Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection
January 26th, 2019
Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.
Traditional double-strand RNA detection assay usually is not sensitive enough to detect double-strand RNA formed in negative-sense RNA virus infection.Therefore the interaction between double-stranded RNA and host pattern recognition receptors has remained largely elusive for these viruses.Utilizing this protocol we are able to visualize and characterize the interaction between viral nuclear protein, double-strand RNA and host a PRRs at a single-cell level for negative-sense RNA virus.This is a multiplex assay that allows for simultaneous staining of double-stranded RNA and two viral or host protein targets in individual cells.Unlike the RNA-FISH assay this technique is RNA sequence independent and usually compatible with antibody detection of protein targets.Begin 24 hours prior to infection by seeding 200, 000 human lung epithelial A549 cells onto Poly-D-Lysine coated glass cover slips in 12-well plates and growing them at 37
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Overview
This study investigates the recognition of double-stranded RNA (dsRNA) produced during RNA virus replication by pattern recognition receptors (PRRs) to induce an innate immune response. The focus is on negative-sense RNA viruses, where the interaction between low-level dsRNA and PRRs is not well understood. A confocal microscopy method has been developed to visualize arenavirus dsRNA and PRRs in individual cells.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- Virology
Background
- Double-stranded RNA is a byproduct of RNA virus replication.
- Pattern recognition receptors play a crucial role in the innate immune response.
- The interaction between dsRNA and PRRs in negative-sense RNA viruses is poorly understood.
- Visualizing dsRNA and PRRs can enhance understanding of viral immune evasion.
Purpose of Study
- To elucidate the interaction between dsRNA and PRRs in negative-sense RNA viruses.
- To develop a method for visualizing these interactions in individual cells.
- To contribute to the understanding of the innate immune response to RNA viruses.
Methods Used
- Confocal microscopy for visualization of dsRNA and PRRs.
- Cellular assays to assess the immune response.
- Analysis of dsRNA levels during viral replication.
- Comparative studies with different RNA viruses.
Main Results
- Successful visualization of arenavirus dsRNA and PRRs in individual cells.
- Identification of specific interactions between dsRNA and PRRs.
- Insights into the immune response mechanisms triggered by dsRNA.
- Clarification of the role of dsRNA in viral pathogenesis.
Conclusions
- The developed confocal microscopy method is effective for studying dsRNA and PRR interactions.
- Understanding these interactions may lead to new therapeutic strategies against RNA viruses.
- This research contributes to the broader field of viral immunology.
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Chapters in this video
0:04
Title
1:05
Preparation of A549 Cells and JUNV (Junn virus) Infection
2:09
Fixation and Immunostaining
5:10
Results: Distribution and Colocalization Between the Retinoic Acid Like Receptors RLRs (RIG-I and MDA-5) and dsRNA in JUNV-infected Cells
5:52
Conclusion
