Effect of recombinant chimeric PKR-APAF-1 protein on PRRS virus infected MARC-145 cell line

Abstract

In swine disease, PRRS virus is recognized as the most significant viral pathogen and causes enormous economic losses to the swine industry worldwide. Although many methods are applied to control and prevent this disease, such as vaccination and biosecurity, outbreaks still occur. In this study, rcPAP that generated according to the human DRACO concept was constructed and produced using bacterial expression system and studied its apoptotic-induction effect on Thai isolated US strain PRRS virus-infected MARC-145 cells. In the first part, the rcPAP was constructed with 3 components of protein TAT HIV part as cell penetration, PKR part as dsRNA detection and CARD domain of Apaf-1 part as apoptotic induction. The rcPAP was produced using two different-expression systems (pET-His6-TEV-LIC and pQE32) and purified to apparent homogeneity under native conditions. The three-dimensional (3D) structure of rcPAP was predicted using I-TASSER software (C-score = -4.27) and was found to be similar to that of human PKR and Apaf-1 proteins, suggesting a similar respective function. The rcPAP produced from the pET-His6-TEV-LIC plasmid was expressed as a soluble protein at a 2.4-fold higher concentration (12.32 mg purified rcPAP per litre of culture) than those from the pQE32 plasmid at a low temperature (25 °C). The optimal condition for rcPAP production was found to be an inoculum cell density at an OD600 of 0.6 induced with 0.75 mM IPTG at 25 °C for 18 h. The mouse anti-rcPAP polyclonal antibodies were produced and found to successfully react with purified rcPAP by indirect immunofluorescence (IFA), western blot and indirect ELISA analyses. In the second part, the in vitro study of the rcPAP (at the concentration of 40, 60, 80 and 120 mg/ml) was studied to treat in MARC-145 cells after 6 h infected with Thai isolated US strain PRRS virus. The enhanced antiviral efficacy (apoptotic induction to kill virus-infected cells) was continued to determine at time course of 24, 48 and 72 hpi. The results demonstrated that rcPAP treated MARC-145 cells alone were no cytotoxicity at a concentration of 40, 60 and 80 mg/ml. The rcPAP concentration of 80 mg/ml at 72 hpi showed maximum apoptotic induction and enhanced antiviral activity in vitro. The rcPAP significantly increases the active caspase-3 levels (16.09 fold (13.36 ± 0.003 vs 0.83 ± 0.003 ng/mg protein) in PRRS virus-infected MARC-145 cells treated with rcPAP (80 mg/ml) when it was compared with no treatment. The PRRS viral RNA copy numbers were significantly reduced 46.08 % (3.86 ± 0.005 vs 7.16 ± 0.009 log10 (copies/ml)) in culture cells and 49.16 % (3.34 ± 0.006 vs 6.57 ± 0.02 log10 copies/ml) in culture supernatants of infected MARC-145 cells treated with rcPAP (80 mg/ml) when it was compared with no treatment. Interestingly, the viral titers were significantly reduced 67.9 % (1.65 ± 0.003 vs 5.15 ± 0.01 log10 (TCID50/ml)) in Thai isolated US strain PRRS virus-infected MARC-145 cells treated with rcPAP (80 mg/ml) when it was compared with no treatment. In addition, the results also showed that the rcPAP was able to lower PRRS virus N protein synthesis and CPE in infected MARC-145 cells. In conclusion, the results from this study suggested that rcPAP successfully expressed, produced and purified and was induced apoptosis in US strain PRRS virus-infected MARC-145 cells and enhanced antiviral activity against virus replication. These results imply that the rcPAP is likely a valuable therapeutic agent against US strain PRRS virus infection.