T-Cell Immunoglobulin and Mucin Domain 1 (TIM-1) Is a Functional Entry Factor for Tick-Borne Encephalitis Virus

ABSTRACT Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection affecting humans. The host factors required for viral entry have yet to be described. Here, we found that T-cell immunoglobulin and mucin domain 1 (TIM-1) acted as the cellular entry factor for TBEV. Using a virus overlay protein binding assay, TIM-1 was identified as a virion-interacting protein. Cells that were relatively resistant to TBEV infection became highly susceptible to infection when TIM-1 was ectopically expressed. TIM-1 knockout and viral RNA bypass assays showed that TIM-1 functioned in the entry phase of TBEV infection. TIM-1 mediated TBEV uptake and was cointernalized with virus particles into the cell. Antibodies for TIM-1, soluble TIM-1, or TIM-1 knockdown significantly inhibited TBEV infection in permissive cells. Furthermore, in TIM-1 knockout mice, TIM-1 deficiency markedly lowered viral burden and reduced mortality and morbidity, highlighting the functional relevance of TIM-1 in vivo. With TIM-1, we have identified a key host factor for TBEV entry and a potential target for antiviral intervention.

endocytosis (6). The process of TBEV entry into a target cell uses host molecules which act as entry factors or cellular receptors. Though a few cell surface molecules have been suggested to play a role in virion attachment (7,8), the molecular interactions mediating TBEV entry are poorly understood, and the host factors involved in TBEV entry have yet to be identified and characterized. In this study, we show that TBEV uses T-cell immunoglobulin and mucin domain 1 (TIM-1) as a cellular entry factor and that this interaction can form a productive infection.

RESULTS
TIM-1 is identified as a TBEV-associated protein.
To identify candidate cell membrane proteins that interact with TBEV, we conducted a virus overlay protein binding assay (VOPBA) followed by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis. VOPBA using TBEV on membrane proteins extracted from permissive A549 cells (9) revealed several bands. The putative virus-associated proteins were excised from the corresponding gels and analyzed using LC-MS/MS. Of the various hits obtained by MS, TIM-1 (Table 1, shown in bold) with a molecular weight of approximately 100 kDa was found to be a potential candidate (Fig. 1A). TIM-1 is a cell surface glycoprotein that binds to phosphatidylserine (PtdSer) on the surface of apoptotic cells and internalizes apoptotic bodies (10). It serves as the receptor for several viruses through viral apoptotic mimicry (11).
To verify the association of TBEV with TIM-1, we conducted a pulldown assay with soluble TIM-1-Fc. TBEV particles were incubated with TIM-1-Fc or IgG1-Fc, followed by protein G Sepharose beads. Binding was evaluated by immunoblotting for TBEV envelope (E) protein using the 4G2 MAb. As shown in Fig. 1B, TBEV bound to the TIM-1 construct, but not to the IgG1-Fc negative-control construct. Similar to previous studies (12,13), soluble TIM-1-Fc immunoprecipitated Ebola virus-like particles (EBOV-VLPs), but not influenza A virus H1N1 (PR8) particles (Fig. S1), confirming the effectiveness of this approach. Additionally, the TBEV-TIM-1 interaction was confirmed by enzymelinked immunosorbent assay (ELISA). We showed that TBEV reacted with the TIM-1-Fc coating on 96-well plates, whereas it did not react with the IgG1-Fc negative control (Fig. 1C). These data demonstrated an interaction between TBEV and TIM-1.
Ectopic expression of TIM-1 facilitates TBEV infection. Next, we explored the involvement of TIM-1 expression in TBEV infection. Previous studies show that the human embryonic kidney cell line 293T does not express TIM-1 (14). Then parental 293T cells were engineered to express TIM-1 (293T-TIM-1; Fig. 2A). Cells were challenged with TBEV, and infection rates were quantified by flow cytometry. Infection of 293T-TIM-1 cells with TBEV resulted in a remarkable increase in the percentage of virus-infected cells compared with the parental 293T cells (Fig. 2B). Through titration of cell-free supernatants collected from cells challenged with TBEV, we found that 293T-TIM-1 cells supported significantly greater virus replication than the parental cells (P , 0.001; Fig. 2C). We next examined whether soluble TIM-1-Fc can inhibit TBEV infection of 293T-TIM-1 cells. As shown in Fig. 2D, virus infection was inhibited by soluble TIM-1-Fc in a dose-dependent manner. In contrast, control IgG1-Fc did not affect TBEV infection of cells. These data indicate that ectopic TIM-1 expression in poorly permissive cells facilitates TBEV infection. TIM-1 binds PtdSer, which can be exposed on the surface of viruses (15,16). To investigate whether the TIM-1 ligand, PtdSer, associates with TBEV, ELISA wells were coated with purified virions and incubated with the specific anti-PtdSer monoclonal antibody (MAb). We found that the anti-PtdSer MAb, but not the isotype control, bound to TBEV-coated ELISA wells (Fig. 2E). To examine whether viral PtdSer correlates with TIM-1-enhanced TBEV infection of 293T-TIM-1 cells, we preincubated TBEV with annexin V (ANX5), a PtdSer-binding protein. ANX5 inhibited TBEV infection of 293T-TIM-1 cells in a dose-dependent manner, suggesting that interaction between PtdSer and TIM-1 could enhance TBEV infection of cells (Fig. 2F). Notably, neither Fc-fused proteins (Fig. S2) nor ANX5 (Fig. S3) treatment led to obvious cytotoxicity. Treatment of cells with Triton X-100 was used as a positive control.
TIM-1 plays a role in the entry phase of TBEV infection. To determine if the requirement for TIM-1 in the TBEV life cycle was at the level of entry, we performed a viral RNA bypass assay. TIM-1 knockout A549 cells (A549-TIM-1-KO) were generated by using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene editing technology (Fig. 3A). We compared the production of progeny viruses between infection (entry-dependent) and viral RNA transfection (entry-independent) in either A549 or A549-TIM-1-KO cells. As shown in Fig. 3B, TBEV infection was significantly inhibited in A549-TIM-1-KO cells compared to A549 cells (P , 0.001). In contrast,  (Fig. 3C). This demonstrates that the requirement for TIM-1 was largely bypassed by viral RNA transfection, suggesting a role for TIM-1 in entry, but not for a step downstream of entry.
TIM-1 mediates TBEV entry and is cointernalized with virus particles into cells. To study the entry of infectious TBEV particles into cells, parental 293T and 293T-TIM-1 cells were incubated with viruses for 1 h at 4°C, followed by 45 min at 37°C to allow endocytosis. Subsequently, these cells were stained for viral E protein using the 4G2 MAb. 293T-TIM-1 cells showed increased intracellular accumulation of viral protein compared with 293T cells (Fig. 3D). Next, total RNA was extracted from virus-infected cells, and viral RNA levels were determined by reverse transcription-quantitative PCR (qRT-PCR). Increased TBEV RNA uptake was observed in 293T-TIM-1 cells compared with the parental 293T cells, suggesting that ectopic TIM-1 enhances virus entry into cells (Fig. 3E).
Then, we performed double immunofluorescence staining for TBEV antigen and TIM-1 to analyze the subcellular localization of the virus and entry factor. Confocal microscopy of 293T-TIM-1 cells incubated with TBEV at 4°C for virus binding without internalization revealed colocalization of TIM-1 and TBEV on the cell surface. When the cells were allowed to internalize the virus at 37°C for 45 min, colocalization of the virus and the TIM-1 was clearly observed within the cytoplasm (Fig. 3F). Similar results were seen in TBEV-infected A549 cells, which express TIM-1 endogenously (17) (Fig. 3G). These data demonstrate that TIM-1 mediates TBEV entry and is cointernalized with virus particles into cells.
Blocking of endogenous TIM-1 inhibits TBEV infection. We then analyzed the effects of neutralizing anti-TIM-1 antibodies (Abs) on TBEV infection in A549 and Vero cells, which endogenously express TIM-1. Treatment with Abs had no effect on cell Next, TIM-1 was silenced by RNA interference in A549 cells. Then, 48 h after TIM-1/ small interfering RNA (siRNA) transfection, TIM-1 expression was decreased compared Total RNA was extracted from infected 293T and 293T-TIM-1 cells, and viral RNA levels were quantified by qRT-PCR with human GAPDH as an endogenous control. Results are expressed as the fold change compared with parental 293T as the calibrator value. Data are presented as the means 6 standard deviation of three independent experiments. (F) 293T-TIM-1 and (G) A549 cells were exposed to TBEV (MOI = 10) at 4°C for 1 h, with or without a shift to 37°C for 45 min. Cells were fixed and stained for nucleus (blue), TBEV E protein (green), and TIM-1 (red). Areas of yellow indicate TBEV-TIM-1 colocalization on the cell surface or within the cytoplasm. Samples were observed under a confocal microscope. Representative images of three independent experiments are shown. Scale bar = 20 mm. The results are presented as the means 6 standard deviation of three independent measurements. ***, P , 0.001. NS, not significant.
TIM-1 promotes TBEV infection in primary cells. To prove that endogenous TIM-1 is important for entry of TBEV in primary cells, mouse primary renal tubular epithelial cells (RTEC) were utilized as a relevant cell type. It was shown that TBEV viral RNA could be detected in kidney and urine samples of humans and other natural hosts even if viremia was not detected, which would suggest infection of renal parenchymal cells (18)(19)(20). TIM-1 expression has been demonstrated in epithelial cells of kidney origin and is upregulated in both mouse and human kidneys after injury (14). We performed flow cytometry analyses to measure the TIM-1 expression level. TIM-1 was detected on the cell surface of RTEC as judged by antibody reactivity (Fig. 5A). Colocalization of TIM-1 and TBEV was detected both on the cell surface and within the cytoplasm of RTEC (Fig. 5B). In blocking experiments, infection of RTEC was inhibited by the TIM-1specific antibodies in a dose-dependent manner (Fig. 5C). Treatment with Abs did not cause obvious cytotoxicity (Fig. S6). These results indicate that TIM-1 promotes TBEV infection in permissive cells naturally expressing the molecule.
TIM-1 deficiency attenuates TBEV infection and pathogenesis in mice with a defective interferon (IFN) system. The contribution of TIM-1 to TBEV infection in vivo was further tested. We first used mice deficient in type I interferon receptor (IFNAR1 KO mice), as these are highly susceptible to flavivirus infection and disease progression (21,22). To explore the impact of TIM-1 on TBEV pathogenesis, homozygous TIM-1 2/2 IFNAR1 2/2 double KO (DKO) mice were generated and used in this study. DKO and IFNAR1 KO mice were challenged with TBEV strain WH2012 via a footpad injection to mimic the primary route of virus transmission. We measured viral RNA loads in serum and infected organs of both DKO and IFNAR1 KO mice. On day 8 postchallenge, the levels of viral RNA in the serum (P , 0.05), brain (P , 0.05), and kidneys (P , 0.05) of DKO mice were significantly reduced compared with those of IFNAR1 KO mice (Fig. 6A). One hundred percent of TIM-1-sufficient IFNAR1 KO mice succumbed to virus by day 12 postinfection. In contrast, DKO mice challenged with TBEV had markedly reduced mortality (Fig. 6B). DKO mice (19.8 6 2.7 days) exhibited a significantly longer mean survival time in comparison with IFNAR1 KO mice (10.4 6 0.9 days) (P , 0.001). Weight loss was observed in both groups; however, the surviving DKO mice almost completely recovered by day 21 postinfection (Fig. 6C). All IFNAR1 KO mice showed severe signs of illness, such as paresis, hind limb paralysis, or tremor. In comparison, only 20% of DKO mice infected with TBEV demonstrated severe neurological symptoms (Fig. 6D).
TIM-1 deficiency reduces viral tissue burden and pathogenicity in immunocompetent mice. In further support, we assessed the impact of TIM-1 on TBEV infection in an immunocompetent mouse model. WT C57BL/6 mice and TIM-1 knockout (TIM-1 KO) littermates were infected with TBEV by the footpad route. Though no deaths were observed in either TIM-1 KO or WT mice during the 21 days after infection of strain WH2012, we observed significantly lower viral RNA loads in the serum (P , 0.001), brain (P , 0.05), and lungs (P , 0.05) of TIM-1 KO mice compared with the WT group (Fig. S7). To validate the contribution of TIM-1 to TBEV infection in vivo, strain Neudoerfl, which displayed virulence in WT mice, was used for the following study. We found that the viral loads in the serum (P , 0.001), brain (P , 0.001), kidneys (P , 0.05), and lungs (P , 0.01) of WT mice were significantly higher than in those of TIM-1 KO mice at day 5 postinfection (Fig. 7A). WT mice were found to be highly susceptible to infection, displaying 100% mortality, compared to 16.7% mortality in TIM-1 KO mice (Fig. 7B). Obvious weight loss was observed in WT mice at the early stage of infection. TIM-1 KO mice lost weight much more slowly, and their body weight bounced back by day 21 postinfection (Fig. 7C). WT mice infected with strain  (Fig. 7D). Taken together, our findings provide evidence that TIM-1 is associated with viral burden and pathogenesis for TBEV in vivo.

DISCUSSION
To date, knowledge regarding TBEV entry into the host cell remains limited, and the host determinants required for TBEV entry have yet to be identified and characterized. Here, we found that TIM-1 is a functional entry factor for TBEV infection in vitro and in vivo.
In this study, we provide several lines of evidence establishing TIM-1 as a key host factor for TBEV entry. Ectopic expression of TIM-1 significantly increased TBEV infection of poorly permissive cells. SiRNA-mediated knockdown of TIM-1 expression or CRISPR/ Cas9-mediated knockout of the TIM-1 gene markedly decreased TBEV entry into permissive cell lines. Therefore, the presence of TIM-1 correlated directly with susceptibility of cells to TBEV infection. Antibodies directed against TIM-1 or soluble TIM-Fc were capable of potently inhibiting TBEV infection. This indicates that blocking viral access to TIM-1 on the cell surface substantially limits TBEV infection, and the role of TIM-1 occurs at the interface of the cell. Moreover, the ability of anti-TIM-1 Abs or PtdSer ligand to inhibit TBEV infection of cells expressing TIM-1 may provide an effective antiviral therapy (16,23). Pharmacological inhibition of such cellular components rather than viral proteins may provide useful antiviral agents and reduce the rate of development of antiviral resistance to therapeutic agents (14,24).
The viral burden was significantly decreased in the organs of TIM-1-deficient mice. Considering the endogenous expression of TIM-1 in keratinocytes (25), lymphocytes (26), human brain tissue (27), kidney cells (10,28), and lung-derived cells (29), the TBEV-TIM-1 interaction may be relevant to viral tissue tropism. We proved that endogenous TIM-1 was important for entry of TBEV in mouse primary RTEC of kidney origin, suggesting the involvement of TIM-1-mediated TBEV entry under physiological conditions. Thus, the TBEV/TIM-1 interaction may be relevant to in vivo TBEV infection and tissue tropism. Future work needs to further clarify the impact of TIM-1 expression on TBEV infection of specific cell types of the susceptible organs.
We have shown that TIM-1 deficiency decreased mortality and morbidity in both IFNAR1 KO and immunocompetent mice when challenged with TBEV. The viral entry (B) Survival was assessed following infection, and the significance for survival curve was determined by the Kaplan-Meier log rank test (n = 6). (C) The body weight change ratio in TBEV Neudoerfl-infected WT and TIM-1 KO mice was calculated for each recording day. Error bars represent standard deviations. (D) Animals were monitored for clinical manifestations. Signs of illness were scored as follows: 0, no symptoms; 1, ruffled fur or hunched posture; 2, asthenia or paresis; 3, lethargy, tremor, or paralysis; 4, moribund or euthanized; and 5, death. Error bars represent standard deviations. *, P , 0.05; **, P , 0.01; ***, P , 0.001. TIM-1 Serves as a Cellular Entry Factor for TBEV ® mediated by TIM-1 may be one of the determinants of TBEV pathogenesis. Other consequences resulting from virus-TIM-1 interaction, such as induction of a massive release of inflammatory mediators, may also contribute to the pathogenesis in mice (30,31). Lower levels of viral loads and milder pathogenesis were observed in mice with a defective TIM-1 after challenge with either strain WH2012 or Neudoerfl, suggesting that the role of TIM-1 in mediating virus infection was not strain-or subtype-specific. Overall, the enhanced survival and reduced viral burden in the TIM-1-deficient mice suggest that TIM-1 serves as a host factor that mediates TBEV infection in vivo. It is noteworthy that treatment with anti-TIM-1 Abs did not completely inhibit TBEV infection, and virus infection levels were not completely abolished in A549-TIM-1-KO cells. Besides, comparable levels of viral burden could be detected in infected spleens of both TIM-1-deficient and -sufficient mice, indicating that possible alternative entry pathways were present. Hence, TIM-1-mediated entry may be one of the main mechanisms, but not the exclusive means, for entry of TBEV.
In summary, the present findings of this study indicate that TIM-1 serves as a host factor for cellular entry of TBEV. Characterization of the TBEV-TIM-1 interaction will shed light on the early events of virus infection and facilitate the development of strategies to intervene and prevent efficient viral entry.

MATERIALS AND METHODS
Ethics statement. This study was conducted in accordance with the guidance of the Institutional Animal Care and Use Committee of Wuhan Institute of Virology, Chinese Academy of Sciences. All surgeries were performed under general anesthesia, and all efforts were made to minimize the number of animals used and reduce their suffering.
VOPBA and MS analysis. To identify putative molecules on the cell plasma membrane involved in virus binding, VOPBA was carried out as described previously (37,38). Briefly, the membrane-associated proteins of A549 cells were extracted using a ProteinExt mammalian membrane protein extraction kit (TransGen Biotech). A549 cells have been found to support robust growth of TBEV (9), as well as other tick-borne flaviviruses (39). Cell membrane proteins were separated on a 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked overnight at 4°C with 5% skim milk and 0.5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and then incubated with purified TBEV in 1% skim milk in TBS overnight at 4°C. After three rinses with TBS, the membrane was reacted with the anti-flavivirus group-specific MAb 4G2 (Millipore), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Sigma-Aldrich). The VOPBA membrane and the SDS-PAGE gel were aligned, and the area in the gel corresponding to the positive band in the VOPBA was dissected and sent to Wuhan Spec-Ally Biotech Co. Ltd. for LC-MS/MS analysis.
TBEV RNA transfection. The full-length DNA (pTNd/c) was linearized and purified as previously described (41). In vitro transcription was carried out to generate viral genomic RNA using a MEGAscript kit, in the presence of a cap analog (Ambion). After extraction via TRIzol LS reagent (Invitrogen), the RNA was quantified by spectrophotometry and stored at 280°C until use. A549 and A549-TIM-1-KO cells were electroporated with viral genomic RNA, and virus titer was determined by the TCID 50 assay at the time points indicated.
Virus pulldown. Virus particles were incubated overnight at 4°C with 2 mg of Fc-chimera protein TIM-1-Fc (AdipoGen) or IgG1-Fc (Sino Biological) in TBS containing 10 mM CaCl 2 . BSA-saturated protein G Sepharose beads (GE Healthcare) were added and incubated for 4 h at 4°C. Beads were washed with TBS containing 10 mM CaCl 2 and 0.05% Tween, and bound material was resolved in Laemmli buffer under nonreducing conditions, followed by loading onto 10% SDS-PAGE gels and electroblotting onto PVDF membranes. PVDF-bound virus particles were detected with the anti-flavivirus group-specific MAb 4G2, anti-EGFP MAb (Abcam) recognizing fused VP40 of EBOV-VLPs, or anti-matrix protein 1 (M1) of influenza A virus H1N1 (PR8) MAb (Sino Biological), followed by incubation with HRP-conjugated rabbit anti-mouse IgG antibodies (Sigma-Aldrich).
ELISA binding assay. Wells (96-well plates) were first coated with Fc-fused proteins TIM-1-Fc or IgG1-Fc (400 ng/well) in TBS supplemented with 10 mM CaCl 2 overnight at 4°C. Wells were washed with PBS with Tween 20 (PBST) and blocked for 2 h at 37°C with TBS containing 10 mM CaCl 2 and 2% BSA. TBEV particles (5 Â 10 5 TCID 50 ) were added and incubated for 2 h at room temperature (RT), followed by rinses with PBST. The 4G2 MAb was added to the wells and incubated for 2 h at RT. After washes with PBST, the plate was incubated with HRP-conjugated goat anti-mouse IgG antibodies (ProteinTech). After five washes with PBST, the plate was incubated with 3,39,5,59-tetramethyl benzidine (TMB; Boster Biotechnology) for 30 min at RT in the dark. The reaction was stopped by addition of 4N H 2 SO 4 . The intensity of yellow color developed by conversion of the substrate was measured at 450 nm with a microplate spectrophotometer reader (BioTek). PtdSer was detected on bound TBEV virions (2 Â 10 6 TCID 50 ) using anti-PtdSer 1H6 MAb (Millipore) and HRP-conjugated goat anti-mouse MAb.
Flow cytometry assays. TIM-1 surface expression was detected by flow cytometry as described previously (42). Briefly, dispersed cells were incubated with anti-human TIM-1, anti-mouse TIM-1, or the corresponding isotype control MAb (BioLegend) for 30 min at 4°C. Cells were washed and pelleted by centrifugation before resuspension in solution containing Alexa 594-conjugated goat anti-mouse or goat anti-rat IgG antibodies (Abcam). Following incubation for 30 min at 4°C in the dark, the cells were washed twice before flow cytometry analysis (fluorescence-activated cell sorter [FACS] LSRFortessa; BD Biosciences). Intracellular viral antigens were stained with the anti-E 4G2 MAb. Infected cells were fixed and permeabilized by prechilled methanol. Then, cells were incubated with primary 4G2 MAb for 1 h at 4°C, followed by two washes. Cells were then incubated with the secondary Alexa 594-conjugated goat anti-mouse Ab for 30 min at 4°C. Cells were washed twice prior to flow cytometry analysis.
Infection inhibition assay. TBEV incubated with the indicated doses of ANX5 (Abcam) or TIM-1-Fc in serum-free medium was used to infect TIM-1-expressing cells, including 293T-TIM-1 and A549, at a multiplicity of infection (MOI) of 1. Virus titers were assessed 48 h later by the TCID 50 assay. The effect of ANX5 and TIM-1-Fc on cellular viability was measured by the lactate dehydrogenase (LDH) assay as described previously (43,44). Treatment with 10 mL of Triton X-100/well served as a positive control for cytotoxicity.
Indirect immunofluorescence and confocal microscopy. Cells were cultured on 35-mm-diameter plastic culture dishes (Nunc) and incubated with TBEV for 1 h at 4°C, with or without a temperature shift to 37°C for 45 min. Cells were fixed with chilled methanol and incubated with primary goat anti-TIM-1 polyclonal Ab (R&D Systems) and mouse anti-E 4G2 MAb. After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 647conjugated donkey anti-goat IgG (Abcam). The cell nuclei were counterstained with Hoechst 33342. Cells were visualized under a PerkinElmer UltraVIEW VoX live-cell imaging system (PerkinElmer) or a Dragonfly spinning confocal system (Andor Technology).
TIM-1 MAb inhibition of infection assay. Cells were preincubated with media containing the indicated quantities of anti-human TIM-1, anti-mouse TIM-1, or the corresponding isotype control MAb (BioLegend) for 30 min prior to infection. Cells were then infected with TBEV at an MOI of 1 or 5. The presence of antibodies in the cell culture medium was maintained throughout the experiment. Supernatants were collected 48 h later, and virus titers were determined by the TCID 50 assay.
RNA interference knockdown. A549 cells were transiently transfected using the Lipofectamine RNAiMax protocol (Life Technologies) with 10 nM siRNAs, according to the manufacturer's instructions. After 48 h, cells were harvested for Western blot analysis or infected with TBEV at an MOI of 5, and infected cell percentages were quantified 24 h postinfection by flow cytometry. Pools of three siRNAs for TIM-1 and one negative-control siRNA were purchased from Cohesion Biosciences. The effect of siRNAs on cell viability was measured by the LDH assay.
Quantitation of viral RNA uptake. Parental 293T and TIM-1-expressing 293T-TIM-1 cells were incubated with TBEV for 1 h at 4°C, followed by 37°C for 45 min. Cells were then treated with proteinase K (Beyotime; final concentration, 1 mg/mL) for 45 min at 4°C to remove noninternalized virus particles. Total RNA was extracted from infected cells using an Omega HP total RNA isolation kit (Omega Bio-Tek, Inc.). The viral RNA level was determined by real-time quantitative PCR (qRT-PCR) with human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the endogenous control. QRT-PCR was carried out using a HiScript II one-step qRT-PCR SYBR green kit (Vazyme) on a Bio-Rad CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.). The following primers were used: TBEV-F (genome position 7656 to 7675 TIM-1 Serves as a Cellular Entry Factor for TBEV ®