The phleboviruses Sandfly Fever Naples virus and Toscana virus uncouple the RNase and kinase activity of IRE-1

Background: Toscana virus (TOSV) and Sandfly Fever Naples virus (SFNV) are the most common phleboviruses in the Mediterranean Basin. Although antigenically related, they differ in pathogenicity: TOSV exhibits pronounced neurotropism and can cause severe or fatal neurological disease, whereas SFNV typically induces a transient, self-limiting, flu-like illness. No vaccine or specific antivirals are currently available against phleboviral infections. Understanding their replication cycles and interactions with host defenses is, therefore, essential. One of the earliest cellular defense mechanisms is the unfolded protein response (UPR), triggered by endoplasmic reticulum stress, which is capable of inducing autophagy or apoptosis. Conversely, viruses exploit host translation and often manipulate the UPR to enhance replication. This study investigates how TOSV and SFNV modulate UPR signaling during infection, providing insight into virus–host interactions and identifying potential targets for therapeutic intervention. Methodology: Human lung carcinoma (A549) cells were infected with TOSV and SFNV and monitored over a 16-hour time course for RNA and protein extraction. The expression of key UPR effectors (BIP, PERK, ATF6, IRE1, ATF4, CHOP, and eIF2α) was analyzed by quantitative PCR and Western Blotting. Unspliced and spliced XBP1 transcripts were detected by PCR to assess IRE1 pathway activation. Furthermore, the impact of pharmacological modulation of the UPR on viral replication was evaluated by determining viral yields in the presence of UPR modulators. Results and Conclusions: The active form of IRE1 was strongly upregulated during the late phase of infection (9-16 hours post-infection). Notably, the spliced form of XBP1 was not detected, suggesting that the virus may interfere with IRE1 RNase activity. Therefore, the kinase activity of IRE1 was assessed through its downstream targets JNK, BCL1, and BCL2 to evaluate the impact on autophagy. These findings reveal the previously unrecognized role of UPR and autophagy in phlebovirus pathogenesis, opening new therapeutic perspectives.