Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May 23;55(1):67.
doi: 10.1186/s13567-024-01316-8.

Ursonic acid from medicinal herbs inhibits PRRSV replication through activation of the innate immune response by targeting the phosphatase PTPN1

Yuanqi Yang  1 , Yanni Gao  1 , Haifeng Sun  1 , Juan Bai  1 , Jie Zhang  1 , Lujie Zhang  1 , Xing Liu  1 , Yangyang Sun  1 , Ping Jiang  2   3
Affiliations

Ursonic acid from medicinal herbs inhibits PRRSV replication through activation of the innate immune response by targeting the phosphatase PTPN1

Yuanqi Yang et al. Vet Res. .

Abstract

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), has caused substantial economic losses to the global swine industry due to the lack of effective commercial vaccines and drugs. There is an urgent need to develop alternative strategies for PRRS prevention and control, such as antiviral drugs. In this study, we identified ursonic acid (UNA), a natural pentacyclic triterpenoid from medicinal herbs, as a novel drug with anti-PRRSV activity in vitro. Mechanistically, a time-of-addition assay revealed that UNA inhibited PRRSV replication when it was added before, at the same time as, and after PRRSV infection was induced. Compound target prediction and molecular docking analysis suggested that UNA interacts with the active pocket of PTPN1, which was further confirmed by a target protein interference assay and phosphatase activity assay. Furthermore, UNA inhibited PRRSV replication by targeting PTPN1, which inhibited IFN-β production. In addition, UNA displayed antiviral activity against porcine epidemic diarrhoea virus (PEDV) and Seneca virus A (SVA) replication in vitro. These findings will be helpful for developing novel prophylactic and therapeutic agents against PRRS and other swine virus infections.

Keywords: PRRSV; Ursonic acid (UNA); antivirals; protein tyrosine phosphatase nonreceptor type 1 (PTPN1).

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Identification of the anti-PRRSV activity of ursonic acid (UNA) in vitro. A High-throughput screening (HTS) assay flowchart. B 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) of UNA in Marc-145 cells. C Relative PRRSV ORF7 mRNA levels in Marc-145 cells determined by RT‒qPCR. GAPDH was used as the internal loading control. D Western blotting of the PRRSV N protein in Marc-145 cells infected with PRRSV and treated with the indicated concentrations of UNA. E Virus titration of samples from Marc-145 cells by TCID50 calculation. F IFA images of Marc-145 cells (PRRSV-infected and UNA-treated) at 48 hpi. The PRRSV N protein is green, and the nuclei are blue. Scale bars, 500 μm. G Viability of PAMs treated with the indicated concentrations of UNA for 24 h. H Relative PRRSV ORF7 mRNA levels in PAMs determined by RT‒qPCR. β-Actin was used as the internal loading control. I Western blot of the PRRSV N protein in PAMs infected with PRRSV and treated with UNA or DMSO at 24 hpi. J Virus titration of samples from PAMs by TCID50 calculation. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 2
Figure 2
UNA suppresses PRRSV replication in various treatment stages in the time-of-addition assay. A Schematic illustration of the time-of-addition experiment. B IFA images of Marc-145 cells (PRRSV-infected and UNA-treated) at 48 hpi. The viral N protein is green, and the nuclei are blue. Scale bars, 500 μm. C–E Western blotting of the PRRSV N protein in Marc-145 cells infected with PRRSV and treated with UNA or DMSO (including pre, co, and post-treatment modes) at 48 hpi. F TCID50 detection for the virucidal activity assay. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 3
Figure 3
In silico target prediction and molecular docking analysis of UNA. A Target prediction for UNA (only the top 10 predicted proteins are listed). B The Ramachandran plot statistics of Chlorocebus sabaeus PTPN1 (chloPTPN1) represent the most favourable, additional allowed, generously allowed, and disallowed regions, with percentages of 91.1, 8.2, 0.7, and 0%, respectively. C The Z score of chloPTPN1 was −8.4. D Docked conformation of homoPTPN1 (PDB: 2F71) with UNA. The compound and protein are represented as lines and cartoons, respectively. UNA is coloured red, and the protein homoPTPN1 is coloured yellow. The binding site is shown as a cavity structure. The binding energy of the UNA-homoPTPN1 complex, which was calculated using Autodock, is marked with an asterisk. E Docked conformation of chloPTPN1 with UNA. UNA is coloured red, and the protein chloPTPN1 is coloured green. The binding site is shown as a cavity structure. The binding energy of the UNA-chloPTPN1 complex, which was calculated using Autodock, is marked with an asterisk. F RMSD values of chloPTPN1 (black), UNA (red), and the UNA and chloPTPN1 complex (blue) over the 25 ns simulation time.
Figure 4
Figure 4
UNA suppresses the phosphatase activity of PTPN1. A PTPN1 expression in Marc-145 cells treated with DMSO or UNA. The histogram on the right is a statistical analysis of the Western blot data. B Protein tyrosine phosphatase (PTP) activity in Marc-145 cells treated with DMSO or UNA. C Expression and purification of chloPTPN1. i Recombinant protein precipitates before (Lane 2) and after (Lane 3) purification were subjected to SDS‒PAGE. Lane 1, Rosetta-pET-28a-chloPTPN1 with IPTG induction. Lane 4, Rosetta-pET-28a with IPTG induction. ii and iii The purified protein was analysed by Western blotting. D Phosphatase activity of chloPTPN1 after incubation with UNA. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 5
Figure 5
PTPN1 is a vital proviral factor in PRRSV infection via its phosphatase activity. A Effect of chloPTPN1 overexpression on PRRSV replication in Marc-145 cells. B IFA images of Marc-145 cells (PRRSV-infected and choPTPN1-transfected) at 36 hpi. The viral N protein is green, and the nuclei are blue. Scale bars, 500 μm. C Virus titration of samples from Marc-145 cells by TCID50 calculation. D Western blot analysis of the effect of PTPN1 knockdown on PRRSV replication in Marc-145 cells. E and F RT‒qPCR analysis of the effect of PTPN1 knockdown on PRRSV replication in Marc-145 cells. G and H Effects of chloPTPN1 phosphatase activity on PRRSV replication determined by RT‒qPCR (G) and Western blotting (H) in Marc-145 cells. The following plasmids were used: chloPTPN1-HA, chloPTPN1-HA-D181A (“substrate-trapping” mutant, D for short), chloPTPN1-HA-C215S (enzyme-inactive mutant, C for short), and chloPTPN1-HA-D181A/C215S (D/C for short). The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 6
Figure 6
PTPN1 inhibits the IFN-β promoter stimulated by poly(I:C) via its phosphatase activity. A Luciferase assay and Western blot analysis for assessing the effect of chloPTPN1 overexpression on IFN-β promoter activity in Marc-145 cells. B RT‒qPCR and Western blot analysis for assessing the effect of chloPTPN1 overexpression on the mRNA levels of IFN-β, ISG15, and ISG56 in Marc-145 cells. C RT‒qPCR and Western blot analysis for assessing the effect of PTPN1 knockdown on the mRNA levels of IFN-β, ISG15, and ISG56 in Marc-145 cells. D Effects of chloPTPN1 phosphatase activity on the IFN-β promoter in Marc-145 cells, as determined by luciferase and Western blot assays. The following plasmids were used: chloPTPN1-HA, chloPTPN1-HA-D181A (“substrate-trapping” mutant, D for short), chloPTPN1-HA-C215S (enzyme-inactive mutant, C for short), and chloPTPN1-HA-D181A/C215S (D/C for short). The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 7
Figure 7
PTPN1 attenuates IFN-β production induced by RLR-mediated signalling. HEK293T cells were transfected with the indicated plasmids, chloPTPN1-myc, and the IFN-β luciferase reporter plasmids for 24 h. The cell lysates were harvested for immunoblotting with the indicated antibodies and for luciferase assays to detect the effect of PTPN1 on the activation of the IFN-β promoter induced by RIG-I A, MDA5 B, MAVS C, TBK1 D, IKKε E, and IRF3 F. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 8
Figure 8
UNA enhances IFN-β promoter activity and inhibits PRRSV replication via PTPN1. A Luciferase assay for assessing the effect of UNA treatment on IFN-β promoter activity in Marc-145 cells. B–D RT‒qPCR for assessing the effect of UNA treatment on the mRNA levels of IFN-β, ISG15, and ISG56 in Marc-145 cells. E and F Luciferase assay and Western blot analysis for assessing the effect of PTPN1 knockdown on UNA-mediated promotion of IFN-β promoter activity in Marc-145 cells. G Western blot analysis for assessing the effect of PTPN1 knockdown on UNA anti-PRRSV activity in Marc-145 cells. The histograms on the right show the results of the statistical analyses of the Western blot data. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 9
Figure 9
UNA exerts anti-PRRSV activity by targeting susPTPN1 in PAMs. A The Ramachandran plot statistics of sus scrofa PTPN1 (susPTPN1) represent the most favourable, additional allowed, generously allowed, and disallowed regions, with percentages of 88.3, 10.5, 0.8, and 0.4%, respectively. B The Z score of susPTPN1 was -8.26. C Comparative analysis of chloPTPN1 and susPTPN1 structures by PyMOL. The structures of chloPTPN1 and susPTPN1 are labelled in green and cyan, respectively. D Docked conformation of susPTPN1 with UNA. The compound and protein are represented as sticks and cartoons, respectively. UNA is coloured red, and the protein susPTPN1 is coloured cyan. The binding site is shown as a cavity structure. The binding energy of the UNA-susPTPN1 complex, which was calculated using Autodock, is marked with an asterisk. E RMSD values of susPTPN1 (black), UNA (red), and the UNA and susPTPN1 complex (blue) over the 25 ns simulation time. F Western blot analysis of the effect of PTPN1 knockdown on PRRSV replication in PAMs. G and H RT‒qPCR analysis of the effect of PTPN1 knockdown on PRRSV replication in PAMs. I Western blot analysis for assessing the effect of PTPN1 knockdown on UNA anti-PRRSV activity in PAMs. The histograms on the right show the results of the statistical analyses of the Western blot data. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).
Figure 10
Figure 10
Broad-spectrum antiviral activity of UNA analysed for various PRRSV strains, porcine epidemic diarrhoea virus (PEDV) and Senecavirus A (SVA). A and D Relative PRRSV S1 or PRRSV FJ1402 ORF7 mRNA levels in Marc-145 cells determined by RT‒qPCR. GAPDH was used as the internal loading control. B and E Western blot analysis of the PRRSV N protein in Marc-145 cells infected with PRRSV S1 or PRRSV FJ1402 and treated with the indicated concentrations of UNA. C and F Virus titration of samples from Marc-145 cells by TCID50 calculation. G and J The mRNA levels of PEDV N in Vero cells G and SVA VP2 in ST cells J determined by RT‒qPCR. H and K Western blot analysis of the PEDV N protein H and SVA VP2 protein K in Vero or ST cells infected with PEDV or SVA and treated with the indicated concentrations of UNA. I and L Virus titration of samples from Vero or ST cells for PEDV I and SVA L detection. M and N Viability of Vero M and ST N cells treated with UNA for 24 h. The results are from one of three independent experiments. The data are presented as the means ± SDs. The asterisks in the figures indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns: not significant).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Rossow KD. Porcine reproductive and respiratory syndrome. Vet Pathol. 1998;35:1–20. doi: 10.1177/030098589803500101. - DOI - PubMed
    1. Helm ET, Curry SM, De Mille CM, Schweer WP, Burrough ER, Zuber EA, Lonergan SM, Gabler NK. Impact of porcine reproductive and respiratory syndrome virus on muscle metabolism of growing pigs1. J Anim Sci. 2019;97:3213–3227. doi: 10.1093/jas/skz168. - DOI - PMC - PubMed
    1. Schulze M, Revilla-Fernandez S, Schmoll F, Grossfeld R, Griessler A. Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report. Acta Vet Scand. 2013;55:16. doi: 10.1186/1751-0147-55-16. - DOI - PMC - PubMed
    1. Olanratmanee EO, Wongyanin P, Thanawongnuwech R, Tummaruk P. Prevalence of porcine reproductive and respiratory syndrome virus detection in aborted fetuses, mummified fetuses and stillborn piglets using quantitative polymerase chain reaction. J Vet Med Sci. 2015;77:1071–1077. doi: 10.1292/jvms.14-0480. - DOI - PMC - PubMed
    1. Adams MJ, Lefkowitz EJ, King AMQ, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR, Nibert M, Sabanadzovic S, Sanfacon H, Siddell SG, Simmonds P, Varsani A, Zerbini FM, Gorbalenya AE, Davison AJ. Changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses (2017) Arch Virol. 2017;162:2505–2538. doi: 10.1007/s00705-017-3358-5. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources