Identification of New Compounds against PRRSV Infection by Directly Targeting CD163

Abstract

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (K_D) values ranging from 28 to 39 μM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Keywords: CD163; PRRSV; SRCR5; antiviral treatment; small molecule compound.

FIG 1

Screen of new compounds that block PRRSV GP2a/GP4-CD163 interaction. (A) Scheme diagram for the BiFC assay constructs between CD163-SRCR5 or PRRSV envelope proteins GP2a/GP4 and the fragments of Venus protein VN155(I152L) or VC155, respectively. (B) BiFC screening images for select positive compounds that inhibit the PPI between SRCR5-VN and GP2a-VC proteins. NC, DMSO Ctrl. Bar = 250 μm. (C) Relative fluorescence intensity of the SRCR5/GP2a BiFC screening results for all 20 positive compounds. Mean ± SD; n = 3. (D) Comparison for the relative fluorescence intensity of the SRCR5/GP2a and SRCR5/GP4 BiFC screening results for the 20 positive compounds. Mean ± SD; n = 3. (E) BiFC screening images for select positive compounds that inhibit the PPI between SRCR5-VN and GP4-VC proteins. NC, DMSO Ctrl. Bar = 250 μm. (F) Chemical structure of the 5 identified strong positive compounds by both BiFC assay.

FIG 2

The Combined GP2a/SRCR5 and GP4/SRCR5 BiFC assays accurately predict potent PRRSV-inhibitory compounds. (A) MTT analysis to the 20 positive compounds in PAMs. NC, DMSO Ctrl. Mean ± SD; n = 3. (B) qRT-PCR for PRRSV in total RNAs extracted from infected PAMs treated with various compounds. Values are normalized with GAPDH of PAMs, and the average of three strains (NADC30, VR-2332, and Lelystad) are plotted. Bars = mean ± SD; n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Titration assay results for PRRSV in the culture media of PAMs treated as described in panel B. Bars = mean ± SD; n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Relative fluorescence intensity of the SRCR5/GP2a BiFC assay for B7, B7-A4, E10, F9, and F12 at different concentrations. Mean ± SD; n = 3. P values are calculated by one-way ANOVA and letters on the top of bars indicate significant differences in Tukey post hoc test. (E) MTT analysis to B7, B7-A4, E10, F9, and F12 at different concentrations in PAMs. NC, DMSO Ctrl. Mean ± SD; n = 3. (F) Titration for PRRSV in the culture media from infected PAMs treated with B7, B7-A4, E10, F9, and F12 at different concentrations. Bars = mean ± SD; n = 3. P values are calculated by one-way ANOVA and letters on the top of bars indicate significant differences in Tukey post hoc test.

FIG 3

Validating of physical interactions between the compounds and SRCR5. (A) Molecular docking (left) and PLIP (right) analyses depicting two potential interaction sites between compounds B7 and CD163-SRCR5 domain. (B) Residues in CD163-SRCR5 with potential interactions with B7 (yellow highlighted), residues forming potential hydrogen bonds with B7 are marked with green. The amino acids in the LBP and loop 5–6 regions are underlined. (C) Two-dimensional ligand-protein interaction diagram by LigPlot+ showing the hydrogen bond and hydrophobic interactions of B7 with SRCR5. (D) Molecular docking analyses by ZDOCK depicting the potential conformation of PPIs between SRCR5 with GP4 (left) or GP2a (right). B7 was placed in position 1 (boxes) to show the potential disturbing of both conformations. (E) Molecular docking analyses by ZDOCK depicting the PPI conformation between SRCR5 with both GP4 and GP2a. B7 was placed in position 2 (box) to show the potential disturbing of this conformation. (F) MST analysis of GFP-fused SRCR5 thermal dynamic association with ligand B7. Values represent Mean ± SD; n = 3. (G) MST analysis of GFP-fused SRCR5 thermal dynamic association with ligand B7-A4, E10, and F9. Values represent Mean ± SD; n = 3. (H) MST analysis of GFP-fused SRCR5 thermal dynamic association with ligand E3. Values represent Mean ± SD; n = 3.

FIG 4

Evaluating compound structure-activity relationship for anti-PRRSV infection. (A) Molecular structures of B7 and B7-A4 analogues. (B) qRT-PCR for PRRSV in total RNAs extracted from infected PAMs treated with B7 and B7-A4 analogues. Values are normalized with GAPDH of PAMs. Bars = mean ± SD; n = 3. P values are calculated by one-way ANOVA, and letters on the top of bars indicate significant differences in Tukey post hoc test. (C) Titration assay results for PRRSV in the culture media of PAMs treated as described in panel B. Bars = mean ± SD; n = 3. P values are calculated by one-way ANOVA, and letters on the top of bars indicate significant differences in Tukey post hoc test.

FIG 5

Evaluating compound structure-activity relationship for anti-PRRSV infection (2). (A) Molecular structures of additional compounds with similarity to B7. (B) qRT-PCR for PRRSV in total RNAs extracted from infected PAMs treated with B7 analogues. Values are normalized with GAPDH of PAMs. Bars = mean ± SD; n = 3. P values are calculated by one-way ANOVA, and letters on the top of bars indicate significant differences in Tukey post hoc test. (C) Titration assay results for PRRSV treated by selected compounds as described in panel B. Bars = mean ± SD; n = 3. P values are calculated by one-way ANOVA, and letters on the top of bars indicate significant differences in Tukey post hoc test.