Tick salivary cystatin Iristatin limits the virus replication in skin of tick-borne encephalitis virus-infected mice

Abstract

Tick-borne encephalitis virus (TBEV) is flavivirus transmitted to the host via tick saliva which contains various molecules with biological impacts. One of such molecules is Iristatin, a cysteine protease inhibitor from Ixodes ricinus that has been shown to have immunomodulatory properties. To characterize Iristatin in the relation to TBEV, we investigate whether this tick inhibitor has any capacity to influence TBEV infection. Mice were intradermally infected by TBEV with or without Iristatin and the viral multiplication was determined in skin and brain tissues by RT-PCR two and 5 days after infection. The viral RNA was detected in both intervals in skin and increased by time. The application of Iristatin caused a reduction in viral RNA in skin but not in the brain of infected mice 5 days post-infection. Moreover, anti-viral effect of Iristatin on skin was accompanied by a significant decline of interferon-stimulated gene 15 gene expression. The effect of Iristatin on TBEV replication was tested also in vitro in primary macrophages and dendritic cells; however, no changes were observed suggesting no direct interference of Iristatin with virus replication. Still, the Iristatin caused a suppression of Erk1/2 phosphorylation in TBEV-infected dendritic cells and had the anti-apoptotic effect. This is the first report showing that a tick cystatin decreases the viral RNA in the host skin, likely indirectly through creating skin environment that is less supportive for TBEV replication. Assuming, that viral RNA reflects the amount of infectious virus, decline of TBEV in host skin could influence the tick biology or virus transmission during cofeeding.

Keywords: Cystatin; Flavivirus; Tick; Tick-borne encephalitis virus; Virus replication.

Fig. 1

The effect of Iristatin on virus multiplication in mice. Ten mice per group were i.d. infected with Hypr ± Iristatin and viral genome RNA loads on indicated days post-infection were determined by RT-qPCR in the skin from inoculation site (a) and in the brain (b). mRNA expression was normalized to the Actb mRNA level. *p ≤ 0.05; ns = not significant

Fig. 2

Iristatin affects the gene expression of interferon-responsive genes in the skin of TBEV-infected mice. Mice were i.d. infected with Hypr ± Iristatin and the gene expressions of ISG15 (a), CXCL-10 (b), CD115 (c), and TCRγ (d) were evaluated on day 5 in the skin of infected mice. mRNA expression was normalized to the Actb mRNA level and non-infected mice. *p ≤ 0.05; ***p ≤ 0.001; ns = not significant

Fig. 3

The effect of Iristatin on virus multiplication and ISG gene expression in primary bone marrow macrophages (BMM; a, b, c) and dendritic cells (DC; d, e, f). BMM and DC were infected with Hypr (MOI 5) and incubated for 24, 48, and 72 h in the presence or absence of Iristatin (6 μM). Viral genome RNA loads (a, d) were determined by RT-qPCR at indicated hours post-infection. mRNA expression of ISG15 (b, e) and CXCL-10 (c, f) were normalized to the Actb mRNA level and non-infected control. The mean of three independent experiments (+ SEM) is shown in all graphs. Differences between groups were not statistically significant

Fig. 4

Iristatin interferes with signaling pathways activation and exerts anti-apoptotic effect in TBEV-infected dendritic cells. DC were activated by imiquimod (IQ; 2 µg/ml) for 3 h in the presence or absence of Iristatin (3 µM) and protein cell lysates were analyzed for the activation of signaling pathways using PathScan intracellular signaling array (a). DC were infected by Hypr at MOI 5 for indicated times in the presence or absence of Iristatin (3 µM) and then Erk1/2 phosphorylation was analyzed by immunoblotting. Membranes were re-probed to determine the level of total Erk1/2 proteins. Proteins were visualized by chemiluminescence and representative blot with relative phosphorylation is shown (b). DC were non-infected or infected by Hypr at MOI 5 in the presence or absence of Iristatin (6 µM) and the percentage of active caspase-3 positive cells was measured by flow cytometry (c). *p ≤ 0.05; ****p ≤ 0.0001 ns = not significant