Porcine Reproductive and Respiratory Syndrome Virus Utilizes Viral Apoptotic Mimicry as an Alternative Pathway To Infect Host Cells

PRRS has caused huge economic losses to pig farming worldwide. Its causative agent, PRRSV, infects host cells through low pH-dependent clathrin-mediated endocytosis and CD163 is indispensable during the process. Whether there exist alternative infection pathways for PRRSV arouses our interest. Here, we found that PRRSV exposed PS on its envelope and disguised as apoptotic debris. The PS receptor TIM-1/4 recognized PRRSV and induced the downstream signaling pathway to mediate viral infection via CD163-dependent macropinocytosis. The current work deepens our understanding of PRRSV infection and provides clues for the development of drugs and vaccines against the virus.

Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in global swine industry since it was first reported in the United States in 1987 (11,12). Currently, loss due to PRRS in the United States is annually estimated $664 million (13). Caused by PRRS virus (PRRSV), the syndrome is characterized by reproductive failures in the late-term gestation of sows and respiratory diseases in pigs of all ages (14). PRRSV is an enveloped single-stranded positive-sense RNA virus with a genome of approximately 15 kb (15,16). All PRRSV isolates are classified into two genotypes, PRRSV-1 and PRRSV-2 (17), which belong to the order Nidovirales, family Arteriviridae, and genus Porartevirus (18).
In the present work, we determined an alternative pathway utilized by PRRSV to infect host cells. First, we found that PRRSV exposed PS on the envelope as viral apoptotic mimicry. Next, we dissected the host cell PSRs recognizing PRRSV as apoptotic mimicry and explored the detailed mechanisms, including the downstream signaling pathways and invasion routes.

RESULTS
PRRSV externalizes PS on the envelope as viral apoptotic mimicry. In order to validate whether PRRSV incorporates PS on its envelope and utilizes viral apoptotic mimicry, we first detected PS on the virions using annexin V, a specific PS-binding protein (28), by flow cytometry (FCM). As shown in Fig. 1A, a typical PRRSV-2 strain, BJ-4, was externalized PS on the envelope. Furthermore, we exploited a commercial antibody against PS (29), a specific antibody against PRRSV major envelope glycoprotein (GP) 5 (30), and dot blotting to confirm that PRRSV BJ-4 did expose PS (Fig.  1B). In addition, highly pathogenic PRRSV (HP-PRRSV) strain HN07-1 and PRRSV-1 strain GZ11-G1 also externalized PS on the envelopes (Fig. 1C to F). All of these results demonstrate that PRRSV incorporates PS on the envelope surface as viral apoptotic mimicry. Since PRRSV-2 strains are predominantly prevalent and PRRSV-1 strains are sporadic in China (31), we only applied PRRSV BJ-4 to the following research.
TIM-1 is identified to recognize PRRSV as apoptotic mimicry in MARC-145 cells. PSRs TIM-1, Stabilin-1/2, and Axl are specially expressed in epithelial cells (7). Here, we sought to identify which host PSRs recognized PRRSV as apoptotic mimicry. Initially, we monitored the transcription of each PSR in MARC-145 cells. Figure 2A shows that TIM-1 and Axl were transcribed in the cells. Expression of TIM-1 and Axl were also demonstrated through immunoblotting (IB) analysis (Fig. 2B). To investigate their specific functions during PRRSV infection, knockdown of TIM-1 and Axl were carried out in MARC-145 cells (Fig. 2C). To measure total PRRSV RNA, a pair of internal primers in the viral open reading frame 7 (ORF7) gene were used to amplify all subgenomic mRNA and genomic RNA by quantitative real-time PCR (RT-qPCR) (32,33). Axl knockdown did not significantly influence the abundance of PRRSV RNA (Fig. 2D). In contrast, PRRSV infection was suppressed, as indicated by decreased viral RNA abundance (3.5-fold) at 12 h postinfection (hpi), infectivity with nucleocapsid (N) protein expression (5-fold) at 24 hpi, and progeny viral titers (2.5-fold) at 48 hpi ( Fig. 2D to F) in the TIM-1 knockdown cells. Figure 2G further shows that knockdown of TIM-1 influenced PRRSV infection during viral binding to MARC-145 cells (4-fold).
Next, we determined whether TIM-1 directly bound to PRRSV. In vitro Fc-pulldown assay with recombinant Fc-fused TIM-1 (TIM-1-Fc) and purified PRRSV indicated that TIM-1 bound to the virions (Fig. 3A). Dot blot analyses further confirmed that TIM-1 interacted with PRRSV (Fig. 3B). Incubation with recombinant TIM-1-Fc showed an interference of PRRSV binding to MARC-145 cells with decreased viral RNA (2.5-fold; Fig.  3C). A blocking experiment using the anti-PS antibody also showed a significant Knockdown of TIM-1 (C) significantly influenced PRRSV RNA abundance (D), infectivity (E), progeny viral titers (F), and viral binding (G). MARC-145 cells were transfected with siTIM-1, siAxl, or siRNA-NC for 36 h and infected with PPRSV (MOI ϭ 10). The infected cells were collected for analyses of PRRSV RNA by RT-qPCR at 12 hpi, N protein expression by immunofluorescence at 24 hpi, viral titers by determining the TCID 50 at 48 hpi, or binding and entry by RT-qPCR. Immunofluorescence images were quantified by counting the number of cells expressing viral N protein. Four random fields were counted per each condition, and the total number of cells per field was determined by DAPI staining. Each experiment was performed three times, and similar results were obtained. Differences between groups were assessed by using a Student t test, and the statistical significance is indicated (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ns, not significant). Scale bars, 50 m. . For the Fc-pulldown assay, the recombinant TIM-1-Fc was bound to protein A/G-beads, whereas the Fc tag served as control. The beads were then incubated with the PRRSV virions. The eluted proteins were then subjected to IB. For dot blotting, TIM-1-Fc or Fc was spotted onto nitrocellulose membranes and detected by anti-human IgG antibody or anti-PRRSV GP5 MAb. After incubation with PRRSV BJ-4 for 4 h, the membranes were eaxmined for PRRSV GP5 detection. For viral binding interference, PRRSV BJ-4 virions were incubated with TIM-1-Fc (5 g) or Fc at 4°C for 4 h, followed by inoculation into MARC-145 cells at 4°C for 1 h. For the blocking experiment, PRRSV BJ-4 virions were pretreated with anti-PS antibody or isotype control antibody at 4°C for 4 h and then inoculated into MARC-145 cells at 4°C for 1 h. The PRRSV RNA was determined by RT-qPCR. Each experiment was performed three times independently. Statistical analysis for the RT-qPCR was carried out using the Student t test (**, P Ͻ 0.01). decrease in PRRSV binding to the cells (3-fold; Fig. 3D), suggesting the specific interaction.
Taken together, these data provide evidence that TIM-1 recognizes and interacts with PRRSV as apoptotic mimicry in MARC-145 cells.
PRRSV induces macropinocytosis via TIM-1 in MARC-145 cells. As apoptotic mimicry, viruses infect host cells via CME and/or macropinocytosis (34,35). To distinguish which routes were involved in PRRSV infection mediated by TIM-1, we performed confocal microscopy in combination with transferrin or dextran during early infection (i.e., at 30 min postinfection [mpi]) in MARC-145 cells. Transferrin is a marker for CME (36), and dextran is a fluid-phase marker which is robustly internalized during macropinocytosis (37). As shown in Fig. 4A, knockdown of TIM-1 greatly influenced macropinocytosis (3.5-fold) but not CME, suggesting that PRRSV induced macropinocytosis via TIM-1. To support this conclusion, we conducted the assay once again with dextran using confocal microscopy for different time periods. In Fig. 4B and C, PRRSV induced macropinocytosis at as early as 15 mpi, while TIM-1 knockdown inhibited PRRSVinduced macropinocytosis (3-to 7-fold). We further demonstrated this conclusion using confocal microscopy simultaneously with the specific antibody against PRRSV N protein and dextran at 30 mpi, which also indicated their colocalization during the process (Fig. 4D).
Macropinocytosis is distinct from other endocytic pathways in extensive actin rearrangements and the formation of protrusions on cellular surfaces (9,10). To determine whether PRRSV induces membrane protrusions, we performed confocal microscopy with phalloidin and monitored actin restructuring. Phalloidin specially binds to the polymerized form of actin (38). As shown in Fig. 4E, PRRSV infection led to depolymerization and distribution changes of actin. More actin-driven membrane protrusions were observed on cell surfaces of MARC-145 cells than that of mockinfected cells (Fig. 4E, white arrows). However, there were decreased membrane protrusions in TIM-1 knockdown MARC-145 cells.
These results illustrate that PRRSV induces macropinocytosis via TIM-1 in MARC-145 cells.
PRRSV utilizes macropinocytosis to infect MARC-145 cells. We analyzed whether PRRSV utilized macropinocytosis to infect MARC-145 cells. We first observed that internalized PRRSV virions colocalized with sorting nexin 5 (SNX5), a marker of specific endosomes for macropinocytosis (macropinosomes, Fig. 5A) (39). The colocalization coefficient was expressed as Manders' overlap coefficient, and the value was Ͼ0.6, indicating an actual overlap of the signals and representing the true degree of colocalization (40). Ethylisopropyl amiloride (EIPA) specifically inhibits Na ϩ /H ϩ exchanger activity and subsequent macropinocytosis (41). As shown in Fig. 5B to E, treatment with EIPA, compared to treatment with dimethyl sulfoxide (DMSO), led to 2to 4-fold reductions in PRRSV RNA abundance, a 5-fold decrease in infectivity, and a 5-fold decrease in viral titers, respectively. Interestingly, the EIPA inhibited PRRSV infection during viral entry rather than binding (Fig. 5F). Since PRRSV infection was previously reported to be mediated by CME (22), we attempted to define the relative contribution of CME and macropinocytosis in PRRSV infection. We pretreated MARC-145 cells with chlorpromazine (CPZ), an inhibitor of clathrin lattice polymerization (42). Treatment with CPZ resulted in a greater decrease in PRRSV RNA abundance than treatment with EIPA (6.3-fold versus 3.5-fold). Simultaneous addition of these two inhibitors almost abolished PRRSV infection (Fig. 5G). All of these results indicate that, in addition to CME, PRRSV infects MARC-145 cells via macropinocytosis as an alternative pathway.

Disruption of actin dynamics inhibits PRRSV infection via macropinocytosis.
Since macropinocytosis requires actin rearrangements (9, 10), we explored whether the disruption of actin dynamics took effect on PRRSV infection. We preincubated MARC-145 cells with cytochalasin D (Cyto D) and latrunculin A (Lat A), respectively, and then inoculated with PRRSV. Cyto D disrupts actin microfilaments (43) and Lat A inhibits actin  (44). As shown in Fig. 6, both inhibitors significantly suppressed PRRSV infection during viral entry in a dose-dependent manner.
PRRSV utilizes macropinocytosis to infect PAMs. Since PAMs are primary in vivo target for PRRSV (19) and undergo constitutive macropinocytosis (45), we determined whether PRRSV utilizes this alternative pathway to infect the cells. TIM-4 is a homolog of TIM-1 specially expressed in macrophages (7). We determined that TIM-4 directly bound to PRRSV as TIM-1 (Fig. 8A). TIM-4 knockdown played a significant inhibitory effect on PRRSV infection (2-to 2.5-fold, Fig. 8B and C). The impacts of EIPA on PRRSV infection were also demonstrated ( Fig. 8D and E). The Rac1/Cdc42-Pak1 signaling pathway was involved in PRRSV infection in PAMs as well (Fig. 8F). These results show that macropinocytosis is utilized by PRRSV in both PAMs and MARC-145 cells.

CD163 is essential for PRRSV infection via TIM-induced macropinocytosis.
It is well established that CD163 is an indispensable receptor for PRRSV infection (24,27). We considered the involvement of CD163 and TIMs in PRRSV infection via macropinocytosis. Figure 9A and B show that expression of TIM-4 alone in baby hamster kidney 21 (BHK-21) cells was not sufficient to support PRRSV infection. In contrast, the expression of CD163 alone conferred susceptibility to PRRSV infection, consistent with a previous study (24). Importantly, the coexpression of TIM-4 and CD163 contributed to PRRSV infection more than did the expression of CD163 alone. Furthermore, CD163 colocalized with PRRSV in SNX5-marked macropinosomes in PAMs, whereas most TIM-4s did not (Fig. 9C). Consequently, CD163 is essential during PRRSV infection via TIM-induced macropinocytosis.

DISCUSSION
Viruses usually exploit multiple strategies to infect host cells and establish infection (1,2,35). Viral apoptotic mimicry has been documented for many enveloped viruses to facilitate viral infections, including alphaviruses, flaviviruses, filoviruses, some arenavi- containing dextran (final concentration, 250 g/ml) and transferrin (final concentration, 10 g/ml) and transferred to 37°C for 30 min. The cells were then fixed, and the nuclei were stained with DAPI. Images were acquired with the same confocal microscope settings. The total fluorescence intensity of transferrin or dextran was calculated using ImageJ software. ***, P Ͻ 0.001; ns, not significant.    (7). In particular, TIMs serve as entry factors or even receptors for dengue virus (29) and Ebola virus (50,51). It is also worth noting that not all PSRs enhance viral entry. For example, the PSRs Stabilin-1/2 and BAI1 do not appear to enhance viral entry (52). Here, we determined that TIM-1/4 interacted with PRRSV virions (Fig. 3 and 8) and induced macropinocytosis (Fig. 4) upon viral infection, whereas Axl did not (Fig. 2). However, TIM-4 was not sufficient to support PRRSV infection and internalized into macropinosomes in PAMs as CD163 (Fig. 9). Consequently, we assume that TIM-1/4 might only function as an attachment factor for PRRSV and inducer of downstream signaling of macropinocytosis. The detailed mechanisms involved in TIM-PRRSV interaction and TIM induction need to be investigated.
Macropinocytosis is usually induced by external stimuli, which may be associated with growth factors-triggered activation of receptor tyrosine kinases (9). Among them, epidermal growth factor receptor (EGFR) has been demonstrated to induce macropinocytosis (53). Although the EGFR-PI3K signaling pathway has been recently reported to be required for actin reorganization and efficient PRRSV entry into MARC-145 cells (54,55), whether PRRSV induces and utilizes macropinocytosis to infect host cells has not been clarified.
To exclude the interference of growth factors and EGFR, we utilized purified PRRSV virions and serum-starved cells throughout our research unless stated otherwise. Here, we found that PRRSV strains externalized PS on their envelopes as viral apoptotic mimicry (Fig. 1). The specific mechanisms by which PRRSV incorporates and exposes PS are our next issue to be studied. Subsequently, we identified that PRRSV is recognized as apoptotic mimicry by TIM-1/4 (Fig. 2, 3, and 8). Moreover, we determined that PRRSV induced macropinocytosis via TIM-1 in MARC-145 cells and utilized the pathway to infect both MARC-145 cells and PAMs (Fig. 4, 5, and 8). All of these results concluded that PRRSV directly induces macropinocytosis via TIM and infects host cells via the pathway. It would be interesting to investigate whether PRRSV exploits other strategies to induce macropinocytosis. We tried to address the individual contribution of macropinocytosis and CME to PRRSV infection and found that CME might contribute more than macropinocytosis during PRRSV infection (Fig. 5). It would be meaningful to define this contribution under actual in vivo conditions in the future.
Other factors, including protein kinase C (PKC) (58) and myosin II (59), are also responsible for macropinocytosis. A recent work has demonstrated that PKC is beneficial to PRRSV replication and infection (60). In addition, nonmuscle myosin IIA, encoded by myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection (61). The cells were collected for assessment of PRRSV RNA abundance by RT-qPCR at 12 hpi. Each experiment was performed three times, and similar results were obtained. Differences between groups were assessed by using a Student t test, and the statistical significance is indicated (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ns, not significant).
Mechanistically, blebbistatin, an inhibitor of myosin II heavy chain activity (62), impairs the viral entry (61). All of these reports strengthen our conclusion that PRRSV utilizes macropinocytosis to infect host cells.
Based on the results stated above, we propose a model to depict PRRSV infection (Fig. 10). In addition to CME, TIM-1/4 recognizes PRRSV as apoptotic mimicry and induces macropinocytosis via the Rac1/Cdc42-Pak1 signaling pathway. PRRSV enters SNX5-macropinosomes via macropinocytosis as an alternative pathway, where CD163 functions as an indispensable receptor.
In conclusion, we demonstrate that PRRSV utilizes viral apoptotic mimicry and TIM-induced macropinocytosis as an alternative pathway to infect host cells. The results we obtained deepen our understanding of PRRSV complicated infection and provide novel opportunities for the development of drugs and vaccines against PRRSV. Each experiment was performed three times, and similar results were obtained. Differences between groups were assessed by using a Student t test, and the statistical significance is indicated (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ns, not significant).  10). The dextran uptake was detected at 30 mpi as stated above. Scale bars, 10 m. The total fluorescence intensity of dextran was calculated using ImageJ software. The infected cells were collected to assess PRRSV RNA abundance by RT-qPCR at 12 hpi, and viral titers were determined from the TCID 50 at 48 hpi. Inhibition of Rac1, Cdc42, and Pak1 all decreased PRRSV RNA abundance (F) and progeny viral titers (G). The serum-starved MARC-145 cells were pretreated with IPA-3 (10 M), nsc23766 (50 M), LY294002 (20 M), pirl-1 (10 M), or DMSO and infected with PRRSV BJ-4 (MOI ϭ 10) for 1 h. The cells were collected for assessment of PRRSV RNA abundance by RT-qPCR at 12 hpi, and viral titers were determined from the TCID 50 at 48 hpi. Each experiment was performed three times, and similar results were obtained. Differences between groups were assessed by using a Student t test, and the statistical significance is indicated (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ns, not significant). The cells were collected for assessment of PRRSV RNA abundance by RT-qPCR at 12 hpi. Each experiment was performed three times, and similar results were obtained. Differences between groups were assessed by using a Student t test, and the statistical significance is indicated (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001). Scale bars, 50 m.

Cells and viruses.
PAMs were prepared from lung lavage of 4-week-old specific-pathogen-free pigs. The experimental procedure for the collection of PAMs was authorized and supervised by the Ethical and Animal Welfare Committee of Key Laboratory of Animal Immunology of the Ministry of Agriculture of China (permit 2017008). PAMs were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA), penicillin (100 U/ml; Gibco), and streptomycin (100 mg/ml; Gibco) in a humidified 37°C, 5% CO 2 incubator. MARC-145 and BHK-21 cells were purchased from Cellbio (Shanghai, China) and maintained in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated FBS and penicillin-streptomycin.
Purified virions in PBS were spotted onto nitrocellulose membranes (Pierce, Rockford, IL), and PBS was spotted onto membranes as a negative control. Membranes were dried and blocked with 5% bovine serum albumin (BSA) in PBS at 4°C overnight. Next, the membranes were incubated with the anti-PS MAb or anti-PRRSV GP5 MAb as a viral loading control for 1 h at 37°C. After three washes with PBS plus Tween Detection of PSR transcription. MARC-145 cells were collected, and the total RNAs were extracted using TRIzol reagent (Invitrogen). The reverse transcription cDNAs were prepared by using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer's instructions. The cDNAs were then subjected to PCR with specific primers for TIM-1, Axl, Stabilin-1, and Stabilin-2 ( Table  1). The PCR products were subjected to agarose gel electrophoresis.
RT-qPCR. Total RNAs were extracted using TRIzol reagent, and the reverse transcription cDNAs were prepared as described above. Then, RT-qPCR was performed using FastStart Universal SYBR green Master (Rox, catalog no. 4913850001; Roche, Basel, Switzerland) on a 7500 Fast RT-PCR system (Applied Biosystems, Foster City, CA). A plasmid containing PRRSV ORF7 was used as the template to generate a standard curve, and the actual viral RNA copies were calculated based on this curve (66). The relative RNA level was evaluated by the 2 -ΔΔCT method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an endogenous control (67). The primers for RT-qPCR are listed in Table 1.
IB. Cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with a cocktail of protease inhibitors (Roche). The lysates were separated by 10% to 15% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked in 5% skim milk for 1 h and probed with the indicated primary antibodies. After incubation with HRP-labeled goat anti-mouse or rabbit IgG antibody as a secondary antibody, the indicated proteins were detected by ECL Plus reagent.
RNA interference. All siRNAs and siRNA negative controls (siRNA-NC) were designed and synthesized by GenePharma (Shanghai, China). In knockdown experiments, PAMs or MARC-145 cells were transfected with the indicated siRNAs at a final concentration of 10 nM using Lipofectamine RNAiMAX according to the manufacturer's instructions for 36 h. After the cell viability was measured by the CellTiter 96 AQueous One Solution assay kit (data not shown), transfected cells were applied for subsequent experiments. The indicated siRNAs are listed in Table 2.
Virus titration assay. The treated cells were inoculated with PRRSV at an MOI of 10 and incubated at 37°C for 3 h. The viruses not entering the cells were then washed away. At 48 hpi, the viral yields were measured by determining a 50% tissue culture infected dose (TCID 50 ) assay in MARC-145 cells (68).
Binding and entry assay. Cells were serum starved for 1 h at 37°C and inoculated with purified virions at an MOI of 10 for 1 h at 4°C, allowing for viral attachment without internalization. The cells were then washed with cold PBS three times so that unbound viruses were removed. The culture medium was replaced with fresh serum-free medium, and the cells were subsequently shifted to 37°C, allowing virus internalization. After 1 h, the cells were washed with citrate buffer solution (pH 3.0) to remove the noninternalized visions and washed with PBS three times. PRRSV RNA abundance was determined by RT-qPCR.
Pulldown assay. The recombinant Fc-fused TIM-1/4 was first bound to protein A/G-beads (Pierce) at 4°C for 4 h. PRRSV virions were subsequently incubated with the beads at 4°C overnight. After extensive washing with PBS, the target proteins were eluted and subjected to IB with the indicated antibodies.
Dot blot assay. TIM-1/4-Fc proteins were spotted onto nitrocellulose membranes. Membranes were dried and blocked with 5% BSA in PBS 4°C overnight. Next, membranes were incubated with the PRRSV virions at 4°C for 4 h. After three washes with PBST, the membranes were incubated with the indicated primary antibodies and detected with HRP-conjugated antibodies.
Confocal microscopy. Cells were grown in 24-well plates on glass coverslips, fixed with 4% paraformaldehyde (PFA) for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Phalloidin (dilution 1:100) or DAPI (4=,6=-diamidino-2-phenylindole) was used to stain actin filaments and nuclei, respectively. Alternatively, cells were stained with the appropriate primary and secondary antibodies. Coverslips were mounted to the glass slides and examined by using a microscope (LSM700; Carl Zeiss AG, Oberkochen, Germany) with the confocal laser scanning setup (20ϫ, 40ϫ, or 63ϫ  (69). Colocalization analyses were carried out according to the method of Zinchuk and Grossenbacher-Zinchuk (40). Manders' overlap coefficient (Ͼ0.6) shows an actual overlap of the signals and is considered to represent the true degree of colocalization. Quantitative analyses of single-channel fluorescence were performed using ImageJ software (70,71). FITC-dextran uptake. MARC-145 cells were grown in 24-well plates on glass coverslips. Prior to FITC-dextran uptake, the cells were serum starved for 2 h. FITC-dextran was incubated with the cells (final concentration, 0.25 mg/ml) in the absence or presence of PRRSV virions at an MOI of 10 for different time periods (15,30, and 45 min). The cells were then washed three times with cold PBS on ice and once with low-pH buffer (0.1 M sodium acetate, 0.05 M NaCl [pH 5.5]), fixed with 4% PFA in PBS, and subsequently permeabilized with 0.1% Triton X-100 in PBS. After mounting, the slides were examined by confocal microscopy.
Cytotoxicity of inhibitors. MARC-145 cells or PAMs were seeded onto 96-well plates and pretreated with indicated inhibitors at 37°C for 4 h. The cell viability was then measured using a CellTiter 96 AQueous One Solution cell proliferation assay. Briefly, the assay was performed by adding CellTiter 96 AQueous One Solution reagent to wells, followed by incubation for 4 h. The absorbance at 490 nm was then recorded with a Bio-Rad microplate reader (data not shown).
Inhibitor treatments. MARC-145 cells and PAMs were serum starved for 1 h and treated with noncytotoxic specific inhibitors or DMSO for 1 h at 37°C in serum-free medium before subsequent experiments.
Plasmid transfection. The optimized cDNA encoding porcine TIM-4 was cloned into the vector pECMV-MCS-FLAG (kept in our laboratory). A construct with complete porcine CD163 cDNA integrated into the PiggyBac transposon system was kindly provided by Enmin Zhou (Northwest Agriculture and Forestry University, China) (72). BHK-21 cells were seeded at a density of 4 ϫ 10 5 cells/ml culture medium overnight. The cells were then transfected with TIM-4 and/or CD163 plasmid using Lipofectamine LTX with Plus reagent according to the manufacturer's instructions. The protein expression was tested by IB as stated above.
Statistical analysis. Three replicates were included in all experiments, and each experiment was independently performed three times. The experimental data are presented as group means and standard deviations (SD) and were analyzed by the unpaired two-tailed Student t test with GraphPad Prism software (v7.0). Statistical significance is indicated in the figures (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ns, not significant).

ACKNOWLEDGMENTS
We thank Hanchun Yang from China Agricultural University for providing PRRSV-2 strain BJ-4 and PRRSV-1 strain GZ11-G1 and Enmin Zhou from Northwest Agriculture and Forestry University for providing the PiggyBac transposon system. This study was supported by grants from the National Natural Science Foundation of China (31972690), the Science-Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (2020YQ01), the Earmarked Fund for Modern Agro-industry Technology Research System of China (CARS-35), and the Special Fund for Henan Agriculture Research System (S2012-06). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
G.Z. and R.L. jointly supervised the work. G.Z., R.L., and X.W. designed the experiments. X.W. performed the studies. X.W. and R.L. analyzed the data and wrote the manuscript. X.W., R.L., S.Q., and X.-X.C. revised the manuscript. G.X. provided technical assistance.
We declare there are no conflicts of interest.