Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production

Abstract

Cellular cholesterol plays an important role in the life cycles of enveloped viruses. Previous studies by our group and other groups have demonstrated that the depletion of cellular cholesterol by methyl-β-cyclodextrin (MβCD) reduces the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine Arterivirus that has been devastating the swine industry worldwide for over two decades. However, how PRRSV infection regulates cholesterol synthesis is not fully understood. In this study, we showed that PRRSV infection upregulated the activity of protein phosphatase 2 (PP2A), which subsequently activated 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the cholesterol synthesis pathway, to increase the levels of cellular cholesterol. By screening the PRRSV-encoded proteins, we showed that nsp4 dominated the upregulation of cellular cholesterol, independently of the 3C-like protease activity of nsp4. A mutation analysis showed that domain I (amino acids 1-80) of PRRSV nsp4 interacted with PR65 alpha (PR65α), the structural subunit, and PP2Ac, the catalytic subunit, of PP2A. Importantly, domain I of nsp4 inhibited Sendai virus-induced interferon β production, and this inhibitory effect was eliminated by Lovastatin, an HMGCR inhibitor, indicating that the upregulation of cellular cholesterol by nsp4 is a strategy used by PRRSV to suppress the antiviral innate immunity of its host. Collectively, we here demonstrated the mechanism by which PRRSV regulates cellular cholesterol synthesis and reported a novel strategy by which PRRSV evades its host's antiviral innate immune response.

Keywords: AMPK; Cholesterol; HMGCR; Interferon; PP2A; PRRSV.

Fig. 1

Cellular cholesterol is upregulated in PRRSV-infected cells. PAMs or PK-15^CD163 cells were infected with PRRSV (MOI = 1.0). At the indicated time points after infection, the cells were harvested for the quantitation of cholesterol with the Amplex™ Red Cholesterol Assay Kit (A, B), or the cells were fixed to detect the cholesterol content with the cholesterol dye filipin (blue) (C, D). The nuclei were counterstained with propidium iodide (red). Fluorescent images were acquired with a confocal laser scanning microscope. The presented results represent the means and standard deviations of data from three independent experiments (*p ≤ 0.05; **p ≤ 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

PRRSV infection activates the PP2A–HMGCR pathway. (A) Model of cholesterol synthesis via the AMPK–HMGCR and PP2A–HMGCR pathways. Level of p-HMGCR (inactive form) was enhanced by AMPK, resulting in the downregulation of cholesterol synthesis, but reduced by PP2A, resulting in the upregulation of cholesterol synthesis. (B–D) PAMs or PK-15^CD163 cells infected with PRRSV (MOI = 1.0) were harvested at 12, 24, and 36 hpi. The expression levels of total HMGCR and phosphorylated HMGCR (p-HMGCR) (B), total AMPK and p-AMPK (C), or total PP2Ac and p-PP2Ac (D) were analyzed with western blotting. PRRSV infection was verified by detecting the expression of viral N protein with anti-N antibody. The β-actin was used as the protein loading control.

Fig. 3

PRRSV nsp4 plays a major role in upregulating cellular cholesterol independently of its protease activity. (A) PK-15^CD163 cells were transfected with expression vectors encoding HA-tagged PRRSV-encoded proteins. The levels of HMGCR and p-HMGCR were determined with western blotting at 36 h post transfection. (B) PK-15^CD163 cells were transfected with different doses of plasmids encoding HA-tagged PRRSV nsp4. The cell lysates were harvested and the expression levels of HMGCR, p-HMGCR, PP2Ac and p-PP2Ac were analyzed with western blotting. (C) PK-15^CD163 cells were transfected with expression vectors encoding HA-tagged nsp4 or nsp4 mutant (H39A, D64A, or S118A). At 36 h post transfection, the cells were harvested to analyze PP2Ac and p-PP2Ac levels with western blotting. (D) PK-15^CD163 cells were transfected with an expression vector encoding HA-tagged nsp4. At 12, 24, and 36 h post transfection, cells were fixed to determine the cholesterol content using the cholesterol dye filipin (blue). The nuclei were counterstained with propidium iodide (red). Fluorescent images were acquired with a confocal laser scanning microscope. (E) PK-15^CD163 cells were transfected with an expression vector encoding HA-tagged nsp4. The cells were harvested for the quantitation of cholesterol with the Amplex™ Red Cholesterol Assay Kit at 12, 24, and 36 h post transfection. The results presented are the means and standard deviations of data from three independent experiments (*p ≤ 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

PRRSV nsp4 interacts with PP2A. (A) PP2A consists of three subunits, including a structural subunit (scaffolding subunit; A subunit, PR65α), a regulatory subunit (B subunit), and a catalytic subunit (PP2Ac, C subunit). (B, C) HEK-293T cells were cotransfected with expression vector encoding Flag-tagged PR65α or PP2Ac and vector encoding HA-tagged PRRSV nsp4. The cells were lysed at 36 h post transfection and immunoprecipitated with anti-Flag (B) or anti-HA (C) antibody. Whole-cell lysates (WCLs) and immunoprecipitation (IP) complexes were analyzed with immunoblotting using anti-Flag, anti-HA, or anti-β-actin antibody. (D) PK-15^CD163 cells were infected with PRRSV (MOI = 1.0). The cells were lysed at 36 hpi and immunoprecipitated with anti-nsp4 antibody. WCLs and IP complexes were analyzed with immunoblotting using anti-nsp4, anti-PR65α, anti-PP2Ac, or anti-β-actin antibody.

Fig. 5

PRRSV nsp4 domain I (amino acids 1–80) interacts with the structural subunit (PR65α) and catalytic subunit (PP2Ac) of PP2A. (A) Schematic diagram of nsp4 truncations. (B–E) HEK-293T cells were co-transfected with expression vectors encoding Flag-tagged PR65α or PP2Ac and EGFP-tagged nsp4, nsp4 domain I, nsp4 domain II, or nsp4 domain III. The cells were lysed at 36 h post transfection and immunoprecipitated with an anti-Flag antibody (B, D) or an anti-GFP antibody (C, E). The WCLs and IP complexes were analyzed with immunoblotting using anti-Flag, anti-EGFP, or anti-β-actin antibody.

Fig. 6

Lovastatin eliminates the IFN-β inhibitory effect mediated by PRRSV nsp4 domain I. (A) PK-15^CD163 cells were transfected with expression vector encoding EGFP-tagged nsp4 or its truncation (domain I, domain II, or domain III), and then the cellular cholesterol levels were determined with the Amplex™ Red Cholesterol Assay Kit. (B) PK-15^CD163 cells were transfected with expression vector encoding EGFP-tagged nsp4 or its truncation (domain I, domain II, or domain III), and then stimulated with Sendai virus (SeV) for 12 h. The total cellular RNAs were extracted to determine the IFN-β mRNA levels with RT–qPCR. (C) PK-15^CD163 cells were incubated with the indicated concentrations of Lovastatin for 36 h and cytotoxicity was detected with the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (D) PK-15^CD163 cells were incubated with various concentrations (2.5, 5, or 10 μΜ) of Lovastatin, and the cellular cholesterol levels were then determined with the Amplex™ Red Cholesterol Assay Kit. (E) PK-15^CD163 cells were pretreated with Lovastatin (Lv; 10 μΜ) for 8 h, and then transfected with an expression vector encoding EGFP-tagged nsp4 or its truncation (domain I, domain II, or domain III), followed by stimulation with SeV for 12 h. The total cellular RNAs were extracted to determine the IFN-β mRNA levels with RT–qPCR. The results presented are ANOVA of data from three independent experiments (**p ≤ 0.01; ***p ≤ 0.001).

Fig. 7

Lovastatin inhibits PRRSV infection. (A) PK-15^CD163 cells were pretreated with 10 μΜ Lovastatin for 8 h before PRRSV infection (MOI = 1.0), and then the cellular cholesterol levels were determined with the Amplex™ Red Cholesterol Assay Kit. (B) PK-15^CD163 cells were pretreated with 10 μΜ Lovastatin for 8 h before PRRSV infection (MOI = 1.0), and then stimulated with SeV for 12 h. The total cellular RNAs were extracted to determine the IFN-β mRNA levels with RT–qPCR. (C–E) PK-15^CD163 cells were pretreated with different concentrations of Lovastatin for 8 h before PRRSV infection (MOI = 1.0). Infected cells were cultured in the presence of the indicated concentrations of Lovastatin and harvested at 24 hpi for a plaque assay (C), RT–qPCR (D), and western blotting (E). The results presented are ANOVA of data from three independent experiments (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).