Human respiratory syncytial virus regulates the expression of interferon-stimulated genes through modulation of fibrillarin

Introduction: Human respiratory syncytial virus (RSV) is one of the main viral agents associated with the development of acute respiratory infections (ARIs), particularly during infancy and early childhood. RSV vaccine have recently been approved, however, are currently limited to older adults and pregnant women, with no approval for young children. In the absence of broadly effective and accessible preventive or therapeutic options for this vulnerable population, understanding the biology of RSV represents a critical alternative strategy. While several viral proteins have been reported to regulate the expression of interferon-stimulated genes (ISGs) to evade the host antiviral immune response, recent studies have shown that some viruses can also recruit host cellular proteins to facilitate their replication or modulate antiviral pathways. In this context, the nucleolus, and its resident proteins, such as fibrillarin (FBL), have been suggested to play a role in the regulation of inflammatory responses and in the activation of genes involved in early antiviral defense mechanisms. Methods: To analyze FBL expression under viral infection conditions, immunofluorescence assays (IFA) and Western blot (WB) analyses were performed. The effects of FBL depletion were evaluated using WB, IFA, RT-qPCR, and lytic plaque assays. Three experimental conditions were established: uninfected A549 cells (mock), RSV-infected cells, and RSV-infected cells with FBL knockdown. To determine the relationship between FBL and interferon-stimulated gene (ISG) expression, RT-qPCR assays were performed to quantify the expression levels of selected ISGs, including OAS1, OAS2, IFIT3, PKR, and RIG-I. Additionally, FBL-knockdown cells were transfected with a GFP-FBL construct to restore FBL expression, and the recovery of RSV infection was evaluated by IFA, RT-qPCR, and plaque-forming unit (PFU) assays. Moreover, the downregulation of ISG expression in cells with restored GFP-FBL was assessed by RT-qPCR. Finally, p53 knockdown assays were performed to evaluate changes in FBL expression and the reduction of RSV infection, as determined by WB. Results: RSV infection was found to induce FBL expression at early stages of infection in A549 cells. Additionally, our data suggest that FBL suppresses the expression of interferon-stimulated genes (ISGs). Conversely, silencing of FBL significantly reduced RSV infection. Importantly, this reduction in viral replication was associated with increased ISG expression in FBL-deficient A549 cells upon RSV infection. Furthermore, exogenous expression of FBL in FBL-knockdown cells restored RSV infection and led to a concomitant reduction in ISG expression following the recovery of FBL protein levels. Finally, p53-knockdown cells reduced viral protein M2-1 levels without affecting FBL expression, pointing to the involvement of other regulatory mechanism controlling FBL during RSV infection. Conclusion: Our data show that RSV infection promotes the expression of the FBL protein, creating an environment devoid of early antiviral response mediators such as ISGS.