Identification of MYH9 Key Domain Involved in the Entry of PRRSV Into Permissive Cells

Figures

• 

  Figure 1. Generation of HEK-293TCD163, BHK-21CD163, and PK-15CD163cell lines and PRRSV infection. HEK-293T, BHK-21, and PK-15 cells were transduced with the indicated lentiviral constructs, and then these cells were selected and subcloned with puromycin. Each cell line was harvested to assess pCD163 expression. (A) Total RNA of each cell line was extracted and reverse transcribed to amplify the full-length gene coding region for pCD163. GAPDH transcripts were amplified to normalize the total amount of input RNA. (B) The expression levels of pCD163 and PRRSV N proteins were confirmed by Western blot in these cell lines infected with PRRSV SD16 at 48 hpi. (C) The progeny virus particles from PK-15CD163, HEK-293TCD163, and BHK-21CD163 at 48 hpi were titrated with MARC-145 cells. (D) These recombinant cell lines were inoculated with PRRSV SD16, JXA1, GD-HD, and VR-2332 strains at 1 MOI, respectively. PRRSV-specific CPEs were detected at 48 hpi with anti-N antibody (6D10) followed by rhodamine-conjugated goat anti-mouse IgG antibody, respectively. The cells were imaged using a fluorescent microscope; scale bar, 100 μm.

• 

  Figure 2. Knockdown of MYH9 of PK-15CD163, BHK-21CD163, and HEK-293TCD163 cell lines reduce PRRSV replication. PK-15CD163 (A), BHK-21CD163 (B), and HEK-293TCD163 (C) cells were transfected with MYH9 siRNA or NC siRNA, respectively, for 12 h, and then infected with PRRSV strain SD16 (1 MOI) for 48 h. PRRSV replication was measured by mRNA and protein levels using qRT-PCR and Western blot analysis. The data shown are representatives from three independent experiments and subjected to Student's t-test. ***P < 0.001 vs. cells treated without siRNA; ##P < 0.01 vs. cells treated without siRNA; ###P < 0.001 vs. cells treated without siRNA.

• 

  Figure 3. PRRSV infection in recombinant cells was inhibited by blebbistatin. PK-15CD163, BHK-21CD163, and HEK-293TCD163 cells were pretreated with the indicated concentrations of blebbistatin or DMSO for 30 min at 37°C, respectively, followed by incubation with PRRSV SD16 or VR-2332 at 1 MOI in the presence of blebbistatin at a concentration of 0, 10, 20, or 40 μM. These cells were fixed and stained with anti-N protein antibody (6D10) to detect PRRSV at 48 h post-infection. Images were captured using Leica microsystems (Leica AF6000, Germany). Scale bar, 100 μm.

• 

  Figure 4. PRA from mouse, human, and monkey species inhibit PRRSV replication in the MARC-145 and PAM cells. MARC-145 cells (A) or PAMs (B) were infected with PRRSV SD16 at 0.1 MOI pre-incubated with indicated PRA protein (1, 2.5, and 5 μM), respectively, and the mRNA and protein levels of PRRSV N gene were measured at 24 hpi by qPCR and Western blot. The data shown are representatives from three independent experiments and subjected to one-way ANOVA. *P < 0.05 vs. cells treated with 0 μM PRA; **P < 0.01 vs. cells treated with 0 μM PRA; and ***P < 0.001 vs. cells treated with 0 μM PRA.

• 

  Figure 5. The key domain of MYH9 for PRRSV inhibition in MARC-145 cells. (A) Schematic diagram of truncation fragments located in the C-terminal of MYH9. MARC-145 cells were inoculated with the mixture of PRRSV 0.1 MOI and truncated protein of PRA (2.5 μM), and the key domain of MYH9 for anti-PRRSV was determined using qPCR (B) and Western blot (C–F). The data shown are representatives from three independent experiments and subjected to one-way ANOVA. ***P < 0.001 vs. PCV2-Cap protein treated cells.

• 

  Figure 6. The blocking effect of MYH91676−1791 against PRRSV SD16 infection in PAMs. PAMs were infected with PRRSV SD16 at 0.1 MOI pre-incubated with MYH91676−1791 (2.5, 5, and 10 μM), respectively, and the mRNA (A) and protein levels (B) of PRRSV N gene were measured by qPCR and Western blot. The data shown are representatives from three independent experiments and subjected to one-way ANOVA. **P < 0.01 vs. 0 μM MYH91676−1791 treated cells; ***P < 0.001 vs. 0 μM MYH91676−1791 treated cells.

• 

  Figure 7. The soluble MYH91676−1791 against PRRSV-2 isolates in MARC-145 and PAM cells. MARC-145 cells (A) or PAMs (B) were infected with PRRSV-2 isolates (JXA1, GD-HD, CH-1a, and VR-2332) at 0.1 MOI pre-mixed with MYH91676−1791 (5 μM), and the mRNA and protein levels of N gene were measured by qPCR and Western blot. The data shown are representatives from three independent experiments and subjected to Student's t-test. **P < 0.01 vs. the cells infected with the same virus and treated with PBS; ***P < 0.001 vs. the cells infected with the same virus and treated with PBS.

• 

  Figure 8. The soluble MYH91676−1791 against PRRSV-1 isolates in PAM cells. MYH91676−1791 (5 μM) was pre-mixed with indicated PRRSV-1 strains (MOI = 0.1) at 37°C for 1 h before infecting PAM cells. Relative PRRSV GZ11-G1 (A) and PRRSV P073-3 (B) N gene mRNA levels were monitored via qPCR at 24 hpi. The data shown are representatives from three independent experiments and subjected to Student's t-test. ***P < 0.001 vs. 0 μM MYH91676−1791 treated cells. Gene copy numbers of PRRSV GZ11-G1 (C) and PRRSV P073-3 (D) N in cell culture supernatant at indicated MYH91676−1791 concentrations of treatment were determined. The data shown are representatives from three independent experiments and subjected to Student's t-test. $$$P < 0.001, in comparison to 0 μM MYH91676−1791 treated cells.

• 

  Figure 9. The polyclonal anti-MYH91676−1791 serum reduces PRRSV infection in MARC-145 and PAM cells. MARC-145 (A) or PAM (B) cells were infected with PRRSV SD16 at an MOI of 0.1 in the presence of various concentrations of mouse anti-MYH91676−1791 polyclonal serum or mouse negative serum, and relative changes in the mRNA level of PRRSV N gene were determined by qPCR at 24 hpi. The data shown are representatives from three independent experiments and subjected to one-way ANONA. *P < 0.05 vs. cells treated with mouse negative serum, ***P < 0.001 vs. cells treated with mouse negative serum. (C) IFA assay results confirmed the PRRSV inhibition in MARC-145 and PAM cells with anti-MYH91676−1791 polyclonal serum (1:20) treated at 24 hpi (left). Scale bar, 100 μm. Histograms represent the percentage of PRRSV-positive cells, performed using ImageJ software (right). The data shown are representatives from three independent experiments and subjected to Student's t-test. &&P < 0.01, &&&P < 0.001.