Fig 2: MERTK promotes CSFV entry. (A) Anti-MERTK antibodies decrease CSFV entry. PK-15 cells were incubated with anti-MERTK antibodies or isotype IgG and were incubated with CSFV-SM (MOI=0.1 or 1) for 2 h in the continuous presence of the antibodies at 37°C for virus entry. Then the cells were subjected to analyze CSFV entry. (B) Soluble MERTKED-His reduces CSFV entry. CSFV-SM (MOI=0.1 or 1) were preincubated with soluble MERTKED-His or BSA for 30 min at room temperature then used to infect PK-15 cells. The infected cells were incubated at 37°C for 2 h. RT-qPCR was performed to determine CSFV genome copies relative to GAPDH. (C) Downregulation of MERTK reduces CSFV entry. PK-15 cells were transfected with MERTK targeting siRNA or siNC and then were analyzed for CSFV entry. Means ± standard deviations (SD) of three technical replicates are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3: MERTK plays a key role in CSFV infection. (A and B) Silencing of MERTK dramatically reduced CSFV infection. PK-15 cells were treated with siRNAs target to MERTK transcript, and then were infected with CSFV-SM (MOI=0.01) for 48 h, the viral genome copies were quantified (A) and virus titers of supernatants were determined (B) at 48 hpi. (C) Immunoprecipitation assay confirmed that the endogenous MERTK protein expression was decreased in siMERTK treated cells. (D) PK-15 cells were transfected with siMERTK or siNC for 48 h, and were incubated with CSFV-SM (MOI=0.01) for 48 h. Immunofluorescence with pig anti-CSFV sera (green) and DAPI (blue) (E) MERTK expose to the surface of PK-15 cells. The cells were stained with anti-MERTK antibodies or isotype IgG, and then were incubated with secondary antibodies and followed by flow cytometric analysis of 10,000 cells per sample. (F) Overexpression of MERTK enhances CSFV infection. PK-MERTK-Myc or PK-ZsGreen1 cells were infected with CSFV-SM (MOI=0.01), the CSFV genomic replication was determined at 24 hpi. Insets display overexpression of MERTK-Myc. Means ± standard deviations (SD) of three technical replicates are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: Soluble MERTK ectodomain reduces BVDV infection in a dose-dependent manner. (A) BVDV-C24 V (MOI=0.01) was incubated with the indicated concentrations of soluble MERTKED-His, or BSA for 30 min at room temperature, and then was incubated with MDBK cells. Total RNA of the cells was extracted at 24 hpi, and relative viral RNA was detected by RT-qPCR. (B) The PRV-TJ (MOI=0.01) was preincubated with various concentrations of soluble MERTKED-His or BSA for 30 min and inoculated into PK-15 cells. Relative PRV genome was determined by qPCR. Mean ± standard deviations (SD) of three technical replicates are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5: Anti-MERTK antibodies or soluble MERTK ectodomain blocks CSFV infection in a dose-dependent manner. (A and B) Antibodies against MERTK reduce CSFV infection. PK-15 cells were pretreated with antibodies against MERTK or isotype IgG at indicated concentrations for 30 min, and then infected with CSFV-SM (MOI=0.01) in the continuous presence of the antibodies. The viral genome copies (A) and progeny viral titers (B) were measured at 48 hpi. (C) Identification of purified MERTKED-His and E2ED-His proteins stained with Coomassie blue. (D) MERTKED-His has no effects on the cell viabilities. (E and F) Soluble MERTKED-His reduces CSFV infection. CSFV-SM (MOI=0.01) was incubated with the indicated concentrations of soluble MERTKED-His or BSA for 30 min at room temperature, and then infected with PK-15 cells. The viral genome copies (E) and progeny viral titers (F) were measured at 48 hpi. (G and H) Anti-MERTK antibodies reduce CSFV-HLJ infection in a dose-dependent manner. PK-15 cells were infected with CSFV-HLJ (MOI=0.01) after incubation with anti-MERTK antibodies for 30 min. The viral genome copies (G) and progeny viral titers (H) were examined at 48 hpi. Means ± standard deviations (SD) of three technical replicates are shown. **, P < 0.01; ***, P < 0.001.