Apoptosis 억제가 viral hemorrhagic septicemia virus (VHSV) 증식에 미치는 영향

Abstract

It has been demonstrated that viral hemorrhagic septicemia virus(VHSV) infection induces apoptosis. Apoptosis occurs through intrinsic and extrinsic pathways, and many viruses exploit apoptosis to facilitate viral proliferation. Although VHSV infection leads to mass mortality of cultured oliver flounder, a little information is available on pathway of apoptosis induced by VHSV infection. In this study, we analyzed the effect of apoptosis suppression on the VHSV replication and explored possible pathway of apoptosis caused by VHSV infection in Epithelioma papulosum cyprini (EPC) cells. Previously, the role of NV protein of novirhabdoviruses in inhibition of apoptosis was reported. Thus, in this study, the effect of apoptosis inhibitors (a p53 inhibitor Pifithrin-α or pan-caspase inhibitor Z-VAD(OMe)-FMK) on the replication was analyzed not only in wild-type VHSV but also in NV gene knock-out recombinant VHSV (rVHSV-△NV-eGFP). EPC cells were treated with pifithrin-α or Z-VAD(OMe)-FMK for 2 hours, then, inoculated with either VHSV or rVHSV-△NV-eGFP. At 24, 36, 48, 60, and 72 h post-infection, cytopathic effect (CPE) was observed, and genomic DNA was isolated for DNA fragmentation analysis. The results showed that both inhibitors dose-dependently delayed CPE and apoptosis by VHSV or rVHSV-△NV-eGFP infection, suggesting that VHSV can induce apoptosis through p53 mediated-pathway and NV gene deletion may not greatly influence on the apoptosis pattern. However, in the semiquantitative RT-PCR analysis, expression of MDM2 that binds to p53 protein for degradation was reduced by VHSV infection. Therefore, further studies are needed to elucidate the involvement of p53 in the apoptosis induced by VHSV infection. Although suppression of apoptosis decreased viral titers of early period (to 3 d post-infection) compared to control groups that were not exposed to apoptosis inhibitors, final titers of VHSV and rVHSV-△NV-eGFP (measured from supernatants isolated when cells showed fully extensive CPE) were increased 2 or 3 times compare to controls. These results suggest that VHSV uses apoptosis for proliferation, and artificial inhibition of apoptosis can extend time for viral replication without budding out from cells, which may lead to increase of the final viral titers in this study.