Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (–R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response.

Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus.

In cardiac myocytes, b-adrenergic stimulation is mainly due to b1and b2receptors (ARs). In ventricular cardiac myocytes, the sub-cellular distribution of specific b-ARs is unclear: immunocytochemistry data and cAMP recording showed different results (between t-tubules (TT) and surface sarcolemma (SS)). Therefore, the functional distribution of b-ARs in ventricular cardiac myocytes (TT versus SS) is still unclear. This study addresses this point. Rat ventricular cells were enzymatically isolated. Detubulation was achieved using osmotic shock as previously described. Intracellular calcium concentration was recorded using fluorescent dye (fura-2 AM) and cell contraction was induced by field stimulation. Selective b1-adrenergic stimulation was achieved by perfusion of isoprenaline (0.1 mM) and ICI 118,551 (0.1 mM). Selective b2-adrenergic stimulation was achieved by perfusion of salbutamol (10 mM) and atenolol (1 mM). In control cells, b1-adrenergic and b2-adrenergic stimulation caused a significant increase in peak calcium transient (peak CaTr; 236.8542.9%, n=29 and 24.654.2%, n=41, respectively), evaluating full b-adrenergic stimulation (i.e. SS þ TT). In detubulated cells, b-adrenergic stimulation had a greater effect on peak CaTr than in control cells (288.1580.6% increase for b1, n=17 and 83.559.0% for b2 n=20; evaluating b-adrenergic stimulation only from SS). From these values, we calculated that the % of increase of peak CaTr from the TT was ~128.3% during b1-adrenergic, and ~0.88% during b2-adrenergic. These data indicates that b1pathway is functional in SS and TT in ventricular cardiac cells. In contrast, b2-adrenegic stimulation have a physiological effect on CaTr via the SS only. These results are in contradiction with the latest report about the localization of b-adrenergic receptors. However, our study focuses on the functional b-ARs response (i.e. CaTr) instead of the response to the increase of cAMP, which may account for the different conclusion. Supported by the Wellcome Trust.

Caveolae are integral to numerous signaling pathways but their function in antiviral signaling is largely unexplored. Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. While it is known that viruses exploit caveolae for entry into host cells, this study instead demonstrates an entry-independent mechanism for virus evasion of host cell defenses through disruption of clusters of signaling molecules organized within caveolae. Super-resolution microscopy has enabled the first single-molecule imaging of interactions between type I interferon receptors (IFN-R) and caveolae. In particular, cav-1b knockdown caused IFN-R clusters to disperse, suppressing 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.

The innate and adaptive arms of immunity work synergistically to mount robust host defenses against invading pathogens. The zebrafish lacks a fully functional adaptive immune system for the first four to six weeks of development, making it an ideal model for studying many aspects of innate immunity, including the Toll-like receptor (TLR) pathways. In mammals, TLRs recognize specific pathogen associated molecular patterns and, acting through adaptor molecules such as MYD88 and TICAM1, elicit an array of pro- and anti-inflammatory responses. In two recent studies we described differences in TLR ligand recognition and downstream signaling in the zebrafish, and proved that sequence homology does not necessarily indicate functional homology. These data support the notion that alternative signaling pathways may have evolved in the zebrafish. To characterize this system, ligands of the zebrafish TLRs and the intracellular protein-protein interactions were studied. Here we report the identification of unique interactors and targeted morpholino knock-down of TLR-associated genes to analyze the impact on signaling of each each protein-protein interaction within zebrafish TLR pathways. The discovery of novel protein interactions in the zebrafish highlights the potential of this model system for studies of innate immune function and further elucidates the evolution of vertebrate immunity.