dependent post-transcriptional mechanism governing TMPRSS2 translation and SARS-CoV-2 entry. It validates RG4 presence in both SARS-CoV-2 and host mRNAs and shows that pharmacological stabilization of RG4s suppresses pseudovirus infection in vitro and in vivo while lowering TMPRSS2 protein levels. The study highlights RG4s as potential therapeutic targets for COVID-19. Future directions include: validating effects with authentic SARS-CoV-2 and primary human lung cells; developing TMPRSS2 RG4-specific ligands or oligonucleotide-based strategies; evaluating efficacy against viral variants; dissecting endogenous regulators of TMPRSS2 RG4; optimizing targeted delivery (e.g., lung-specific) and assessing long-term safety and pharmacology.

• Use of a pseudovirus model assesses entry but not the full SARS-CoV-2 life cycle; confirmation with authentic virus and primary patient-derived cells is needed.
• PDS is a non-specific G4 stabilizer and may affect multiple host and viral G4s; target specificity remains unresolved, and TMPyP4 can destabilize some RG4s.
• Evidence of PDS toxicity is limited to short-term mouse studies; comprehensive pharmacokinetics and safety are pending.
• AAV9-hACE2 approach exhibited pulmonary and hepatic targeting, complicating tissue-specific interpretation; lung-specific vectors may refine conclusions.
• Small number of human lung specimens limits generalizability of TMPRSS2 upregulation findings.
• Global translation attenuation by PDS could confound interpretation; though mutant analyses support RG4 specificity for TMPRSS2, broader translational effects warrant caution.