Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory
Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after contamination by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important functions in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity. (-)-Gallocatechin gallate pontent inhibitor IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host’s immune response and induces prolonged contamination. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of (-)-Gallocatechin gallate pontent inhibitor Gpc2 T cell proliferation and differentiation when expressed as (-)-Gallocatechin gallate pontent inhibitor soluble CD83 (sCD83). In this study, we found that PRRSV contamination induces sCD83 expression in porcine MoDCs via the NF-B and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These (-)-Gallocatechin gallate pontent inhibitor findings provide new insights into the molecular mechanism of immune suppression by PRRSV. axis) value is shown for each analyzed viral strain. (D) Culture supernatants were collected, and sCD83 was analyzed by ELISA. (E) RT-PCR analysis was conducted to measure CD83 mRNA levels, expressed as 2?values. Actin was used as a reference gene, and the untreated sample was utilized for calibration. Data are expressed as means and standard errors of the means (SEM) for the fold changes with respect to expression levels in untreated cells. ELISA and qRT-PCR data are representative of one of three impartial experiments. PRRSV induces CD83 expression in a time- and dose-dependent manner. MoDCs were inoculated with live or UV-inactivated HP-PRRSV BB0907 at a multiplicity of contamination (MOI) of 1 1 and then harvested for CD83 analysis at 6, 12, 24, 36, and 48 h postinfection (hpi). Cells treated with TNF- were used as a positive control. mCD83 and CD83 mRNA expression levels increased strongly as a result of TNF- treatment and PRRSV contamination over time, but UV inactivation of HP-PRRSV (-)-Gallocatechin gallate pontent inhibitor abolished this effect. sCD83 levels increased significantly only in cells infected with PRRSV (Fig. 2A to ?toC).C). PRRSV titers in infected MoDCs peaked at 36 hpi (Fig. 2D), suggesting that CD83 induction is dependent on PRRSV replication. Open in a separate windows FIG 2 PRRSV increases CD83 expression in a time- and dose-dependent manner. MoDCs were inoculated with live or UV-inactivated HP-PRRSV BB0907 at an MOI of 1 1, and MoDCs were treated with PBS (1 mM) and TNF- (50 ng/ml) as negative and positive controls, respectively. (A) At 6, 12, 24, 36, and 48 hpi, cells were collected, and surface CD83 (mCD83) expression was detected by circulation cytometric analysis. Culture supernatants were collected, and sCD83 was analyzed by ELISA (B) and qRT-PCR (C). (D) PRRSV contamination kinetics were measured in the supernatants of infected MoDCs by TCID50 assay. MoDCs were infected with live or UV-inactivated PRRSV at an MOI of 0, 0.1, 1,.