Characterization of a flavivirus-inhibitory compound implicating Reticulon 3 as a potential host proviral factor

Viral infections remain among the most formidable challenges to global health, particularly due to the continuous emergence of new or re-emerging viruses. Within this context, the Flaviviridae family, which includes major human pathogens such as Zika virus (ZIKV), West Nile virus (WNV), and Dengue virus (DENV), represents a critical threat due to its capacity for rapid evolution, vector-borne transmission, and lack of effective antiviral therapies. The development of direct-acting antivirals has increased treatment options for a number of RNA viruses, but this approach is often limited by the rapid onset of viral resistance. As an alternative, host-targeted antivirals (HTAs) have gained increasing attention because they interfere with host cellular pathways essential for viral replication, offering the potential for broad-spectrum activity and reduced resistance. The present work aimed to identify and characterize novel antiviral compounds with activity against flaviviruses and other viruses of medical importance, while identifying possible host factors involved in their mechanism of action. An initial screening of 24 chemically diverse compounds, previously reported to inhibit other RNA viruses, was performed using ZIKV as a model flavivirus. Among these, the pyrido[2,3-g] quinoxalinone derivative (PS1097) emerged as the most potent antiviral candidate, exhibiting high selectivity and low cytotoxicity. Subsequent assays demonstrated that PS1097 inhibited viral replication across multiple virus families, indicating a potentially broad-spectrum effect. To determine the mechanism of action of PS1097, a series of virological and biochemical assays were performed, including time-of-addition (TOA) experiments, virucidal tests, and protein interaction studies. These analyses revealed that PS1097 does not act directly on viral particles but rather interferes with intracellular replication stages, suggesting a host-targeted mechanism. Further investigation identified significant and selective downregulation of the endoplasmic reticulum (ER)-associated protein Reticulon 3 (RTN3) following PS1097 treatment. RTN3 4 belongs to a conserved family of ER-shaping proteins involved in the formation of replication organelles (ROs), specialized membrane structures induced by many positive-sense RNA viruses to facilitate replication and shield viral RNA from immune detection. Functional assays and protein stability studies indicated that PS1097 does not promote RTN3 degradation through the proteasomal pathway. PS1097 did not exhibit virucidal properties nor interfere with viral entry, further supporting the hypothesis that its target lies within host cellular machinery. Collectively, these findings suggest that RTN3 may act as a proviral host factor exploited by flaviviruses to remodel ER membranes and establish replication organelles. The modulation of RTN3 expression by PS1097 provides a novel insight into the interplay between viral replication and ER dynamics, identifying a previously underexplored therapeutic target. Future directions include generating RTN3-overexpressing and RTN3- knockout cell lines to validate its functional role and to confirm whether PS1097- mediated RTN3 downregulation is the primary determinant of its antiviral effect. In conclusion, this study contributes to the understanding of virus–host interactions centered on ER remodeling and establishes PS1097 as a promising lead compound for the development of host-directed, broad-spectrum antivirals. By uncovering the potential proviral role of RTN3, this research highlights a new cellular vulnerability that could be exploited for future antiviral strategies, particularly against flaviviruses and other emerging RNA pathogens.