Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling. Caveolae are cell membrane domains integral to numerous signaling pathways. A mechanism for virus evasion of host cell defenses through disruption of clusters of signaling molecules organized within caveolae is demonstrated.
Visualization of caveolae has previously been hampered by limitations of spatial resolution imposed by diffraction in light microscopy. A novel imaging technique has circumvented the diffraction limit to obtain near-molecular level data. Using repeated cycles of activation, localization, and bleaching of single photoactivatable fluorescent molecules, fluorescence photoactivation localization microscopy (FPALM) and related methods have achieved sub-diffraction resolution.
FPALM enabled the first single-molecule imaging of interactions between interferon antiviral signaling receptors (IFN-R) and caveolae. Strikingly, knockdown of Caveolin-1, the primary protein component of caveolae, caused IFN-R clusters to disperse. Dispersal of IFN-R clusters led to a suppressed antiviral immune response through abrogation of downstream signaling, a response strongly suggesting that IFN-R organization within caveolae is critical for IFN-mediated antiviral defense.
We demonstrate that a functional consequence of caveolar disruption is the dispersal of antiviral molecules and inhibition of the host antiviral response. This has broader implications for the role of caveolae in antiviral immunity. To further apply this model and technique to human disease, an influenza A virus infection was characterized in zebrafish, where it can infect and replicate, causing a disease state. Zebrafish embryos infected with influenza virus exhibit an increased viral burden over time and eventually succumb to death, further suggesting that influenza is replicating within the zebrafish host. Expression of IFN is upregulated as a result of infection. Influenza infection in zebrafish provides a new model to study antiviral immunity in a living organism using an important human pathogen. To perform experiments in a physiologically relevant system with sufficient resolution, we have extended FPALM to the level of a living zebrafish embryo. This is the first instance of applying super-resolution microscopy in vivo in a vertebrate organism. Understanding the complex mechanisms through which viruses modulate immune function should provide insight into a range of potential targeted antiviral therapies.
|Advisor:||Kim, Carol H., Hess, Samuel T.|
|School:||The University of Maine|
|School Location:||United States -- Maine|
|Source:||DAI-B 74/01(E), Dissertation Abstracts International|
|Keywords:||Antiviral response, Caveolae, Innate immunity|
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