Introduction

Chikungunya virus (CHIKV) is an arbovirus of the Togaviridae family, Alphavirus genus, transmitted mainly by Aedes aegypti and Aedes albopictus. It was first isolated in Tanzania in 1952, and currently, CHIKV cases have been reported in more than one hundred countries worldwide. Most patients suffer from polyarthralgia and myalgia that can persist for months or years, impacting their quality of life. Since no licensed drugs or vaccines are currently available, treatment of CHIKV infection focuses on supportive care, and prevention on mosquito vector control strategies and personal protection against mosquito bites1,2. During a viral infection, the host innate immune response is triggered as an early defense mechanism to restrict viral infection. Pattern-recognition-receptors (PRRs) in the infected cells such as toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I) receptors, recognize viral components and trigger a downstream signaling cascade that further promotes expression and phosphorylation of several transcription factors involving Interferon regulatory factors (IRFs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and activator protein 1 (AP-1), resulting in the production of interferons (IFNs) and other cytokines3,4. Type I interferon signaling pathway is of the essential innate immune pathways that protects against viral infection through the expression of interferon-stimulated genes (ISGs) with antiviral activity5. Among ISGs, the immune-related interferon-induced transmembrane proteins (IFITMs), including IFITM1, IFITM2, and IFITM3, are transmembrane proteins that have been demonstrated to be upregulated following influenza A virus (IAV) infection in dendritic cells6, as well as infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human patients7,8suggesting their role in the human immune response against viral infection9,10. IFITM1 was first demonstrated to exert antiviral activity against vesicular stomatitis virus (VSV) in 199611; since then, a wide range of viruses have been reported to be inhibited by IFITM1, IFITM2, and/or IFITM3 proteins. However, the underlying mechanism of the antiviral activity of IFITMs is still unclear. They have been demonstrated to disrupt viral infection at the early stage through several means, including decreased membrane flexibility12, induced lipid accumulation13and being incorporated in the viral envelope, resulting in a reduction of virion infectivity14. IFITMs have been shown to exert different inhibitory effects in different cell types15,16, suggesting a cooperation between IFITMs and other cell type-specific proteins during viral infection. Some reports studying IFITMs-deficient mice revealed the modulatory effect of IFITMs on cytokines that regulate cellular immunity, including NK cells and T helper 2 (Th2) cells17,18. However, studies of the effect of IFITMs on the human innate immune response remain limited.

This work aims to study the effects of IFITM proteins on CHIKV infection and their impact on the human antiviral innate immune response during CHIKV infection. This study used human skin fibroblast as a model. Fibroblasts are located in the basal membrane layer, which is the site of virus inoculation and are permissive to CHIKV19,20. As expected, IFITM1, IFITM2, and IFITM3 have antiviral effects against CHIKV infection in human skin fibroblasts. Furthermore, our study on the impact of IFITMs on the innate immune pathways in response to viral infection revealed a substantial upregulation of TLRs genes and a downregulation of DHX58 gene in TLR and RLR signaling pathways, as well as enhanced expression of IFNA1, IFNA2 and STAT1 in the IFN pathway. Moreover, investigation of the effect of IFITMs on another arbovirus infection, Zika virus (ZIKV), demonstrated the broad-spectrum antiviral properties of IFITMs in human skin fibroblasts.

Results

IFITMs overexpression in human skin fibroblasts

HFF-1 cells were transfected with IFITM1, IFITM2 and IFITM3 plasmids at various concentrations. Cells transfected with plasmids at concentrations of 0.01 µg and 0.1 µg showed viability above 80%, whereas at a concentration of 1 µg, showed low cell viability compared to the control (Supplementary Fig. S1A). RT-qPCR and western blot analysis showed that all IFITM proteins were overexpressed in HFF-1 cells at both RNA and protein levels (Supplementary Fig. S1B and S1 C).

IFITMs exert antiviral properties against Chikungunya virus infection

To examine the effect of IFITMs on CHIKV infection, cells were transfected with IFITM plasmids and subsequently infected with CHIKV at MOI of 1. The inhibition of viral infection was analyzed by quantifying viral RNA and virus particles. Both viral RNA and virus particle production were completely abrogated (~ 100%) in all IFITMs overexpressing cells compared to plasmid control cells at 24- and 48-hour post-infection (hpi) (Fig. 1A and B). The data indicated that IFITM1, IFITM2, and IFITM3 equally constrain CHIKV infection in human skin cells.

Fig. 1
figure 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.

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Deficit of IFITMs elevates Chikungunya virus infection

The effect of IFITMs on CHIKV infection was investigated in IFITM1, 2, or 3 knockdown cells. Endogenous IFITMs were silenced using a non-toxic concentration of 80 nM of specific siRNA (Fig. 2A). To confirm the expression level of IFITM1, 2, or 3 knockdown cells in both 24 and 48 h, RT-qPCR was used to quantify the RNA level compared to siControl knockdown cells (Supplementary fig. S3). Although the expression levels of all IFITMs were not different, RT-qPCR and plaque assay results showed that siIFITM-1 did not affect CHIKV replication, while siIFITM-2 and − 3 transfected cells had a significant increase in CHIKV viral RNA and infectious viral particles compared to control cells (Fig. 2B and C).

The outcomes derived from both extrinsically overexpressed and knockdown cell models indicate that, despite IFITM1 exhibiting the highest expression level among all IFITMs, their antiviral properties against CHIKV are uniformly effective. Conversely, the silencing of IFITM1 in cells did not exert any observable impact on CHIKV replication. These findings collectively suggest that IFITM-2 and − 3 play a pivotal role in the inhibition of CHIKV. Furthermore, based on the outcomes obtained through qRT-PCR and plaque assays, we can infer that IFITMs have an impact on both viral genome replication and the late stages of viral infection.

Fig. 2
figure 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.

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Expression of IFITMs modulates the human innate immune response

Next, the impact of IFITM overexpression on the innate immune system was investigated using PCR array. RNA was collected from plasmid control, IFITM1, 2, or 3 HFF-1-overexpressing cells before RT-PCR and PCR array was performed. The results were analyzed and compared with plasmid control- overexpressing HFF-1 cells (Fig. 3A). The results show that all IFITM1, 2, and 3 markedly upregulated TLR3, TLR7, TLR8, and TLR9 genes and their downstream signaling molecules such as TRADD, IRAK1, TRAF6, and MAP3 K7 (Fig. 3B). In the RLR signaling pathways, the overexpression of IFITMs markedly downregulated the expression of DHX58 gene which encodes LGP2 protein, a negative regulator of retinoic acid-induced gene I (RIG-I). Conversely, it promoted the expression of DDX58gene which encodes RIG-I, a receptor in RLR signaling pathways. Moreover, transcription factors including interferon regulatory factor (IRF) 3/5/7, which are downstream signaling of both TLR and RLR signaling pathways3,4, were upregulated in IFITMs overexpressing cells as well (Fig. 3C). For the IFNs signaling pathway, IFNA1, IFNA2 and STAT1 expression was enhanced by IFITMs, while the expression of other ISGs such as MX1 decreased (Fig. 3D). Altogether, these results imply that IFITMs modulated innate immune response mainly through at least TLR, RLR and Type I IFNs signaling pathways on human foreskin cells, as shown in Fig. 4.

Fig. 3
figure 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.

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Fig. 4
figure 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/).

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Extrinsic-overexpressed IFITMs inhibit CHIKV infection via modulating innate immune responses

To examine the effects of IFITM overexpression in CHIKV-infected cells, we compared gene expression patterns of IFITM overexpressing cells infected with CHIKV with CHIKV-infected plasmid control- overexpressing cells (Fig. 5A). In contrast, CHIKV infection in upregulation of all IFITM1, 2, and 3 markedly downregulated TLR3, TLR7, TLR8, and TLR9 genes as well as down-regulated other gene-encoded downstream signaling protein of TLR signaling pathways, for example, TRADD and TRAF6 (Fig. 5B). On the other hand, in RLR signaling pathways, all CHIKV-infected IFITMs-overexpressing cells markedly downregulated the expression of DHX58 gene while slightly increasing expression of DDX58 gene. Additionally, the downstream signaling of both TLR and RLR signaling pathways, IRF 3/5/7, was reduced in expression after CHIKV infection (Fig. 5C). All genes involved in IFNs signaling pathway, IFNA1, IFNA2, STAT, MX1 and CXCL1, markedly declined in their expression after cell infection (Fig. 5D). These data suggest that CHIKV attempts to impair the immune response to favor its replication mainly through TLR, RLR and Type I IFNs signaling pathways.

Fig. 5
figure 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.

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IFITMs extend the antiviral properties to inhibit ZIKV infection

ZIKV is another important arbovirus that circulate in several areas worldwide, like CHIKV21. The prior study by Monel et al., has documented that the overexpression of IFITM3 in primary human fibroblasts leads to a reduction in ZIKV infection22. However, the present investigation endeavors to validate and broaden the spectrum of antiviral properties associated with IFITM1, IFITM2, and IFITM3 against ZIKV, utilizing the HFF-1 cell lines. The HFF-1 cells transfected with either plasmid control, IFITM1, IFITM2, or IFITM3 were infected with ZIKV and quantified for viral RNA and virus particles. Interestingly, all IFITMs exert antiviral properties against ZIKV infection in human skin fibroblasts demonstrated through the reduction of viral RNA and virus particles in IFITM transfected cells compared to plasmid control- overexpressing cells. Noticeably, using RT-qPCR, IFITM2 and IFITM3 showed a stronger inhibitory effect than IFITM1 on ZIKV infection (Fig. 6A). In support of this finding, silencing of IFITM expression in HFF-1 cells enhanced ZIKV infection. Viral RNA and virus particles of ZIKV significantly increased in IFITM2 and IFITM3 knockdown cells when compared to the control (Fig. 6B). These data indicated that IFITM2 and IFITM3 extend the antiviral role to ZIKV infection.

Fig. 6
figure 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.

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Discussion

CHIKV is an arbovirus transmitted mainly by Aedes spp. during bite on human skin23. Despite being a global health risk, no specific antiviral drugs and vaccines against the virus are available. However, several molecules have been studied as potential antiviral agents, including suramin24, picolinic acid25, imipramine26as well as ISGs, such as IFI1627and IFITMs28,29. The immune-related interferon-induced transmembrane proteins, or IFITMs, have been previously reported to inhibit several viruses, including alphaviruses. Ectopic expression of IFITM2 and IFITM3 in A549 cells (human epithelial cells from lung tissue) restricted replication of alphaviruses, including Semliki Forest virus (SFV) and Sindbis virus (SINV)30. Additionally, IFITM3 has been shown to play an essential role in restricting CHIKV infection both in vitro and in vivo28,29.

In this study, we examined the role of IFITM1, IFITM2, and IFITM3 in CHIKV infection in human skin fibroblasts, which are among the cells initially infected in the setting of natural infection. Our results indicate that the overexpression of IFITM1, IFITM2, and IFITM3 restricts CHIKV infection in HFF-1 cells. The reduction in both viral RNA and virus particle production suggests that all IFITMs interfere with the CHIKV replication cycle. These findings are supported by the report of Lin and collaborators, which demonstrated that IFITM1 decreases host membrane fluidity with increasing interruption of the IAV-induced cell-to-cell fusion field31. Concomitantly, it has been reported that IFITM3 oligomers inhibit virus-cell fusion by enhancing membrane rigidity32. Changes in membrane fluidity have been shown in other studies that overexpression of IFITM proteins induced changes in the lipid packaging order of host membranes, resulting in less membrane fluidity31,33. Cholesterol has also been reported to interact with IFITM proteins, altering their conformation, which is vital for the antiviral activity against IAV, SARS-CoV-2 and EBOV34,35. IFITM3 has been shown to interact with vesicle-associated membrane protein-associated protein A (VAPA), disrupting cholesterol homeostasis and resulting in cholesterol accumulation in late endosomes. This accumulation inhibits endosomal fusion with the viral envelope and viral entry, restricting infections by viruses such as VSV and IAV36. These observations are consistent with our previous study, demonstrating that cholesterol accumulation in the late endosome/lysosome inhibits CHIKV replication in human skin fibroblasts26. These data suggest that IFITMs might inhibit CHIKV infection through the interaction with VAPA and inducing cholesterol accumulation in late endosomes, thereby inhibiting viral fusion.

Surprisingly, when HFF1 cells were deficient in IFITMs, only IFITM2 and IFITM3 could enhance the virus replication, but not IFITM1. This may be explained by the cellular localization of IFITM1 which is mainly at the plasma membrane with some distribute in cytoplasm, while IFITM2 and IFITM3 are dominantly located at the endo/lysosomal compartments (Supplementary fig. S4)37, where CHIKV and ZIKV penetrate and uncoat38. Thus, IFITM1 may exert a limited antiviral effect and exhibit negligible impact on viral infection when knocked down, likely due to its plasma membrane localization. In contrast, IFITM2 and IFITM3’s localization within endo/lysosomal compartments, crucial for CHIKV and ZIKV entry and uncoating, suggests their greater antiviral potential. However, further investigation is warranted.

Here, we demonstrate that the overexpression of IFITMs alters the expression of several proteins involved in antiviral innate immune response, particularly increasing the expression of TLRs while decreasing the expression of DHX58, which encodes laboratory of genetics and physiology 2 (LGP2) expression. TLRs are PRRs that sense viral RNA. Activation of TLR signaling pathways leads to the expression and phosphorylation of transcription factors, including IRF3/5/7, NF-κB, and AP-1, which further resulted in upregulated the expression of IFNs and several inflammatory cytokines3,4. As demonstrated by our study, the expression of IFITMs markedly upregulates the expression of TLR3, TLR7, TLR8 and TLR9 in HFF1-infected cells. This upregulation of TLRs is associated with the upregulation of IRF3/5/7, which can potentially affect the expression of IFNs and cytokines. Moreover, the upregulation of NF-κB, another downstream signaling protein in TLR pathway that acts as a transcription factor responsible for the expression of several inflammatory genes, has also been observed39,40. These effects result in the upregulation of TNF and IL-8 in IFITMs overexpressed cells. Altogether, these data suggest that IFITMs promote the antiviral immune response to restrict CHIKV infection through activation of TLR signaling pathway. In contrast with our finding, Jiang et al. suggested that IFITM3 promotes the degradation of IRF3, limiting the type-I interferon response to Sendai virus infection41. However, it is important to note that the study used Hela cells, which originate from the human cervix, may exhibit different responses to different types of virus infections.

The overexpression of IFITMs was also demonstrated to substantially reduce the expression of LGP2, which has been reported to act as a negative regulator of RIG-I through an unclear mechanism. It has been proposed that LGP2 interferes RIG-I signaling via competitively binding to ligands of RIG-I, binding directly to RIG-I repressor domain, or interfering with the recruitment of mitochondrial antiviral signaling (MAVS), a downstream signaling protein of RIG-I, by forming a protein interaction42. Therefore, downregulation of LGP2 expression could suggest increasing antiviral response through RIG-I and RIG-I-like receptor (RLR) signaling pathways. In the RLR signaling pathway, not only does LGP2 respond to IFITMs expression, but RIG-I is also shown to be upregulated. RIG-I is one of the receptors in RLR signaling pathway that can recognize viral RNA and trigger downstream signaling to respond to viral infection, resulting in the expression of transcription factors involving IRF3/7 and NF-κB, and ultimately stimulating IFNs and cytokines production3,4.

For the IFNs signaling pathway, in this finding, interferon alpha-1 and − 2 (IFNA1 and IFNA1) are upregulated in all IFITM overexpressing cells, which might be due to the increasing antiviral response through TLR and RLR signaling pathway. Our discovery is substantiated by a recent study conducted by Chen and colleagues, demonstrating that IFITM2 elicits an antiviral response through augmentation of the type I interferon (IFN) signaling pathway43. The secreted IFNs from infected cells can result in the phosphorylation of tyrosine kinase 2, JAK1 and STAT1 and 2 proteins. The activated STAT1 and STAT2 further interact with interferon regulatory factor 9 (IRF-9) to form IFN-stimulated genes factor 3 (ISGF-3) complex, which then binds to interferon-stimulated response elements (ISREs) on the promoter of IFN-stimulated genes (ISGs), resulting in expression of ISGs that exhibit antiviral activity through various mechanisms5. The increase in gene expression of IFNA1, IFNA2, and STAT1 reported in this study could suggest the promotion of IFN signaling, which finally resulted in the upregulation of many ISGs, such as CXCL9, aiding in restricting viral infection. While Shi and colleagues found that IFNA is suppressed when IFITM2 is overexpressed, and the gene is enhanced when IFITM2 is knocked out in Huh7 cells44. These discrepancies may have occurred because the study utilized a different model of cell line and virus. However, some ISGs, including MX1, were demonstrated to be downregulated in IFITMs overexpress cells via unknown mechanism. Since we investigated the effect of IFITMs on only a few ISGs and studies on the impact of IFITMs on other ISGs remain limited, further studies on this aspect are still required to better understand and confirm IFITMs’ effect on promoting the IFNs signaling pathway.

Considering the modulation of gene expression in the innate immune response by IFITMs, it was shown that some genes have effects at different types and levels of alteration between each IFITM, indicating various activities and properties among these immune-related IFITM proteins. These may raise a reason that CHIKV was inhibited at a different level in each IFITM-overexpressed cell. However, the mechanism underlying the alteration is one of the interesting points that should be further investigated.

Conclusion

In conclusion, this study reports the antiviral activity of IFITMs against CHIKV in human skin fibroblast. Based on our findings, we propose several antiviral mechanisms of IFITMs on CHIKV infection in human fibroblasts (Fig. 7). IFITMs might inhibit the virus replication through the inducing accumulation of cholesterol to block viral fusion and act as a host-targeting antiviral agent to modulate the expression of genes involved in antiviral innate immune response, especially TLRs and LGP2 which could help in the promotion of TLR and RLR signaling resulting in IFNs production. Moreover, IFITMs expression also promotes STAT1 expression which could activate type I IFN signaling pathway, leading to increased transcription of ISGs in human skin fibroblast. We also demonstrated the inhibitory effect of IFITMs on another arbovirus, ZIKV, in these cells. However, the exact mechanism of IFITM-induced viral fusion blocking needs to be investigated. Our study provides an additional aspect of IFITMs’ properties to act as antiviral agent against arbovirus infection.

Fig. 7
figure 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/).

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Materials and methods

Cells and viruses

The Vero cells line (African green monkey kidney cells; ATCC® CCL-81™) and HFF-1 cells line (Human skin fibroblast cells; ATCC® SCRC-1041™) were cultured in High Glucose Dulbecco’s modified Eagle’s Minimum Essential Medium (DMEM; Cytiva, Logan, Utah) supplemented with 10% and 15% heat-inactivated fetal bovine serum (FBS; Gibco, Paisley, UK), respectively. The C6/36 (Aedes Albopictus cells; ATCC® CRL-1660™) grown in L-15 Medium (Leibovitz) supplemented with 10% tryptose phosphate broth (TPB, Sigma-Aldrich, MO, USA) and 10% FBS. Vero and HFF-1 cells were maintained in a humidified 37 ºC incubator with 5% CO2while C6/36 were maintained in 28 ºC incubator. CHIKV, LR2006_OPY1 strain (isolated from a viremic patient in La Réunion Island in 2006), was received from Dr. Philippe Desprès (PIMIT, Inserm U1187, St Clotilde) while the clinical isolate PF-25013-18of ZIKV has been previously described20. The CHIKV and ZIKV were grown in C6/36 cells at 28 °C and kept at −80 °C until used.

Plasmids and reagents

Plasmids encoding IFITM1, IFITM2 and IFITM3 proteins were kind gifts from Pr P. Kellam (Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, UK). Small interfering RNA (siRNA) of IFITM1, 2, and 3 (siIFITM1, 2, and 3) and non-silencing control siRNA (siControl) were purchased from QIAGEN (Maryland, USA). The target sequence of siControl, IFITM1 IFITM2, and IFITM3 are shown in Table 1.

Table 1 Target sequence of SiControl, IFITM1, IFITM2 and IFITM3.
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Overexpression and small interfering RNA (siRNA) transfection

After seeding of HFF-1 cells in six-well plates for 24 h, plasmids containing IFITM1, IFITM2, or IFITM3 were transfected with Lipofectamine™ 3000 (Invitrogen, CA, USA) according to the manufacturer’s protocol to overexpress each gene in HFF-1 cells while small interfering RNA (siRNA) of IFITM1, 2, and 3 (siIFITM1, 2, and 3) at a concentration of 80 nM in the final volume of transfection were transfected with Lipofectamine™ RNAiMAX (Invitrogen, CA, USA) following the manufacturer’s protocol to knockdown each IFITMs. Then, the transgene expressions were evaluated by Western blot analysis and RT-qPCR.

Viability assay

The MTT-based assay was used to determine cell viability. Briefly, cells were transfected with IFITMs plasmids using Lipofectamine™ 3000 according to the manufacturer’s protocol and incubated at 37 °C, 5% CO2 for 24 h. Then, cells were washed with phosphate-buffered saline (PBS; Gibco, Paislay, UK) and incubated with 100 µL MTT. After 2 h, MTT was removed, 50 µL of DMSO was added and incubated at 37 °C for 10 min. Finally, the absorbances were measured at 570 nm in order to determine cell viability.

Gene expression by quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Total RNA was extracted from human fibroblasts. The reactions were carried out using 0.25 µg of total RNA as a template. The reaction was carried out using 1 µg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2×) (Thermo Scientific) was used in qPCR experiment. Each reaction of 25 µL contained 400 nM of each primer, 200 nM of specific probe and 1× MaximaTM Probe/ROX qPCR Master Mix. Primers sequences are listed in Table 2. The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). GADPH was used as endogenous control for calculation of relative fold change.

Table 2 Primers of IFITM1, IFITM2 and IFITM3.
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Western blot analysis

Total proteins were collected from human fibroblasts using a RIPA buffer supplemented with a protease inhibitor cocktail solution (Calbiochem, MA, USA). Protein concentrations were determined by using a NanoDrop Spectrophotometer. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF (polyvinylidene difluoride) membrane for immunoblotting. Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline with 0.05% Tween 20 (PBST) for 2 h and incubated overnight with primary antibodies followed by washing with PBST and incubated in the dark with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Inc) at room temperature for 1 h. The proteins were then visualized using a SuperSignal™ West Pico chemiluminescent substrate kit (Thermo Scientific, IL, USA). Finally, the membrane was stripped and re-probed with an anti-β-actin antibody to ensure that equivalent protein levels were loaded in each lane.

Viral infection

Cells were washed with PBS and the cells were incubated with CHIKV or ZIKV at the desired multiple of infection (MOI) for 2 h at 37 °C with 5% CO2 while gently agitating the plates. Then, the inoculum was removed, the cells were washed with PBS and DMEM supplemented with 15% FBS was added. Plates were incubated at 37 °C with 5% CO2 for 24 and 48 h before collecting supernatants and cell lysis for virus particle and viral genome quantification, respectively.

Plaque assay

Confluent Vero cells monolayer were prepared in 24-well tissue culture plates at 2.5 × 105 cells/well for 24 h. Viruses were diluted by making a serial 10-fold dilution with 1X DMEM. Each virus concentration was transferred to monolayer cells and incubated at 37 ºC with 5% CO2 for 90 min for ZIKV and 2 h for CHIKV, continuously mixing for viral adsorption. The inoculums were discarded then infected cells were washed with 1X PBS and overlayed with 2.4% carboxyl methyl cellulose, subsequently incubated at 37 ºC with 5% CO2 for 7 days for ZIKV and 3 days for CHIKV. Plaques were counted after fixing with 3.7% formaldehyde for 30 min and staining with 0.5% Crystal violet in 20% ethanol for 1 h. The virus titer will be calculated and presented as a plaque-forming unit per milliliter (PFU/mL).

Viral RNA quantification by RT-qPCR

2 µL of total RNA extracted from human fibroblasts were used as a template for RT-qPCR using the KAPA SYBR FAST One-Step (KAPA Biosystem, Cape town, South Africa). Each reaction of 10 µL contained 2 µM of each primer (Table 3), 5 µL of KAPA SYBR FAST qPCR, and 0.2 µL of RT Mix. The amplification conditions were 42 °C for 10 min and 95 °C for 3 min followed by 45 amplification cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 30 s. Reactions were performed in a CFX96 Real-Time System, C1000 Touch Thermal cycler (Bio-Rad). Viral RNA was quantified by comparing each virus’s threshold cycle (Ct) values with the RNA standard curve.

Table 3 Primers for viral detection used in this study.
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RT2 profiler PCR array

Total RNA extracted from human fibroblasts was reverse transcript to cDNA using the iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad, USA) according to the manufacturer’s instructions. Then cDNA was analyzed by RT2Profiler PCR Arrays Antiviral response (PAHS-122Z; QIAGEN, Maryland, USA). The 8 µL of cDNA was mixed with 650 µL of 2x RT SYBR Green Mastermix and 642 µL of RNase-free water before adding into the 384-well RT2 Profiler PCR Array plate under the supporting of 384EZLoad Cover for profiling expression of total 84 genes of human antiviral response, as described in the manufacturer’s handbook. The DNA amplification was carried out with the Roche LightCycler 480 real time cycler using a cycling program of 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min before a melting curve acquisition step. The fold changes of gene expression in each condition were calculated in comparison to the values of controls.

Data analysis and statistical methods

Data are presented as means ± SD. The significance of the differences among groups was evaluated using the two-way ANOVA. Statistical significance was accepted at p < 0.05.