Briefing Document: Pan-Antiviral Activity of the Hsf1-sHsp Cascade in Mosquito Cells

Source: Qu, J., Schinkel, M., Chiggiato, L., Machado, S. R., Overheul, G. J., Miesen, P., & van Rij, R. P. (2025). The Hsf1-sHsp cascade has pan-antiviral activity in mosquito cells. Nature Communications Biology, 8(1), 74. https://doi.org/10.1038/s42003-024-07435-4

Date: Received - 05 July 2023 | Accepted - 20 December 2024 | Published - 25 January 2025

1. Executive Summary:

This research paper investigates the antiviral immune responses in Aedes aegypti mosquitoes, a primary vector for several arboviruses. Through multi-omics data integration, the study identifies a novel early-responsive antiviral cascade involving heat shock factor 1 (Hsf1) and a cluster of eight small heat shock protein (sHsp) genes. This "Hsf1-sHsp cascade" demonstrates pan-antiviral activity against multiple arboviruses, including chikungunya, Sindbis, dengue, and the insect-specific Agua Salud alphavirus in Ae. aegypti cells, and also against chikungunya and O’nyong-nyong viruses in other mosquito species (Aedes albopictus and Anopheles gambiae cells). The findings suggest that Hsf1 could be a promising target for developing novel intervention strategies to limit arbovirus transmission by mosquitoes.

2. Main Themes and Important Ideas:

• Identification of a Novel Antiviral Cascade: The study reveals a previously uncharacterized early immune response in Ae. aegypti cells triggered by chikungunya virus (CHIKV) infection. This response involves the transcription factor Hsf1 activating the expression of eight sHsp genes located within a single topologically associated domain (TAD) in the mosquito genome.
• "By integrating multi-omics data, we find that heat shock factor 1 (Hsf1) regulates eight small heat shock protein (sHsp) genes within one topologically associated domain in the genome of the Aedes aegypti mosquito."
• Hsf1 Drives Early sHsp Expression: RNA-seq and chromatin profiling data indicate that Hsf1 binds to the promoter regions of these eight sHsp genes, leading to their rapid upregulation upon CHIKV infection. RNAi-mediated silencing of Hsf1 significantly diminished the induction of all eight sHsps, confirming its regulatory role.
• "We found the DNA binding motif of the transcription factor heat shock factor 1 (Hsf1) to be highly enriched at promoter regions of these eight sHsp genes..."
• "Hence, the Hsf1-sHsp cascade functions as an early transcriptional response to CHIKV infection in Aag2 cells."
• The Hsf1-sHsp Cascade Exhibits Anti-CHIKV Activity: Silencing either Hsf1 or all eight sHsp genes using RNAi resulted in elevated CHIKV RNA levels in Ae. aegypti cells, indicating that this cascade plays a role in suppressing CHIKV replication.
• "To explore the antiviral potential of the identified Hsf1-sHsp axis, we silenced Hsf1 using RNAi and observed elevated CHIKV RNA levels at 8 hpi..."
• "...we therefore used two sets of dsRNA targeting the conserved Hsp20 domain to knock down all eight sHsp genes and observed significantly increased CHIKV RNA copies at 8 hpi..."
• Modulation of Hsf1 Activity Impacts CHIKV Replication: Using a pharmacological inhibitor (KRIBB11) of Hsf1 led to increased CHIKV replication, while an activator (hsfa1) resulted in a significant reduction in viral RNA levels. Interestingly, DTHIB, reported as a human Hsf1 inhibitor, acted as an activator in mosquito cells and also repressed CHIKV replication, further supporting the antiviral role of the sHsps.
• "KRIBB11 treatment significantly downregulated most sHsp genes after 12 h...led to more than 5-fold increase of viral RNA levels in Aag2 cells..."
• "Hsfa1 treatment significantly increased Hsf1 expression and strongly activated all eight sHsp genes, leading to about a 25-fold reduction in CHIKV copies at 12 hpi..."
• The Cascade Targets an Early Post-Entry Stage of Viral Replication: Time-of-addition assays with the Hsf1 activator and inhibitor suggest that the Hsf1-sHsp cascade interferes with an early stage of the CHIKV replication cycle, occurring around 4 hours post-infection, after virus entry but before significant RNA replication.
• "When the Hsf activator hsf1a was added at time points before inoculation and up to 4 hpi, CHIKV replication was strongly inhibited..."
• "This indicates that the Hsf1-sHsp cascade interacts with the early stage of CHIKV RNA replication but does not affect subsequent stages."
• Pan-Antiviral Activity Across Multiple Arboviruses and Mosquito Species: The study demonstrates that the Hsf1-sHsp cascade is not specific to CHIKV. Activation of Hsf1 with hsfa1 reduced replication of Sindbis virus (SINV), Agua Salud alphavirus (ASALV), and dengue virus (DENV) in Ae. aegypti cells. Furthermore, activating the cascade showed antiviral effects against CHIKV in Aedes albopictus cells and the RNAi-deficient C6/36 cells, and against O’nyong-nyong virus (ONNV) in Anopheles gambiae cells.
• "Consistently, KRIBB11 treatment led to increased virus replication for SINV and ASALV, whereas hsfa1 treatment significantly reduced virus replication for SINV, ASALV and DENV..."
• "The fact that the heat shock response was antiviral in both U4.4 cells and the RNAi-deficient C6/36 cells indicates that the Hsf1-sHsps cascade functions independently of RNAi."
• Hsf1 as a Potential Target for Intervention: The broad antiviral activity of the Hsf1-sHsp cascade across different arboviruses and mosquito vectors highlights Hsf1 as a promising target for developing novel strategies to block arbovirus transmission at the mosquito stage.
• "Overall, our work uncovers an antiviral cascade Hsf1-sHsp and identifies Hsf1 as a potential target for developing transmission-blocking strategies against arboviruses at the mosquito stage."
• "Considering the pan-antiviral activity of the Hsf1-sHsp cascade, our findings may open the way for the development of a broad-spectrum drug to prevent arbovirus transmission."
• Potential Mechanism of Action: The study suggests that sHsps, known for maintaining protein homeostasis by preventing protein aggregation, might exert their antiviral effects by interacting with conserved viral or host protein complexes involved in early viral replication. Proteomic approaches are proposed to identify sHsp client proteins and elucidate the underlying mechanism.
• "Our time-of-addition assays suggest that the response inhibits an early post-entry stage of the viral replication cycle."
• "It has been suggested that sHsps maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation."

3. Implications and Future Directions:

• The discovery of the Hsf1-sHsp cascade adds a new layer to our understanding of mosquito antiviral immunity, complementing the well-established role of RNAi and other innate immune pathways.
• Targeting Hsf1 in mosquitoes could offer a novel, broad-spectrum approach to reduce the transmission of multiple medically important arboviruses. This strategy could potentially be less susceptible to viral resistance mechanisms compared to targeting specific viral proteins.
• Further research is needed to validate the in vivo relevance of this cascade in whole mosquitoes and to understand the precise molecular mechanisms by which sHsps inhibit viral replication.
• Investigating the interaction between the Hsf1-sHsp cascade and other m...