Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish

10.1371/journal.pone.0068759PLoS ONE87-POLN

Zebrafish MDA5 is Essential for a Vigorous Antiviral Response to Rhabdovirus Infection

[Description from paper\u27s Discussion, pp 50-53] ...The zebrafish is becoming a well established model for studying not only development but also immunity. Drosophila has been used as a model for studying all aspects of science including development and immunity, however, its evolutionary 50 divergence from mammals has made transitions of findings into mouse and human models difficult. On the other hand, although the mouse model is well suited for molecular studies pertinent to human disease, cost and low breeding numbers have put restraints on the usability of mice. The zebrafish encompasses the benefits of both model systems in that cost is low, breeding cycles and number of offspring is high, and it appears that the zebrafish contain many of the same proteins present in the mammalian system... ...This work presents the first functional data demonstrating the presence of a MDA5-like signaling cascade in zebrafish. Data showing the ability of zMDA5 to recognize rhabdo virus infection suggests a new role for MDA5. It has been suggested that existed prior to RIG-I. It is quite possible that earlier in evolution, MDA5 was involved in the recognition of a wider array of viruses (48). Preliminary data suggest that SHRV has already devised a mechanism to evade MDA5 signaling, reinforcing the notion that zMDA5 plays a role in recognition of SHRV. Further studies of the MDA5 signaling cascade may give new insight into how viruses have evolved to evade immune recognition and how vertebrates have evolved to combat them. The use of the zebrafish model provides us with an evolutionarily more primitive, and theoretically less complex, system which may provide clues as to how the immune system has evolved over time to deal with changes in the environment and pathological communities

<i>Cav-1b</i> expression is modulated during virus infection, and Cav-1b knockdown leaves morphants susceptible to infection.

<p>A) Quantitative RT-PCR results revealed fold changes in the expression levels of <i>Cav-1b</i> in infected embryos when compared to uninfected embryos. Zebrafish were exposed seven dpf to 1×10⁶ TCID₅₀/mL virus. Total RNA was isolated from at 12, 24, and 48 hours post infection and reverse transcribed to cDNA (n = 20 fish per time point). All expression values have been normalized to the zebrafish β-actin gene. Error bars represent SEM of three replicates. B) Zebrafish embryos that were injected with Cav-1b morpholino (MO) to knock down the expression of Cav-1b or control MO were infected 48 hpf with 1×10⁶ TCID₅₀/ml virus and monitored for mortality. Results are representative of three separate experiments. Statistical analysis (Wilcoxon test) of the Kaplan-Meier curve was performed (*, p = 0.008). C) Zebrafish embryos that were injected with Cav-1b MO to knock down the expression of Cav-1b or control MO were infected by static immersion 48 hpf with 1×10⁶ TCID₅₀/ml virus. The graph indicates that early in infection (0–12 hpi), there is no difference in viral burden between Cav-1b morphants and controls. However, by 24–48 hpi, Cav-1b morphants have a higher viral burden. Figure is representative of three experiments; error bars are standard error of the mean (*, p<0.05). D) Western blot showing efficacy of MO knockdown in zebrafish. Zebrafish embryos from Control and Cav-1b MO, and Control MO with SHRV infection were compared for cav-1b expression at the 72 hpf developmental stage. At this time, infected fish were 24 hpi. Membranes were re-probed with antibody against β-actin to control for protein loading.</p

Decrease of Cav-1b expression negatively affects the IFN pathway.

<p>A) <i>Stat1</i> gene expression was assessed by qRT-PCR in Control MO and Cav-1b MO embryos that were either SHRV infected or uninfected. Total RNA was extracted from 10 fish per treatment, cDNA synthesized and <i>Stat1</i> mRNA expression assessed by qRT-PCR 24 hpi. The data are representative of three individual experiments and error bars indicate SEM. Each bar represents the mean fold induction of SHRV-infected embryos over corresponding controls. All expression values were normalized to zebrafish 18s. B) ISRE promoter activity is dampened in Cav-1b knockdown ZFL cells upon SHRV infection. ZFL cells were transfected with 250 ng of zISRE-luc construct along with 250 ng of cav-1b MO or control MO. Twenty four hours post transfection the ZFL cells were infected with SHRV at an MOI of 0.01. Cells were harvested for luciferase measurements 24 hpi. The graph shows relative luminescence units of control uninfected cells compared to cav-1b MO or control infected cells. Error bars are representative of SEM for two experiments. (**, p<0.001).</p

Crosslinking CRFB1 keeps receptor molecules clustered despite caveolin depletion.

<p>ZFL cells were co-transfected with MO and expression plasmid via nucleofection and allowed to recover/adhere to cell culture plates for ∼6 hr prior to addition of crosslinking reagent. The crosslinking reaction was performed according to the manufacturer’s procedures. Cells were subsequently replenished with media and returned to the incubator for 24 hr post crosslinking. Scale bars, 1 µm. A) Cells transfected with Cav-1b MO/CRFB1 without crosslinking treatment show dispersed receptor molecules. B) Cells transfected with Control MO/CRFB1 with crosslinking clearly show clustered receptor molecules. C) Cells transfected with Cav-1b MO/CRFB1 with crosslinking. This demonstrates that despite depletion of Cav-1b, receptor molecules remain clustered. D) Pair correlation analysis confirms that with crosslinking, CRFB1 remains clustered despite Cav-1b depletion. Values of g(r) in cells with Cav-1b KD are similar to that for Controls (n≥8 cells per treatment).</p

Cav-1b colocalizes with the zebrafish homolog of IFN-R and is positively correlated.

<p>ZFL cells (n≥10) were transfected with Cav-1b-PAmCherry (red) and with CRFB1-dendra2 (green). For all images, 60×/1.2 NA magnification. Scale bars, 1 µm. Shown is the plasma membrane of one cell representative of the experiment (<b>A</b>) and a magnification (<b>B</b>) of the region marked by the white box in A. The image shows that Cav-1b and CRFB1 colocalize in the cell membrane. (<b>C</b>) Measurements of Cav-1b and CRFB1 show a positive pair correlation value g(r) greater than one, confirming that the two species are colocalized together. Pair correlation calculations were performed as described in Methods; briefly, g(r) >1 indicates positive correlation/clustering, and g(r) = 1 indicates a random distribution. (<b>D</b>) Pair correlation measurements of CRFB1 were calculated for the receptor, control morpholino (MO), and Cav-1b MO. CRFB1 is more prone to random distribution when Cav-1b is knocked down. Error bars SEM (n ≥8 cells).</p

MxA expression is retained with rescue of Cav-1b depletion.

<p>ZFL cells were transfected and rescued as described in Methods. Shown is the fold difference in gene expression of MxA, an interferon stimulated gene. MxA transcript levels in Cav-1b depleted cells treated with crosslinking reagent (A) or rescued with cav-1b plasmid (B) show that rescuing caveolar disruption negates the depletion of caveolae which keeps CRFB1 molecules clustered under normal conditions. When Cav-1b is depleted and caveolae are not maintained with crosslinking reagent or cav-1b plasmid, minimal MxA expression is measured. The knockdown of CRFB1, CRFB2, and CRFB5 is the negative control; data indicate that there is low induction of MxA without IFN receptor subunits. When Cav-1b is depleted and CRFB1 is kept clustered, MxA expression remains at the same level as in the controls, demonstrating that the clustering of the receptor is essential for downstream signaling and that caveolin-1 plays a critical role in keeping the receptor clustered. Representative of 3 experiments; error bars indicate SEM (*, p<0.05). These results indicate dampening of MxA transcript production between control MO and Cav-1b MO prior to rescue of caveolae domains, and no significant difference of MxA transcript levels between Control MO and Cav-1b MO after rescue of caveolae domains.</p