石斑魚雙股RNA激活蛋白激酶對野田病毒複製與細胞自噬之影響

Abstract

雙股RNA激活蛋白激酶 (Double-stranded RNA-activated protein kinase, PKR) 為干擾素誘導基因之一，在脊椎動物先天免疫中扮演重要角色。PKR的典型抗病毒機制，為透過磷酸化eIF2α、抑制蛋白質轉譯，在抑制病毒蛋白表現的同時，也引發細胞自噬避免病毒複製。此反應途徑已在哺乳動物及部分真骨魚類中證實；但在石斑魚中，PKR在病毒感染下的功能仍所知有限。本次自點帶石斑魚 (Epinephelus coioides) 細胞中分離出PKR同源物，並命名為GPKR；實驗旨在探討其於神經壞死病毒 (Nervous Necrosis Virus, NNV) 感染下的表現模式與功能，並進一步驗證其是否會影響細胞自噬。GPKR基因包含長度為1659 bp的ORF，對應552個胺基酸，在系統發育樹分析中，與龍膽石斑魚PKR最相近。GPKR蛋白具有N端的兩個dsRBM (Double-stranded RNA binding motif) 及C端的Serine/Threonine激酶結構域；因與其他脊椎動物PKR具有相似的結構域，因此推測GPKR可能也具有相似的功能。為探討GPKR在病毒感染下於活體中的表現，首先以107 TCID50之NNV攻毒石斑魚，並在感染前後以qPCR測定不同組織的GPKR mRNA表現量。結果顯示，GPKR廣泛存在於所有觀察的組織中，並在感染前就有基本表現量，感染後則在心臟、肝臟與脾臟中顯著上調。在細胞實驗中，以NNV感染石斑魚腦細胞 (cGB) 與鰭細胞 (GF-3)，觀察到GPKR的顯著上調，證實其會受病毒感染引發。此外若在細胞中轉染poly (I:C)，同樣能刺激GPKR的表現；進一步顯示其受dsRNA誘發的特性。若以siRNA敲落GPKR基因，則病毒NNV RNA2的表現量會在感染後72小時下降，顯示GPKR可能參與NNV的複製過程。為探討GPKR是否參與自噬反應，本次利用MDC染劑標記自噬小體，並觀察其數量變化。結果顯示自噬小體數量會隨病毒感染增多，但GPKR敲落並不會對自噬反應造成影響。本研究在點帶石斑魚中，首次分離並定義PKR同源蛋白：GPKR。有別於脊椎動物PKR常見的抗病毒功能，GPKR似乎參與了NNV的複製，且其作用機制與自噬反應無關。

Double-stranded RNA-activated protein kinase (PKR) is an interferon-stimulated gene that plays a crucial role in the innate immune response of vertebrates. Its classical antiviral mechanism involves phosphorylation of eIF2α, which inhibits protein translation, thereby suppressing viral protein synthesis and triggering autophagy to prevent viral replication. While this pathway has been well-documented in mammals and certain teleost species, the functional role of PKR during viral infection in groupers remains largely unknown. In this study, we identified and cloned a PKR homolog: GPKR from orange-spotted grouper (Epinephelus coioides) and investigated its expression pattern and potential function during nervous necrosis virus (NNV) infection. Its possible involvement in autophagy was also examined. GPKR gene contains an ORF of 1659 bp, encoding a protein of 552 amino acids. Phylogenetic analysis showed that GPKR is most closely related to giant grouper PKR. GPKR contained two N-terminal double-stranded RNA-binding motifs (dsRBMs) and a C-terminal serine/threonine kinase domain. This result indicated functional domain conservation and suggesting potential functional similarity between GPKR and other vertebrate PKRs. To evaluate the in vivo expression of GPKR, groupers were challenged with NNV at a dose of 10⁷ TCID₅₀. Tissue-specific GPKR mRNA levels were measured using qPCR before and after infection. Results showed that GPKR was found to be constitutively expressed in all examined tissues pre-infection and significantly upregulated in heart, liver, and spleen post-infection. In vitro, NNV infection of grouper brain cells (cGB) and fin cells (GF-3) resulted in upregulation of GPKR, confirming its inducibility by viral infection. Similarly, transfection with poly(I:C) also elevated GPKR levels, suggesting that its upregulation is responsive to double-stranded RNA stimulation. Knockdown of GPKR via siRNA led to a reduction in NNV RNA2 levels at 72 hours post-infection, indicating a potential role in supporting viral replication. To assess whether GPKR is involved in autophagy, MDC staining was used to monitor autophagosome formation. The results showed an increase in autophagosome number following viral infection; however, no significant difference of autophagosome number was found in GPKR knockdown cells. This is the first study to characterize a new PKR homolog: GPKR in E. coioides. Unlike the typical antiviral function of vertebrate PKRs, GPKR appears to play a role in NNV replication, possibly through mechanisms that are independent of autophagy.