Potential role of porcine reproductive and respiratory syndrome virus structural protein GP2 in apoptosis inhibition

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells. In addition, we found significantly higher activities of transcriptional factors (NF- κ B and AP-1) in both cell lines as compared to MARC-145 (parent cells). Overall, our data suggest that, although both stable cell lines activate NF- κ B and AP-1, GP2 triggers the antiapoptotic process through an intermediate step that needs to be further investigated.

Figure 1

Characterization of GP2 and N expressing stable cell lines. (a) Agarose gel analysis. DNA was extracted from both cell lines and subjected to PCR amplification with primers specific to the 2a and N genes. Template amplification with no DNase and no reverse transcription (RT) treatment (lanes 2 and 6); template amplification with DNase treatment and no RT (lanes 3 and 7); template amplification with DNase and RT treatment (lanes 4 and 8); no template controls (lanes 5 and 9). Lane 1 represents the GeneRuler 1 kb Plus DNA Ladder from Fermentas. (b) Immunofluorescence staining of MARC-N cells to show evidence of the N protein expression. (c) Immunofluorescence staining of MARC-145 parent cells.

Figure 2

Inhibition of apoptosis in MARC-2a cells. After incubation for 24 h with 1 μM staurosporine, MARC-145, MARC-2a, and MARC-N cells were stained with FITC-annexin V and PI (a) or Hoechst 33342 (b). The percentage of viable cells was determined by MTT assay (c). The data represent the results of three independent experiments in triplicates, and error bars indicate standard deviations of the means. P < 0.05 (*); P < 0.01 (**).

Figure 3

NF-κB and AP-1 activity in MARC-145, MARC-2a, and MARC-N cells. Cells were transfected with the secreted alkaline phosphatase (SEAP) reporter vectors: pNFκB-SEAP and pAP1-SEAP. The culture supernatants were collected and analyzed for SEAP activity and represented as relative fluorescence unit (RFU) per mL. The data represent the results of three independent experiments in triplicates, and error bars indicate standard deviations of the means. P < 0.05 (*); P < 0.01 (**).

Figure 4

Western blot analysis of NF-κB and AP-1 activation. Equal amounts of nuclear protein fractions were separated on 10% SDS-PAGE. Cytoplasmic fraction contamination was evaluated by α-tubulin antibody (data not shown). Fibrillarin antibody was used as a nuclear marker and also as a loading control. Fold changes in protein amounts are plotted. Western blot with (a) NF-κB rabbit monoclonal antibody and (b) c-Jun mouse monoclonal antibody. Arrow shows the position of the 48 kDa band corresponding to the phosphorylated form of the c-Jun protein.