Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1β to promote viral replication by regulating K48 ubiquitination

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (Nsp1β) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1β, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1β, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Keywords: Nsp1β; PRRSV; USP1; deubiquitination; immune escape.

Fig 1

ML323 inhibited PRRSV replication. (A) The cells treated with 10 different small-molecule compounds were collected for 48 h and detected by flow cytometry. (B) Western blot assay for ML323 (10 µM) was performed after treatment with HEK293T, PAM-Tang, and MARC-145 and detected with antibodies against USP1 and β-actin. (C) After pretreatment with ML323, PRRSV BJ-4-infected PAM-Tang cells were identified using 0, 1, 3, and 10 µM ML323, and reverse transcription-quantitative PCR (RT-PCR) was used to evaluate the level of ORF7 mRNA in the cells. (D) MARC-145 cells were pretreated with ML323 at doses of 0, 1, 3, and 10 µM for 4 h and infected with BJ-4 and rSD16 PRRSV (MOI = 10). qPCR was used to detect the PRRSV ORF7 mRNA levels in MARC-145 cells. (E) The treated cells were collected after 48 h, and Western blot analysis was performed with USP1 antibody and PRRSV-N antibody. (F) PRRSV BJ-4 and rSD16 viruses were infected into ML323-pretreated MARC-145 cells, and subviral titers were assayed. (G) The 48-h treated cells were collected and examined by fluorescence microscopy and Western blot. (H) The treated cells collected at 48 h were examined by flow cytometry. The data were presented with significant differences (note: P > 0.05 ns, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.000 1).

Fig 2

USP1 can enhance PRRSV infection. (A) MARC-145 cells were transfected using the monkey USP1-Flag overexpression plasmid, and Western blot was used to detect USP1 and Flag expression. (B) USP1-Flag-transfected MARC-145 cells were infected with PRRSV BJ-4 (MOI = 10), and qPCR was used to detect the mRNA load of ORF7. (C) PRRSV N protein expression test by Western blot. (D) PRRSV BJ-4 and rSD16 viruses were each infected into USP1-Flag-transfected MARC-145 cells, and the titer of the daughter viruses was found. (E and F) Fluorescence microscopy, Western blot, and flow cytometry were performed to detect the rSD16 virus during overexpression of USP1, and the fluorescence content of viral green fluorescent protein (GFP) was compared with that of controls. Data were displayed with noticeable differences (note: P > 0.05 ns, *P < 0.05, ***P < 0.001, and ****P < 0.000 1).

Fig 3

Knockdown of USP1 inhibits PRRSV infection. (A) MARC-145 cells were transfected with siNC and USP1siRNA#1/2/3, and USP1 protein expression was detected after 36 h. (B) Simultaneous qPCR was performed to detect USP1 mRNA expression. (C and D) PRRSV BJ-4 (MOI=10) was infected with siNC and USP1siRNA#3 after transfection for 24 h, qPCR was used to detect the mRNA load of ORF7 (C). Western blot assay to detect PRRSV N and USP1 (D). (E) Twenty-four hours after transfection, PRRSV BJ-4 and PRRSV rSD16 (MOI=10) were infected, and infectious zygotic viral load was determined by the TCID50 assay. (F and G) The detection of PRRSV-GFP virus with fluorescence microscopy and Western blot and flow cytometry during the USP1 iRNA#3 (F). The fluorescence content of viral-GFP was compared with the control (G). The data were presented with significant differences (P > 0.05 ns, *P < 0.05, **P < 0.01, and ****P < 0.0001).

Fig 4

Knockdown of USP1 inhibits PRRSV infection. (A) USP1 protein expression in wild-type (WT) and USP1-deficient (USP1 KO) MARC-145s was analyzed by immunoprecipitation. (B) When USP1 KO MARC-145 cells were infected with PRRSV BJ-4 (MOI = 10) WT, PRRSV ORF7 transcription was determined by qPCR. (C) The protein levels of PRRSV N in WT and USP1 KO MARC-145 cells. (D) Loads of infectious PRRSV BJ-4 and rSD16 were determined by TCID₅₀. (E and F) Fluorescence microscopy, Western blot, and flow cytometry were used to determine the PRRSV GFP virus in WT and USP1 KO MARC-145 cells. The fluorescence content of viral GFP was compared with that of the WT. The data were presented with significant differences (note: P > 0.05 ns, *P < 0.05, **P < 0.01, and ****P < 0.0001).

Fig 5

USP1 interacts with Nsp1β and stabilizes its protein. (A) Viral protein expression plasmids (Nsp1a, Nsp1β, Nsp2, Nsp4, Nsp7, Nsp9, Nsp10, Nsp11, Nsp12, ORF5, and ORF7-HA) were transfected in WT and USP1 KO MARC-145 cells. Western blot detects virus protein expression. (B) After MARC-145 cells were pretreated with DMSO and ML323, Nsp1β-Flag transfection was performed for 24 h, and the related protein expression was then discovered. (C) Western blot detection of protein expression in MARC-145 cells after transfection with Nsp1β-Flag for 24 h after transfection with siNC and siUSP1#3. (D) Western blot detection of different concentrations of USP1-Flag (0, 1, and 2 µg) after transfection of MARC-145 cells with Nsp1β-Flag (1 µg). (E) HEK293T cells were co-transfected with PRRSV (Nsp1a, Nsp1β, Nsp2, Nsp4, Nsp7, Nsp9, Nsp10, Nsp11, Nsp12, ORF5 and ORF7) expression plasmids and USP1-Flag for 24 hours. Cell samples were lysed to carry out immunoprecipitation with an anti-Flag pAb. (F) HEK293T cells were cotransfected with USP1-Flag and Nsp1β-HA. Cells for Co-IP assay were taken 24 h after transfection. (G) Nsp1β-HA-transfected cells were observed by confocal microscopy 24 h after transfection. Anti-USP1 and anti-HA antibodies were used to identify endogenous USP1 and Nsp1β, respectively. (H) Immunoprecipitation analysis of USP1-deficient MARC-145 cells transfected with Nsp1β-Flag.

Fig 6

Nsp1β K48-linked ubiquitination is formed at lysine position 45. (A) HEK293T cells were cotransfected with vector, WT-Ub-HA, K48-Ub-HA, and K63-Ub-HA plasmids and Nsp1β-Flag, and the immunoprecipitation detection assay was performed 24 h later. (B) HEK293T cells were pretreated with DMSO and ML323, transfected with Nsp1β-Flag and K48-Ub-HA plasmids for 24 h, and then detected by Co-IP assay. (C) HEK293T cells were transfected with the indicated plasmids for 24 h and then detected by Co-IP.

Fig 7

USP1 modulates Nsp1β stabilization by deubiquitinating Nsp1β. (A) Diagram showing Nsp1β and its lysine-mutant variants. (B) Nsp1β-NL-Flag was cotransfected with the K48-Ub-HA plasmid in HEK293T cells, and a Co-IP assay was performed 24 h later. (C) Nsp1β-PC-Flag was cotransfected with the K48-Ub-HA plasmid in HEK293T cells, and a Co-IP assay was performed 24 h later. (D) Nsp1β-Flag, Nsp1β-NL-Flag, Nsp1β-PC-Flag, and K48-Ub-HA were cotransfected into HEK293T cells, and a Co-IP assay was performed after 24 h. (E) The Nsp1β-NL-terminal mutant plasmid was cotransfected into HEK293T cells and K48-Ub-HA. (F) Nsp1β-K45R-Flag was cotransfected into HEK293T cells with Nsp1β-Flag and K48-Ub-HA, and Co-IP assay was performed 24 h later for the Co-IP assay.

Fig 8

In vitro replication of the PRRSV rSD16-K45R virus. (A) MARC-145 was infected with rSD16 and rSD16-K45R (MOI = 10) at different time points (0, 6, 12, 24, 36, and 48 h), and the viral life cycle was determined by the viral titer. (B) Western blot was used to detect the expression of N protein in MARC-145 cells infected with rSD16 and rSD16-K45R (MOI = 10) for 48 h. (C) MARC-145 cells transfected with the empty plasmid and USP1-Flag plasmid were infected with rSD16-K45R and rSD16 (MOI = 10) for 48 h, and the change of PRRSV N was detected by Western blot. The ratio of PRRSV-GFP to β-actin was analyzed by ImageJ. (D) MARC-145 cells transfected with ML323 were infected with rSD16-K45R and rSD16 (MOI = 10) for 48 h, and the expression of PRRSV N was detected by Western blot. (E) rSD16-K45R and rSD16 were infected with siUSP1#3-transfected MARC-145 cells (MOI = 10), and PRRSV N protein expression was found 48 h later. (F) The N protein expression of rSD16-K45R and rSD16 (MOI = 10) in USP1 KO MARC-145 cells was detected by Western blot.