Role of RNA G‑Quadruplexes in the Japanese Encephalitis Virus Genome and Their Recognition as Prospective Antiviral Targets

G-quadruplexes (GQs) have been primarily
studied in the context
of cancer and neurodegenerative pathologies. However, recent research
has shifted focus to their existence and functional roles in viral
genomes, revealing GQ-regulated key pathways in various human pathogenic
viruses. While GQ structures have been reported in the genomes of
emerging and re-emerging viruses, RNA viruses have been understudied
compared to DNA viruses, including notable examples such as human
immunodeficiency virus-1, hepatitis C virus, Ebola virus, Nipah virus,
Zika virus, and SARS-CoV-2. The flavivirus family, comprising the
Japanese encephalitis virus (JEV), poses a significant global threat
due to recurring outbreaks yet lacks approved antivirals. In this
study, we identified and characterized eight putative G-quadruplex-forming
motifs within essential genes involved in genome replication, assembly,
and internalization in the host cell, conserved across different JEV
isolates. The formation and stability of these motifs were validated
through a multitude of biophysical and cell-based assays. The interaction
and binding affinity of these motifs with the known GQ-binding ligand
BRACO-19 were supported by biophysical assays, confirming the capability
of these motifs to form GQ structures. Notably, BRACO-19 also exerted
antiviral properties through reduction of viral replication and infectious
virus titers as well as inhibition of viral protein expression, as
evaluated by the cell-based assays. This comprehensive molecular characterization
of G-quadruplex structures within the JEV genome highlights their
potential as promising antiviral targets for intervention strategies
against JEV infection through GQ-specific ligands.