The role of the Unfolded Protein Response in HSV-1 infection: interplay with NF-κB signaling and viral replication in neuroblastoma cells

Herpes Simplex Virus type-1 (HSV-1) is a neurotropic DNA virus which establishes a lifelong infection in the host by alternating between lytic replication and latency. During infection, HSV-1 relies heavily on the host's cellular machinery to synthesize and mature viral proteins, particularly in the ER. This process places a significant strain on ER homeostasis, resulting in the activation of the Unfolded Protein Response (UPR), which is a cellular stress response aimed at restoring proteostasis. The UPR is mediated by three main signaling branches, governed by PERK, IRE1 and ATF6, which contribute differently to cellular adaptation or stress-induced cell fate decisions. This study aimed to investigate the role of UPR activation during HSV-1 infection, and to analyses the contribution of individual UPR branches to viral replication. Additionally, the study aimed to explore the interaction between UPR signaling and the NF-κB pathway, a key regulator of innate immune responses, to improve our understanding of how ER stress responses influence antiviral defense mechanisms. A neuronal cell model was used to investigate UPR activation pharmacologically with thapsigargin, and HSV-1 replication was assessed over the course of an infection. The results demonstrate that global UPR activation significantly reduces HSV-1 replication, indicating an overall antiviral effect The role of NF-κB signaling was evaluated during HSV-1 infection under conditions where the UPR was activated. Although UPR activation increased NF-κB transcription, immunofluorescence analyses revealed dynamic, time-dependent regulation of NF-κB nuclear translocation. Early in the infection process, NF-κB activation appeared to be limited; however, at later stages, NF-κB contributed to the antiviral effect of the UPR. Pharmacological inhibition of NF-κB restored viral replication at intermediate and late time points, showing that NF-κB plays a functional role in mediating UPR-induced antiviral responses. Importantly, experiments using UVC-inactivated HSV-1 demonstrated that viral entry alone is insufficient to modulate NF-κB signalling, indicating that a replication-competent virus is required for the observed regulatory effects. Together, these findings emphasize the dynamic interplay between UPR signaling and NF-κB activation. In this context, NF-κB acts as a pivotal mediator, linking ER stress responses to the host immune response during infection. Selective inhibition of individual UPR branches revealed a more complex, differential contribution from each pathway. Inhibiting PERK signaling using 4-phenylbutyric acid led to a marked increase in viral replication, highlighting the central antiviral role of this branch, which is likely mediated through translational attenuation and stress-induced signaling. Conversely, inhibiting IRE1 and ATF6 using 4μ8C and Ceapin-7, respectively, resulted in decreased viral replication. This suggests that these pathways support viral propagation by enhancing the ER folding capacity and maintaining the proteostasis required for efficient viral protein production. Overall, this study supports a model in which the activation of the UPR exerts an antiviral effect through the coordinated action of its three branches. PERK acts as a major antiviral pathway, while IRE1 and ATF6 provide pro-viral functions. NF-κB emerges as a critical node in this network, contributing to the antiviral defense response at later stages of infection and linking stress signaling to immune activation. These findings shed new light on the complexity of host–virus interactions and suggest that selectively modulating UPR pathways could be a promising strategy for controlling HSV-1 infection. Further research is needed to elucidate the molecular mechanisms underlying the interaction between UPR signaling and NF-κB, particularly in relation to PERK activation. It is also necessary to determine the relevance of this regulatory network in more physiologically relevant neuronal models, as well as during HSV-1 latency and reactivation. Additionally, investigating whether similar mechanisms operate in other DNA and RNA viruses will help establish whether this is a conserved antiviral strategy or a virus-specific adaptation.