Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus

doi:10.1128/jvi.00146-24

. 2024 Apr 16;98(4):e0014624.

doi: 10.1128/jvi.00146-24. Epub 2024 Mar 5.

Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus

Junhuang Wu^{1

2}, Wenping Yang^{1

2}, Lingxia Li³, Jingyan Wu^{1

2}, Jijun He^{1

2}, Yi Ru^{1

2}, Jingjing Ren^{1

2}, Yong Wang⁴, Haixue Zheng^{1

2}, Youjun Shang^{1

2}, Dan Li^{1

2}

Affiliations

¹ State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
² Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou, China.
³ College of Agriculture and Animal Husbandry, Qinghai University, Xining, China.
⁴ College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

PMID: 38440983
PMCID: PMC11019896
DOI: 10.1128/jvi.00146-24

Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus

Junhuang Wu et al. J Virol. 2024.

. 2024 Apr 16;98(4):e0014624.

doi: 10.1128/jvi.00146-24. Epub 2024 Mar 5.

Authors

Junhuang Wu^{1

2}, Wenping Yang^{1

2}, Lingxia Li³, Jingyan Wu^{1

2}, Jijun He^{1

2}, Yi Ru^{1

2}, Jingjing Ren^{1

2}, Yong Wang⁴, Haixue Zheng^{1

2}, Youjun Shang^{1

2}, Dan Li^{1

2}

Affiliations

¹ State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
² Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou, China.
³ College of Agriculture and Animal Husbandry, Qinghai University, Xining, China.
⁴ College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

PMID: 38440983
PMCID: PMC11019896
DOI: 10.1128/jvi.00146-24

Abstract

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Keywords: Peste des petits ruminants virus; VISA; fusion protein; plasminogen activator urokinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig 1**
PPRV inhibits the expression of goat PLAU. (**A, B and C**) The effect of PPRV infection on mRNA, protein and concentration levels of goat PLAU in different doses. GAMs (2 × 10⁶) were infected with PPRV at different doses [multiplicity of infection (MOI) = 0, 0.1, 0.5, and 1.0] for 48 h. The levels of goat PLAU mRNA and protein were measured, and the PLAU concentration was measured using a Plasminogen Activator/Urokinase enzyme-linked immunosorbent assay ELISA KIT. (**D, E and F**) The effect of PPRV infection on goat PLAU mRNA, protein and concentration levels at different time points. GAMs (2 × 10⁶) were infected with PPRV (MOI = 1.0) for 0, 12, 24, and 48 h. The levels of goat PLAU mRNA and protein were measured, and the PLAU concentration was measured using a Plasminogen Activator/Urokinase ELISA KIT. Western blot analysis was performed using anti-PLAU antibodies. Data are means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

**Fig 2**
Goat PLAU interacts with F. (A) Goat PLAU interacts with PPRV H and F proteins. GAMs (2 × 10⁷) were infected with PPRV for 0, 24, and 48 h before the silver staining proteins after SDS electrophoresis in polyacrylamide gels and Co-IP were performed. (B) HA-PLAU interacts with Flag-F in HEK293T cells. HA-PLAU (5 µg) was co-transfected with Flag-F (5 µg) and Flag-H (5 µg) in HEK293T cells (2 × 10⁶), respectively. Co-IP with anti-HA antibody was performed, followed by western blot analysis. (C) Goat PLAU interacts with endogenous F in GAMs. GAMs (2 × 10⁷) were infected or not infected with PPRV (MOI = 5) for 48 h, and the cells were harvested. Co-IP with anti-PLAU antibody was performed, followed by western blot analysis. (D) Colocalization of HA-PLAU protein with Flag-F in HEK293T cells. HA-PLAU (0.5 µg) or Flag-F (0.5 µg) were individually transfected into HEK293T cells (4 × 10⁵) or co-transfected with HA-PLAU (0.5 µg) and Flag-F (0.5 µg) for 24 h. The cells were fixed overnight at 4°C and subjected to indirect immunofluorescence to detect HA-PLAU (green) and Flag-F (red) with mouse anti-HA and rabbit anti-Flag antibodies. The position of the nucleus is indicated by 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining in the merged image. (E) Goat PLAU and F were colocalized in GAMs. After infection or non-infection with PPRV (MOI = 1.0) for 48 h in GAMs (2 × 10⁶). The cells were fixed overnight at 4°C and subjected to indirect immunofluorescence to detect PLAU (green) and F (red) with rabbit anti-PLAU and mouse anti-F antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. (F) Goat PLAU has no function of cutting PPRV F protein. The HEK293T cells (4 × 10⁵) were transfected with Myc-PLAU (2 µg) and Flag-F (0.5 µg) for 24 h, followed by western blot analysis.

**Fig 3**
Goat PLAU inhibits the replication of PPRV. (**A to D**) Effect of goat PLAU overexpression on PPRV replication in Vero cells. In Vero cells (4 × 10⁵), HA-PLAU plasmid or EV (1 µg) was transfected for 24 h. Then, cells were followed by PPRV infection (MOI = 1.0) for 24 and 48 h. The levels of PPRV H gene mRNA (A), genomic copy numbers (B), virus titers (C), and N protein levels (D) were measured. (**E and F**) The effect of siRNA on goat PLAU expression level. GAMs (2 × 10⁶) were transfected separately with PLAU-RNAi-#1, PLAU-RNAi-#2, PLAU-RNAi-#3, and Control-RNAi (Coni) for 48 h. Then, the goat PLAU mRNA (E) and protein levels (F) were measured. (G–J) The effect of knocking down goat PLAU on PPRV replication in GAMs was investigated. The transfection mode of siRNA is shown in Fig. 3E. Then, cells were followed by PPRV infection (MOI = 1.0) for 24 and 48 h. The levels of PPRV H gene mRNA (G), genomic copy numbers (H), virus titers (I), and N protein levels (J) were measured. (**K and L**) The effect of siRNA on goat PLAUR expression level. The GAMs (2 × 10⁶) were transfected with PLAUR-RNAi-#1, PLAUR-RNAi-#2, PLAUR-RNAi-#3, and Coni for 48 h, respectively. Then, the goat PLAUR mRNA (K) and protein levels (L) were measured. (**M and N**) The effect of knocking down goat PLAUR on PPRV replication in GAMs was investigated. GAMs (2 × 10⁶) were transfected with PLAUR-RNAi and Coni for 48 h, and cells infected with PPRV (MOI = 1.0) for 24 or 48 h. PPRV virus titers (M) and N protein levels (N) were measured. (O) The effect of Flag-PLAU on PLAUR expression. Flag-PLAU (0, 0.5, 1, and 2 µg) was transfected into GAMs (2 × 10⁶) for 24 h, followed by western blot analysis. Data are means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

**Fig 4**
Goat PLAU enhances the activation of RLR-mediated signaling. (**A and B**) Effects of overexpression of goat PLAU on SeV or poly(I:C)-triggered IFN-β promoter activation. HEK293T cells (1 × 10⁵) were transfected with the IFN-β reporter (0.1 µg) and the indicated expression (0.1 µg) plasmids for 24 h. The cells were infected with SeV for 12 h or transfected with poly(I:C) for 18 h before luciferase assays were performed. (**C and D**) Dose-dependent effects of goat PLAU on SeV or poly(I:C)-triggered activation of IFN-β promoter. HEK293T cells (1 × 10⁵) were transfected with the IFN-β reporter (0.1 µg) and the indicated expression (0, 50, 100 and 200 µg) plasmids for 24 h. The cells were infected with SeV for 12 h or transfected with poly(I:C) for 18 h before luciferase assays were performed. (E) Goat PLAU promotes the induction of downstream antiviral genes triggered by SeV. HEK293T cells (4 × 10⁵) were transfected with HA-PLAU plasmid (1 µg) or EV plasmid (1 µg) for 24 h, followed by SeV infection. Subsequently, the transcription levels of downstream antiviral genes were detected using RT-PCR. (F) The effect of goat PLAU overexpression on the phosphorylation of IKBα, P65, IRF3, and TBK1 induced by SeV. The cells were treated similarly as in Fig. 4E, and western blot analysis was performed using specific antibodies. Data are means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

**Fig 5**
Goat PLAU interacts with VISA. (A) Goat PLAU targets upstream of TBKI. Effects of goat PLAU on IFN-β promoter activation by various signaling components. HEK293T cells (1 × 10⁵) were transfected with IFN-β promoter (0.1 µg) and expression plasmids for goat PLAU and the indicated proteins (0.1 µg each). Luciferase assays were performed 24 h after transfection. (B) The Flag-PLAU and HA-VISA interact with each other in HEK293T cells. Flag-PLAU (5 µg) and HA-VISA (5 µg) were co-transfected in HEK293T cells (2 × 10⁶). After 24 h of transfection, the cells were harvested. Co-IP was performed using anti-Flag antibody, followed by western blot analysis. (C) Endogenous associations between goat PLAU and VISA proteins in GAMs. GAMs (2 × 10⁷) were infected or not infected with PPRV (MOI = 5) for 48 h, and the cells were harvested. Co-IP was performed using anti-PLAU antibody, followed by western blot analysis. (D) Colocalization of Flag-PLAU protein with HA-VISA in HEK293T cells. Flag-PLAU (0.5 µg) or HA-VISA (0.5 µg) were individually transfected into HEK293T cells (4 × 10⁵) or co-transfected with Flag-PLAU (0.5 µg) and HA-VISA (0.5 µg) for 24 h. The cells were fixed overnight at 4°C and subjected to indirect immunofluorescence to detect Flag-PLAU (green) and HA-VISA (red) with mouse anti-Flag and rabbit anti-HA antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. (E) Both the domains of Flag-PLAU (1–218 aa) and Flag-PLAU (219–433 aa) interact with VISA. Flag-PLAU (1–218 aa) (5 µg) and Flag-PLAU (219–433 aa) (5 µg) were co-transfected with HA-VISA (5 µg) in HEK293T, respectively. Co-IP was performed using anti-Flag antibody, followed by western blot analysis. (F) Flag-PLAU interacts with HA-VISA (360–540 aa). Flag-PLAU (5 µg) is used in HA-VISA (1–540), HA-VISA (1–180), HA-VISA (1–360), HA-VISA (100–504), and HA-VISA (360–540) (5 µg), respectively. Co-IP was performed using anti-Flag antibody, followed by western blot analysis. Data are means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

**Fig 6**
Goat PLAU exerts its antiviral function through the domain PLAU (219–433 aa). (A) The effect of Flag-PLAU on HA-VISA expression. The HEK293T cells (4 × 10⁵) were transfected with Flag-PLAU (0, 0.5, 1, and 2 µg) and HA-VISA (0.5 µg) for 24 h, followed by western blot analysis. (B) The effect of Flag-PLAU on endogenous VISA expression. Flag-PLAU (0, 0.5, 1, and 2 µg) was transfected into GAMs (2 × 10⁶) for 24 h, followed by western blot analysis. (C) The effect of goat PLAU-knockdown on endogenous VISA expression. SiRNA-treated cells were similar as in Fig. 3E. Then, cells were infected with PPRV (MOI = 1.0) for 24 or 48 h, followed by western blot analysis with specific antibodies. (D) Effects of inhibitors on the goat PLAU-mediated stability of VISA. The HEK293T cells (4 × 10⁵) were co-transfected with Flag-PLAU (1 µg) and HA-VISA (0.5 µg), and after transfection for 18 h, the cells were treated with the specific inhibitor for 6 h, and then western blot analysis was performed. (**E and F**) The effect of goat PLAU domains on VISA expression. Flag-PLAU (1–218 aa) or Flag-PLAU (219–433 aa) (0, 0.5, 1, and 1.5 µg) were co-transfected with HA-VISA (0.5 µg) in HEK293T cells (4 × 10⁵) for 24 h, followed by western blot analysis. (**G and H**) The effect of Flag-PLAU (1–218aa) on PPRV replication. Vero cells (4 × 10⁵) were transfected with Flag-PLAU (1–218 aa) (1 µg) for 24 h, followed by PPRV (MOI = 1.0) infection for 24 and 48 h. The levels of PPRV N protein and H gene mRNA were then measured. (**I and J**) The effect of Flag-PLAU (219–433aa) (1 µg) on PPRV replication. It is processed in the same way as Fig. 6G and H.

**Fig 7**
Goat PLAU, VISA, and PPRV F proteins interact with each other. (A) Myc-PLAU, HA-VISA, and Flag-F proteins interact with each other. The HEK293T cells (2 × 10⁶) were transfected with Myc-PLAU (5 µg), HA-VISA (5 µg), and Flag-F (5 µg). After 24 h of transfection, the cells were harvested. Co-IP was performed using anti-HA antibody, followed by western blot analysis. (B) VISA can interact with goat PLAU and F endogenous. GAMs (2 × 10⁷) were infected or not infected with PPRV (MOI = 5) for 48 h, and the cells were harvested. Co-IP used anti-VISA, followed by western blot analysis. (C) Colocalization of HA-VISA with Flag-F in HEK293T cells. HA-VISA (0.5 µg) or Flag-F (0.5 µg) were individually transfected into HEK293T cells (4 × 10⁵) or co-transfected with HA-PLAU (0.5 µg) and Flag-F (0.5 µg) for 24 h. The cells were fixed overnight at 4°C and subjected to indirect immunofluorescence to detect HA-VISA (green) and Flag-F (red) with mouse anti-HA and rabbit anti-Flag antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. (D) VISA and F were colocalized in GAMs. After infection or non-infection with PPRV (MOI = 1.0) for 48 h in GAMs (2 × 10⁶). The cells were fixed overnight at 4°C and subjected to indirect immunofluorescence to detect VISA (green) and F (red) with rabbit anti-VISA and mouse anti-F antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. (E) The effect of Flag-F on HA-VISA expression. Flag-F or HA-VISA (0, 0.5, 1, and 1.5 µg) were co-transfected with HA-VISA (0.5 µg) in HEK293T cells (4 × 10⁵) for 24 h, followed by western blot analysis. (F) Goat PLAU can restore the degradation of VISA protein by F. Immunoblotting analysis of HEK293T cells (4 × 10⁵) co-transfected with plasmids expressing F (0.5 or 1 µg; wedges), PLAU protein (2 µg; black rectangular box), and VISA (0.5 µg) (+) or empty vectors (−).

**Fig 8**
The inhibitory effect of goat PLAU on PPRV replication is weakened in VISA-knockdown GAMs. (**A and B**) The effect of siRNA on goat VISA expression level. The GAMs (2 × 10⁶) were separately transfected with VISA-RNAi-#1, VISA-RNAi-#2, VISA-RNAi-#3, and Coni for 48 h. Then, the VISA mRNA (A) and protein levels (B) were measured. (**C to H**) The inhibitory effect of goat PLAU on PPRV replication is weakened in VISA-knockdown GAMs. VISA-RNAi was transfected in GAMs (2 × 10⁶) for 48 h, followed by Flag-PLAU transfection for 24 h. Then, the cells infected PPRV (MOI = 1.0) for 24 and 48 h, respectively. The levels of PPRV N protein levels (**C and D**), genomic copy numbers (**E and F**), and virus titers (**G and H**) were measured. Data are means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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References

1. Shaila MS, Shamaki D, Forsyth MA, Diallo A, Goatley L, Kitching RP, Barrett T. 1996. Geographic distribution and epidemiology of Peste des petits ruminants virus. Virus Res 43:149–153. doi:10.1016/0168-1702(96)01312-3 - DOI - PubMed
1. Zahur AB, Ullah A, Hussain M, Irshad H, Hameed A, Jahangir M, Farooq MS. 2011. Sero-epidemiology of Peste des petits ruminants (PPR) in Pakistan. Prev Vet Med 102:87–92. doi:10.1016/j.prevetmed.2011.06.011 - DOI - PubMed
1. Muthuchelvan D, Sanyal A, Sreenivasa BP, Saravanan P, Dhar P, Singh RP, Singh RK, Bandyopadhyay SK. 2006. Analysis of the matrix protein gene sequence of the Asian lineage of Peste-des-petits ruminants vaccine virus. Vet Microbiol 113:83–87. doi:10.1016/j.vetmic.2005.10.014 - DOI - PubMed
1. Liu F, Li J, Li L, Liu Y, Wu X, Wang Z. 2018. Peste des petits ruminants in China since its first outbreak in 2007: A 10-year review. Transboundary and emerging diseases 65:638–648. doi:10.1111/tbed.12808 - DOI - PubMed
1. Kumar N, Maherchandani S, Kashyap SK, Singh SV, Sharma S, Chaubey KK, Ly H. 2014. Peste des petits ruminants virus infection of small ruminants: a comprehensive review. Viruses 6:2287–2327. doi:10.3390/v6062287 - DOI - PMC - PubMed

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[1] Shaila MS, Shamaki D, Forsyth MA, Diallo A, Goatley L, Kitching RP, Barrett T. 1996. Geographic distribution and epidemiology of Peste des petits ruminants virus. Virus Res 43:149–153. doi:10.1016/0168-1702(96)01312-3 - DOI - PubMed

[2] Shaila MS, Shamaki D, Forsyth MA, Diallo A, Goatley L, Kitching RP, Barrett T. 1996. Geographic distribution and epidemiology of Peste des petits ruminants virus. Virus Res 43:149–153. doi:10.1016/0168-1702(96)01312-3 - DOI - PubMed

[3] Zahur AB, Ullah A, Hussain M, Irshad H, Hameed A, Jahangir M, Farooq MS. 2011. Sero-epidemiology of Peste des petits ruminants (PPR) in Pakistan. Prev Vet Med 102:87–92. doi:10.1016/j.prevetmed.2011.06.011 - DOI - PubMed

[4] Zahur AB, Ullah A, Hussain M, Irshad H, Hameed A, Jahangir M, Farooq MS. 2011. Sero-epidemiology of Peste des petits ruminants (PPR) in Pakistan. Prev Vet Med 102:87–92. doi:10.1016/j.prevetmed.2011.06.011 - DOI - PubMed

[5] Muthuchelvan D, Sanyal A, Sreenivasa BP, Saravanan P, Dhar P, Singh RP, Singh RK, Bandyopadhyay SK. 2006. Analysis of the matrix protein gene sequence of the Asian lineage of Peste-des-petits ruminants vaccine virus. Vet Microbiol 113:83–87. doi:10.1016/j.vetmic.2005.10.014 - DOI - PubMed

[6] Muthuchelvan D, Sanyal A, Sreenivasa BP, Saravanan P, Dhar P, Singh RP, Singh RK, Bandyopadhyay SK. 2006. Analysis of the matrix protein gene sequence of the Asian lineage of Peste-des-petits ruminants vaccine virus. Vet Microbiol 113:83–87. doi:10.1016/j.vetmic.2005.10.014 - DOI - PubMed

[7] Liu F, Li J, Li L, Liu Y, Wu X, Wang Z. 2018. Peste des petits ruminants in China since its first outbreak in 2007: A 10-year review. Transboundary and emerging diseases 65:638–648. doi:10.1111/tbed.12808 - DOI - PubMed

[8] Liu F, Li J, Li L, Liu Y, Wu X, Wang Z. 2018. Peste des petits ruminants in China since its first outbreak in 2007: A 10-year review. Transboundary and emerging diseases 65:638–648. doi:10.1111/tbed.12808 - DOI - PubMed

[9] Kumar N, Maherchandani S, Kashyap SK, Singh SV, Sharma S, Chaubey KK, Ly H. 2014. Peste des petits ruminants virus infection of small ruminants: a comprehensive review. Viruses 6:2287–2327. doi:10.3390/v6062287 - DOI - PMC - PubMed

[10] Kumar N, Maherchandani S, Kashyap SK, Singh SV, Sharma S, Chaubey KK, Ly H. 2014. Peste des petits ruminants virus infection of small ruminants: a comprehensive review. Viruses 6:2287–2327. doi:10.3390/v6062287 - DOI - PMC - PubMed

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Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus

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Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus

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