IFITMs exhibit antiviral activity against Chikungunya and Zika virus infection via the alteration of TLRs and RLRs signaling pathways

Abstract

Chikungunya virus (CHIKV) poses a significant challenge as there are currently no targeted antiviral drugs or vaccines to combat this infection. Here, we demonstrate that interferon-induced transmembrane proteins (IFITMs), including IFITM1, IFITM2, and IFITM3, which are interferon-stimulated genes (ISGs), inhibit CHIKV infection in human skin fibroblasts. Overexpression of IFITMs in cells restricts viral infection, whereas knockdown of IFITMs enhances viral infection. IFITMs overexpression causes a substantial upregulation of antiviral genes, namely TLR3, TLR7, TLR8, and TLR9, and their downstream signaling molecules such as TRADD, IRAK1, TRAF6, and MAP3K7, involved in TLRs signaling pathways. Furthermore, the DHX58 gene encoding the LGP2 protein, a negative regulator of RIG-I in RLRs signaling pathways, was downregulated in the overexpressed cells. Transcription factors including interferon regulatory factors (IRF) 3/5/7, which are downstream signaling components of both TLR and RLR signaling pathways, were also upregulated, resulting in enhanced IFNs signaling. IFITMs not only inhibits the early and late stages of viral infection but can also alter the antiviral innate-immune response to restrict CHIKV infection in human skin fibroblasts. Additionally, IFITMs exhibit their antiviral activity against Zika virus (ZIKV). Altogether, these results show the broad-spectrum antiviral property of IFITMs against arboviruses in foreskin cells.

Keywords: Arbovirus; Chikungunya virus; IFITMs; TLRs and RLRs signaling pathways; Zika virus.

Fig. 1

IFITMs exhibit antiviral properties to CHIKV infection. HFF-1 cells were transfected with 0.1 µg of the plasmid containing either plasmid control, IFITM1, IFITM2, or IFITM3 gene to overexpress each gene in HFF-1 cells. The cell was then infected with CHIKV at an MOI of 1 for 24 and 48 h before harvested cells lysate for quantifying (A) the viral RNA using RT-qPCR and supernatant for quantification of (B) virus particles using plaque assay. Data are presented as means ± SD from three independent experiments. The significance of the differences among groups was evaluated by using two-way ANOVA. Statistical significance was accepted at p < 0.05. **=p < 0.01, ***=p < 0.001, ****=p < 0.0001.

Fig. 2

Knockdown of IFITMs present antiviral properties of IFITMs against CHIKV infection. HFF-1 were transfected with 80 nM siControl and siIFITM1, siIFITM2, and siIFITM3 to knockdown the IFITMs gene. Transfected cells were (A) visually inspected to detect putative morphological changes and cell death (10x). The cells were then infected with CHIKV at a MOI of 2 for 24 and 48 h before harvested cells lysate for quantifying (B) the viral RNA using RT-qPCR and supernatant for quantification of (C) virus particles using plaque assay. Data are presented as means ± SD from three independent experiments. The significance of the differences among groups was evaluated by using two-way ANOVA. Statistical significance was accepted at p < 0.05. ****=p < 0.0001.

Fig. 3

Exogenous overexpressed of IFITMs modulates the human innate immune response. plasmid control and IFITM1, 2, and 3 transfected were collected for RNA extraction, RT-PCR and PCR array. (A) The results present as fold change of gene expression compared plasmid control- overexpressing HFF-1 cells. The upregulation was shown as orange to red while the downregulation was shown as blue. Three main innate immune pathways which demonstrated to be modulated by IFITMs are separately shown as (B) TLR signaling pathway, (C) RLR signaling pathway, and (D) IFNs signaling pathway.

Fig. 4

Summary of genes modulated by the expression of IFITM1, 2, and 3 in HFF-1 cells. Three main pathways of the antiviral innate immune response, TRL signaling pathway, RLR signaling pathway, and IFNs signaling pathway were shown. The up arrow represents the upregulation of the gene while the down arrow represents downregulation and – represents the gene with no effect. Purple arrow state gene effect by IFITM1, brown arrow state gene effect by IFITM2, and red arrow state gene effect by IFITM3. Figure was created using Keynote version 9.1 (https://www.apple.com/keynote/).

Fig. 5

CHIKV infection impairs the innate immune response in IFITMs-overexpressed HFF-1 cells. Plasmid control and IFITM1, 2, and 3 overexpressed cells were infected by CHIKV. After 48 h, the cells were collected for RNA extraction, RT-PCR and PCR array. (A) The results present as fold change of gene expression compared to infected-plasmid control cells. The upregulation was shown as orange to red while the downregulation was shown as blue. Three main innate immune pathways were demonstrated to be imbalanced by CHIKV including (B) TLR signaling pathway, (C) RLR signaling pathway, and (D) IFNs signaling pathway. - = uninfected cells, + = infected cells.

Fig. 6

IFITMs inhibit ZIKV infection on human skin cells. HFF-1 were seed and transfected with (A) plasmid containing either IFITM1, IFITM2, or IFITM3 gene and (B) siIFITM1, siIFITM2, or siIFITM3 before challenging with ZIKV at MOI of 1 and quantified the viral RNA using RT-qPCR and virus particles using plaque assay at 24 and 48 hpi. Data are presented as means ± SD from three independent experiments. The significance of the differences among groups was evaluated by using two-way ANOVA. Statistical significance was accepted at p < 0.05. ***=p < 0.001, ****=p < 0.0001.

Fig. 7

Proposed mechanism of IFITMs on CHIKV infection in human dermal cells. IFITMs expression in response of viral infection downregulated the expression of LGP2 and upregulated the expression of TLRs resulting in increasing type I interferon expression. IFITMs also upregulated the expression of STAT1 in IFNs signaling pathway which could further induced ISGs expression that can act against viral infection. Moreover, IFITMs in corresponding with cholesterol could inhibit the fusion step of viral replication restricted the viral infection. Figure was created using Keynote version 9.1 (https://www.apple.com/keynote/).