HeLa, BHK-21, and COS7 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagles Medium (DMEM; Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen) and 1% Penicillin-Streptomycin (Gibco/Invitrogen) at 37°C and 5% CO₂. Aedes albopictus C6/36 cells (ATCC) were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibco/Invitrogen) supplemented with 10% FBS and 1% Penicillin-Streptomycin at 28°C. The Zika Virus strain Z16006 (GeneBank: no. KU955589.1) was kindly provided by the Institute of Microbiology, Guangdong Provincial Center for Disease Control and Prevention, and was propagated in C6/36 cells.

The ZIKV NS3 was cloned into pcDNA3.1(+) vector using a forward primer containing a KpnI restriction site and a reverse primer containing an XbaI restriction site, with a 3×FLAG tag fused at the C-terminal (The primers were listed in Supplementary Table 1 ). Complementary DNA (cDNA) was obtained by reverse transcription of RNA from the HeLa cells infected with ZIKV and used as the template for PCR amplification of ZIKV NS3 gene fragment. The amplified product was digested with restriction enzymes, ligated into the plasmid vector, and used to construct the recombinant expression plasmid. The resulting recombinant plasmids were confirmed by DNA sequencing (Tsingke Biotechnology, Beijing, China). The pcDNA3.1(+)-S100A6-HA and pcDNA3.1(+)-S100A6-TurboID-HA plasmids were previously constructed (Zhou et al., 2021) and stored in our laboratory at −20°C.

The antibodies used in this study were anti-β-Actin antibody (Abcam, Cambridge, UK, Cat# ab179467), anti-ZIKV NS3 antibody (Genetex, CA, USA, Cat# GTX133309), anti-ZIKV E antibody (Genetex, CA, USA, Cat# GTX133314), anti-S100 alpha 6 antibody (Selleck, Houston, TX, USA, Cat# A5890), anti-HA antibody (Cell Signaling Technology, Boston, MA, USA, Cat# 3724), anti-FLAG M2 antibody (Sigma, St. Louis, MO, USA, Cat# F1804), goat anti-mouse IgG-HRP (ABclonal, Wuhan, China, Cat# AS003), goat anti-rabbit IgG-HRP (Abclonal, Wuhan, China, Cat# AS014), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA, Cat# A32740), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA, Cat# A11001). The antibodies were used following the manufacturers’ guidance.

Proteasome inhibitor MG132 (APExBIO, Houston, TX, USA) was diluted in DMSO and added to the cell culture medium to a final concentration of 10 μM. Similarly, Lysosomal acidification inhibitor NH₄Cl (aladdin, Shanghai, China) was diluted in sterile water and added to the cell culture medium to a final concentration of 30 mM.

Cells were collected and lysed on ice for 30 minutes using cell lysis buffer (Beyotime, Shanghai, China) containing 1mM phenylmethanesulfonyl fluoride (PMSF, Dingguo, China). After centrifugation at 14000×g, 4°C for 10 minutes, the supernatant was collected and the concentration was measured by bicinchoninic acid (BCA) method. For the S100A6-siRNA experiments, 100 μg of total protein was used, while for the other experiments, 50 μg of total protein was taken. The samples were then diluted in 6 × loading buffer, boiled for 10 minutes, then quickly placed in ice water. After a brief spin, the protein samples were loaded onto 10% or 15% SDS-PAGE gels for separation, and subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% bovine serum albumin (BSA) dissolved in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4) in a 37°C shaker for two hours, and then incubated with the primary antibody at 4°C overnight. After washing three times with TBST, the membrane was incubated with an HRP-labeled secondary antibody at 37°C for two hours. Finally, the protein bands were visualized using Clarity Western ECL Substrate (Bio-Rad) and photographed with a ChemiDoc Touch Imaging System (Bio-Rad).

The ZIKV copies, the knockdown and overexpression efficiency of S100A6, and the NS3 RNA copies were detected by qRT-PCR. Total RNA was isolated using TRIzol Reagent (Invitrogen) and the viral RNA in the supernatant of cell cultures was extracted according to the instructions of EasyPure Viral DNA/RNA Kit (TransGen, Beijing, China). Then the RNAs was reverse-transcribed to cDNAs using HiScript All-in-one RT SuperMix (Vazyme, Nanjing, China). The cDNA was subjected to quantitative PCR using Hieff qPCR SYBR Green Master Mix (Low Rox; Yeasen, Shanghai, China) on QuantStudio 6 real-time PCR system. The S100A6 transcript and NS3 RNA levels were normalized to the transcript level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using comparative Ct method. Absolute quantification for ZIKV copies were implemented by comparison to a standard curve. The fragment corresponding to nucleotides (10,409 to 10,554) of ZIKV (Z16006) was used as an amplification target. Standard curves were prepared using 10-fold serial dilutions of known quantities of ZIKV fragment. All assays were performed in triplicate. The primers used to amplify the target genes are listed in Supplementary Table 2 .

HeLa cells were grown in a 12-well plate to about 70% confluence, and then transfected with pcDNA3.1(+)-S100A6-HA or control plasmid for 24 hours, using Lipofectamine 3000 Reagent (Invitrogen, USA) following the manufacturer’s instruction. For the virus binding assay, the cells were then infected with ZIKV at an MOI of 10 and incubated at 4°C for 1 hour. After the supernatant was discarded, the cells were washed with phosphate buffered saline (PBS) for three times. The cells were harvested and total RNA was extracted by TRIzol and the amount of the viral RNA was determined by qRT-PCR. For the entry assay, after one hour of ZIKV binding at 4°C, the cells were washed with PBS and incubated at 37°C for additional 1 hour. Subsequently, the supernatant was discarded and the cells were harvested following washing with PBS for three times. TRIzol was used to extract total RNA, and qRT-PCR was used to detect the effect of S100A6 on ZIKV entry. The efficiency of S100A6 overexpression was verified by WB.

BHK cells were seeded in 12-well plates until they reached approximately 70% confluency, and were transfected with either 3.0 ug siRNA-NC or siRNA-S100A6 per well for 24hours. Following transfection, cells were infected with ZIKV at an MOI of 0.1 and incubated for 5 or 10 days. The cells were then fixed in 10% paraformaldehyde for 5 min, stained with 0.1% crystal violet for 30min, followed by washing with PBS for 3 times. After that, the plaques on the plate bottom were photgraphed under a light microscope with 100× or 40× magnification.

BHK-21 cells were grown on coverslips in a 12-well plate to about 70% confluence, and then transfected with 1 μg of plasmids using Lipofectamine 3000 Reagent (Invitrogen) following the manufacturer’s instruction for 24 hours. The cells were separated into two groups, and then infected with ZIKV for 24 hours or left uninfected for control. Afterward, cell cultures were aspirated, and cells were fixed and permeabilized with 100% precooled methanol for 8 minutes at −20°C. Then the methanol was aspirated and the cells were blocked with 10% BSA dissolved in PBS for one hour at room temperature.

After aspiration of the blocking buffer, the coverslips were incubated with the primary antibody at 4°C overnight, at the recommended concentration diluted in 10% BSA according to the manufacturer’s instruction. After washing three times with PBS, the coverslips were incubated with Alexa Fluor 594 goat anti-rabbit IgG (1:1500, Invitrogen) and/or Alexa Fluor488 goat anti-mouse IgG (1:1500, Invitrogen), diluted in 10% BSA for one hour at room temperature. Subsequently, the coverslips were washed with PBS for three times, rinsed with ddH₂O and mounted with 4’,6-diamidino-2-phenylindole (DAPI) Fluoromount-G (Southern Biotech, USA). The samples were visualized and photographed using a fluorescence microscope (Nikon, Tokyo, Japan). Ten fields of view were randomly selected from the up, down, left, right and middle sections of each coverslip, and the total number of cells and the number of ZIKV positive cells were counted using Image J software. The virus infection rates of the two groups were calculated and compared.

HeLa cells were grown in T25 flasks to 70% confluence, and then, each flask was transfected with 3.0 µg pcDNA3.1(+)-S100A6-TurboID-HA or control plasmid for 24 hours using Lipofectamine 3000 Reagent (Invitrogen), following the manufacturer’s instruction. Afterward, the medium was replaced with DMEM containing 160 μM D-Biotin (Sigma-Aldrich, St. Louis, MO, USA) or no D-biotn supplemented, and the cells were infected with ZIKV at an MOI of 1, or left uninfected for another 24 hours. Then the cells were washed with PBS for three times, and lysed with cell lysis buffer (Beyotime) containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Cell lysates were centrifuged at 14,000×g, 4°C for 10 minutes, and the supernatant was collected and incubated with Streptavidin magnetic beads (Thermo Fisher, Waltham, MA,USA) at 4°C overnight with gentle rotation. The beads were collected with a magnetic stand to remove the supernatant, and then, 500 μl precooled Washing Buffer (TBST, 0.1% Tween 20) was added and mixed gently with the beads. After washing three times, the magnetic beads were collected and resuspended in 50 μl PBS and 10 μl 6 × SDS loading buffer. The samples were boiled for 5 minutes and subjected to WB detection.

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). A two-tailed unpaired Student’s t test was used to compare differences between each group, and one-way Analysis of Variance (ANOVA) with Tukey’s multiple comparisons test was used to compare differences among multiple groups. P < 0.05 was considered statistically significant. Data were represented as mean ± SEM, and all experiments were repeated at least three times.

HeLa cells were infected with ZIKV at a multiplicity of infection (MOI) of 1 for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. Cells were collected at the corresponding time points, and the expression of S100A6 was detected. The results showed that the S100A6 expression in ZIKV-infected cells was significantly increased. With the prolongation of infection time, the level of S100A6 increased in a time-dependent manner ( Figures 1A, B ). The faint NS3 signal observed in the Mock group represents background due to non-specific antibody binding. Subsequently, S100A6 levels were detected 24 hours post infection (hpi) with ZIKV at MOIs of 0, 0.1, 1 and 5 in HeLa cells. It was found that S100A6 expression was significantly upregulated with increasing MOIs in a dose-dependent manner ( Figures 1C, D ). These results demonstrated that ZIKV infection upregulated S100A6 protein levels in host cells, suggesting that S100A6 may be involved in the regulation of ZIKV infection.

Given the increased expression of host S100A6 protein following ZIKV infection, we hypothesized that S100A6 might regulate the life cycle of ZIKV. The entire life cycle of ZIKV requires the interaction between viral and host proteins. During ZIKV infection, the attachment of viral envelope protein E to host receptors mediates its internalization into the host cell (Agrelli et al., 2019). Following the fusion of the virus membrane protein with host endosomal membrane, the viral genome is released into the cytoplasm to initiate viral genome replication and particle assembly.

To investigate whether S100A6 affects ZIKV’s binding and entry into host cells, HeLa cells were transfected with or without the pcDNA3.1(+)-S100A6-HA plasmid. To evaluate the impact of S100A6 on virus binding, cells were infected with ZIKV at 4°C for 1 h, washed three times with PBS and then harvested for qRT-PCR ( Figure 2A ). To determine whether S100A6 affected the virus entry, the HeLa cells were infected with ZIKV at 4°C for 1 h, then the cell culture medium was replaced with fresh medium, and the cells were incubated at 37°C for the internalization of the binding viruses ( Figure 2B ). The overexpression of the S100A6 protein was verified by WB ( Figure 2C ). There was no significant difference in viral copy number between the S100A6 overexpression group and the control group ( Figures 2A, B ), indicating that S100A6 did not affect virus binding and entry into cells.

Since S100A6 did not affect the binding and entry of ZIKV into host cells, we next explored whether it affected virus replication. RNAi was performed in HeLa cells to inhibit S100A6 expression, after which virus replication was detected. HeLa cells were transfected with si-S100A6 and si-NC for 24 hours, then infected with ZIKV for another 24 hours before being harvested for WB and qRT-PCR. The results showed that S100A6 expression level and transcription level were significantly reduced, confirming successful knockdown ( Figures 3A, B ). In cells transfected with si-NC, ZIKV infection increased S100A6 mRNA levels, which is consistent with the upregulation of S100A6 protein expression observed in Figure 1 . After the knockdown of S100A6, the copy number of ZIKV in both the cell lysates and cell culture supernatant significantly increased ( Figures 3C, D ), suggesting that host S100A6 plays a role in inhibiting ZIKV replication.

To further verify this, we performed plaque assays. At 5 days post infection (dpi), distinct patterns of cytopathic cell foci formation were observed across groups. Specifically, the si-S100A6 + ZIKV group exhibited the most pronounced foci, followed by the siRNA-NC + ZIKV group. No cytopathic foci were detected in the uninfected controls (siRNA-NC - ZIKV and si-S100A6 - ZIKV) ( Figure 4A ). At 10 dpi, the plaques formed in siRNA-S100A6 transfected cells were significantly larger than those in siRNA-NC transfected cells ( Figures 4B, C ).

Given that knockdown of S100A6 expression in host cells would lead to increased viral replication, to further explore the effect of S100A6 on viral replication, the pcDNA3.1-S100A6-HA plasmid was transfected into COS-7 cells, which were subsequently infected with ZIKV. S100A6 overexpression in cells was verified by WB with anti-HA antibody ( Figure 5C ), and ZIKV replication was detected by qRT-PCR. The copy numbers of ZIKV in both cell lysates and cell culture supernatants were significantly decreased after S100A6 overexpression ( Figures 5A, B ). We further confirmed the inhibitory effect of S100A6 on viral replication in BHK-21 cells by IFA. S100A6 overexpression was verified by WB with anti-HA antibody ( Figure 5F ). The nucleus was stained with DAPI (blue) and the virus was stained with a ZIKV E protein antibody (red). The total number of cells and ZIKV positive cells were counted by ImageJ software, and virus infection rates were calculated and compared. ZIKV infection rate in the S100A6 overexpression group was significantly lower than that in the control group ( Figures 5D, E ), consistent with the qRT-PCR results. These results further confirmed that the host protein S100A6 played a role in inhibiting ZIKV replication.

NS3 is an essential protein for ZIKV replication. In our previous experiments, we found that S100A6 knockdown in HeLa cells resulted in a significantly higher NS3 protein level compared to the control group ( Figure 3B ). Conversely, overexpression of S100A6 led to a significant downregulation of the viral protein NS3 ( Figures 5C, F ). Furthermore, HeLa cells were transfected with a gradient of S100A6-HA plasmid and then infected with ZIKV for 24 hours. As S100A6 protein expression increased, ZIKV copies decreased significantly in a dose-dependent manner ( Figure 5A ). Meanwhile, we also found that the level of ZIKV NS3 protein and ZIKV genome copies decreased significantly with the increase of S100A6 ( Figures 6A, B ).

Therefore, we hypothesized that S100A6 might inhibit viral replication through its effect on the NS3 protein. We further constructed the pcDNA3.1(+)-NS3-FLAG plasmid, and co-transfected it with pcDNA3.1(+)-S100A6-HA into HeLa cells. It was found that the exogenous NS3 protein level decreased with an increased amount of S100A6 ( Figures 6C, D ), indicating that ZIKV NS3 levels were negatively correlated with S100A6 protein amounts and that S100A6 promotes the degradation of ZIKV NS3.

S100A6 downregulated the NS3 protein level, but it was not clear whether this was regulated at RNA or protein level. We overexpressed S100A6 in HeLa cells, then infected them with ZIKV( Figures 7A, B ), or simultaneously overexpressed both S100A6 and NS3( Figures 7C, D ). We found that the protein level of NS3 was significantly decreased compared to the control group without S100A6 expression in both conditions ( Figures 7B, D ). However, the genome copy (represented by NS3-RNA copy), as detected by qRT-PCR, was not significantly different from the control group ( Figures 7A, C ), indicating that the downregulation of NS3 protein level by S100A6 occurred at the post-translational level rather than the RNA level.

In eukaryotic cells, proteins are mainly degraded through the ubiquitin-proteasome pathway and lysosome pathway. To explore which pathway S100A6 utilized to degrade NS3, we co-expressed S100A6-HA and NS3-FLAG in HeLa cells. After 36 hours of transfection, the cells were treated with either the proteasome inhibitor MG132 or the lysosomal acidification inhibitor NH₄Cl for 12 h, and the level of NS3 protein was detected by WB. NS3 degradation was still observed in the MG132-treated group, whereas NH₄Cl restored the decrease in NS3 level caused by S100A6 ( Figures 7E, F ). These results indicated that overexpression of S100A6 promoted NS3 protein degradation through the lysosomal pathway.

Host S100A6 degraded ZIKV NS3 through the lysosomal pathway. We further investigated whether this degradation process was facilitated by the interaction between S100A6 and NS3. HeLa cells were infected with ZIKV following S100A6 overexpression, and the cells were lysed for Co-IP. WB results showed that S100A6 interacted with ZIKV NS3 ( Figure 7G ). Next, we used IFA to detect the co-localization of S100A6 and NS3 in cells. BHK-21 cells were transfected with S100A6-HA, infected with ZIKV, and stained with a fluorescent HA antibody (green) and NS3 antibody (red). Co-localization was indicated by yellow fluorescence. We observed co-localization of S100A6 and NS3 in the cytoplasm ( Figure 7H ). Therefore, we concluded that S100A6 co-localized and interacted with ZIKV NS3, inducing NS3 degradation through the lysosomal pathway.