Galectin 3-binding protein suppresses PRRSV replication via Cullin3-mediated ubiquitination degradation of non-structural protein 12

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to the global swine industry, yet effective antiviral strategies remain limited. This study identifies galectin 3-binding protein (LGALS3BP) as a critical host factor inhibiting PRRSV infection through targeting the viral conserved non-structural protein 12 (nsp12), a key component of the viral replication-transcription complex. Overexpression of LGALS3BP significantly suppressed PRRSV replication, while its knockdown enhanced viral replication. Mechanistically, LGALS3BP recruits the Cullin3 E3 ubiquitin ligase via its BACK domain to mediate the ubiquitination of nsp12 at lysine residue 91, leading to proteasomal degradation. This process disrupts nsp12-dependent synthesis of viral subgenomic RNA, thereby disrupting replication. Additionally, LGALS3BP enhances antiviral innate immunity by upregulating interferon (IFN)-β and IFN-stimulated genes (ISGs). The antiviral effect of LGALS3BP is conserved across diverse PRRSV strains, highlighting its broad-spectrum potential. These findings reveal a dual mechanism whereby LGALS3BP restricts PRRSV through direct degradation of a critical viral enzyme and modulation of host immune responses, highlighting LGALS3BP as a promising therapeutic avenue for PRRSV control.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains a major challenge to global swine production due to its genetic diversity, rapid mutation rate, and ability to evade host immunity. The nsp12 is highly conserved across PRRSV strains and plays a crucial role in viral RNA synthesis. This study identifies LGALS3BP as a critical host factor that inhibits PRRSV infection by targeting nsp12 via the ubiquitin-proteasome pathway. By uncovering this novel antiviral mechanism, the research highlights LGALS3BP as a promising therapeutic target for PRRSV control. Moreover, it contributes to our understanding of how host factors modulate viral replication and immunity, opening new avenues for developing host-targeted antiviral strategies. These findings have the potential to mitigate PRRSV-driven economic losses and improve swine health worldwide.

Keywords: LGALS3BP; Nsp12; PRRSV; antiviral activity; ubiquitination.

Fig 1

PRRSV infection induces the upregulation of LGALS3BP expression in the lungs. (A) Heatmaps show the core genes of the PPI network in the lungs of piglets infected with HuN4 and SD53. Samples are represented on the horizontal axis, and genes are represented on the vertical axis; red indicates high expression, and blue indicates low expression. (B and C) RT-qPCR analysis of LGALS3BP mRNA expression in lung homogenates at 3 dpi. Lung tissues of pigs infected with PRRSV-HuN4 (B) or PRRSV-SD53 (C) were collected to assess LGALS3BP mRNA levels by RT-qPCR. (D and E) ELISA analysis of LGALS3BP protein levels in lung tissues from pigs infected with PRRSV-HuN4 (D) or PRRSV-SD53 (E). Data are presented as the mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The P value was calculated using a Student’s t-test (two-tailed).

Fig 2

LGALS3BP overexpression suppresses PRRSV replication. (A–C) Marc-145 cells were transfected with EV or different doses of LGALS3BP, and then infected with PRRSV HuN4 (MOI = 0.1) for 24 h. The cell lysates and culture supernatants were collected to analyze PRRSV N protein expression and viral titers with RT-qPCR (A), Western blot (B), and TCID₅₀ assay (C). (D–F) Marc-145 cells were transfected with EV or LGALS3BP and then infected with PRRSV (MOI = 0.1) for 12, 24, and 36 h. The cell lysates and culture supernatants were collected to analyze PRRSV N protein expression and viral titers with RT-qPCR (D), Western blot (E), and TCID₅₀ assay (F). (G–I) Marc-145 cells were transfected with EV or LGALS3BP and then infected with PRRSV at MOI = 0.1 or 1 for 24 h. PRRSV N protein expression and viral titers were assessed with RT-qPCR (G), Western blot (H), and TCID₅₀ assay (I). (J) Marc-145 cells were transfected with LGALS3BP or EV for 24  h and then infected with the indicated PRRSV strains at 0.1 MOI for an additional 24 h. The cell lysates were collected to analyze PRRSV RNA levels. (K–N) PAMs were incubated with LGALS3BP lentivirus mixed with polybrene for 12 h and then infected with PRRSV for 24 h. The cell lysates and culture supernatants were collected to analyze LGALS3BP RNA levels (K), PRRSV RNA levels (L), PRRSV protein expression (M), and viral titers (N). Data are represented as the mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Fig 3

Knockdown of LGALS3BP enhances PRRSV infection. (A–D) Marc-145 cells were transfected with NC or siRNA-LGALS3BP for 24 h and then infected with PRRSV (MOI = 0.1) for another 24  h. The cell lysates and culture supernatants were collected to analyze LGALS3BP RNA levels (A), PRRSV RNA levels (B), viral titers (C), and PRRSV protein expression (D). (E–G) iPAMs were transfected with NC or siRNA-LGALS3BP for 24 h and then infected with PRRSV (MOI = 0.1) for another 24  h. The cell lysates and culture supernatants were collected to analyze LGALS3BP RNA levels (E), PRRSV RNA levels (F), and PRRSV protein expression (G). Data are presented as the mean ± SD from three independent experiments. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Fig 4

LGALS3BP regulates PRRSV infection at the stage of biosynthesis. (A–C) The overexpression of LGALS3BP does not affect virus attachment and entry. Marc-145 cells were transfected with EV or LGALS3BP for 24 h, incubated with PRRSV (0.1 MOI) at 4°C for 1 h (A), and then incubated at 37°C for 1 to 3 h (B) or 6 to 24 h (C). PRRSV RNA levels were determined by RT-qPCR. (D–F) Marc-145 cells were transfected for 24 h. The cell lysates were collected to analyze IFN-β (D), ISG15 (E), and IFIT1 (F) RNA levels. (G–I) iPAMs were transfected for 24 h. The RNA levels of IFN-β (G), ISG15 (H), and IFIT1 (I) were determined by RT-qPCR. Data are presented as the mean ± SD from three independent experiments. Statistical significance is indicated as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01.

Fig 5

LGALS3BP specifically degrades the PRRSV nsp12. (A) HEK-293T cells were transfected with PRRSV nsps and LGALS3BP for 24 h. The cell lysates were collected to analyze PRRSV nsps protein expression. (B) HEK-293T cells were transfected with different doses of LGALS3BP (0.1, 0.2, 0.4, 0.8, and 1.6 µg) and nsp12 for 24 h. The cell lysates were collected to analyze nsp12 protein expression. (C) HEK-293T cells were transfected with LGALS3BP and nsp12 for 12, 24, 36, and 48 h. The cell lysates were collected to analyze nsp12 protein expression. (D) HEK-293T cells were transfected with nsp12 and LGALS3BP for 12 h, and then treated with MG132 (0.1 µM), or 3-methyladenine (3-MA) (1 mM) for 24 h. (E) HEK-293T cells were transfected with nsp12 and LGALS3BP for 12 h and treated with or without MG132 (0.1 µM) for 24 h. Cells were collected to analyze by Co-IP assay. (F) HEK-293T cells were transfected with LGALS3BP and nsp12 for 24 h. Cells were collected to analyze by Co-IP assay. (G) HEK-293T cells were transfected with nsp12 and LGALS3BP for 24 h, and double stained with a rabbit anti-HA antibody and a mouse anti-Flag antibody, followed by fluorescein Alexa Fluor-conjugated anti-rabbit IgG (green) and Alexa Fluor-conjugated anti-mouse IgG (red). Cell nuclei were counterstained with 1 µg/mL of 4’, 6’-diamidino-2-phenylindole.

Fig 6

LGALS3BP degrades nsp12 through BACK domain. (A) Schematic diagram of truncated constructs of LGALS3BP. (B) HEK-293T cells were transfected with nsp12 and truncated mutant plasmids for 24 h. The cell lysates were collected to analyze nsp12 protein expression. (C) HEK-293T cells were transfected with LGALS3BP domain plasmids and nsp12 plasmids for 24 h. Cells were collected to analyze by Co-IP assay. (D) HEK-293T cells were transfected with LGALS3BP and nsp12 lysine site mutant plasmids for 24 h. The cell lysates were collected to analyze nsp12 protein expression. (E) Quantitative analysis results of the gray values in panel D. Statistical significance is indicated as follows: ***, P < 0.001.

Fig 7

LGALS3BP degrades nsp12 by recruiting the Cullin3 E3 ubiquitin ligase. (A) HEK-293T cells were transfected with Cullin3-specific siRNAs for 24 h, cells were collected to analyze the Cullin3 mRNA levels by RT-qPCR. (B) HEK-293T cells were transfected with Cullin3-specific siRNA for 24 hours, followed by co-transfection with LGALS3BP and nsp12 plasmids for another 24 hours. The cell lysates were collected to analyze nsp12 protein expression. (C and D) HEK-293T cells were transfected with Cullin3 and LGALS3BP (C) /nsp12 (D) plasmids for 36 h. Cells were collected to analyze by Co-IP assay. (E) HEK-293T cells were transfected for 12 h, then treated with MG132 (0.1 µM) or DMSO for another 24 h. Cells were collected to analyze by Co-IP assay. (F and G) HEK-293T cells were transfected and stained with mouse anti-HA (F) antibody or mouse anti-Flag (G) antibody, followed by Alexa Fluor-conjugated anti-mouse IgG (red). Cell nuclei were counterstained with 1 µg/mL of 4′,6′-diamidino-2-phenylindole. Statistical significance was determined by one-way ANOVA (****, P < 0.0001).

Fig 8

LGALS3BP inhibits the synthesis of PRRSV subgenomic RNA. (A–C) Marc-145 cells were transfected with LGALS3BP or EV for 24 h, followed by infection with PRRSV (0.1 MOI) for 4 h. Cells were collected to analyze PRRSV genomic RNA levels (A), PRRSV plus-strand subgenomic RNA levels (B), and PRRSV minus-strand subgenomic RNA levels (C). Data are presented as the mean ± SD from three independent experiments. ns, P > 0.05; **, P < 0.01, ***, P < 0.001. The P value was calculated using a Student’s t-test (two-tailed).

Fig 9

Schematic diagram of the inhibitory effect of LGALS3BP on PRRSV infection.