魚類結病毒在金目鱸腦細胞株之持續性感染及金目鱸Mx蛋白抗病毒之機制

Abstract

從神經壞死症病毒 (nervous necrosis virus, NNV) 感染後存活下來之金目鱸腦組織建立的BB細胞株有NNV持續性感染。為了釐清干擾素 (interferon, IFN) 是否在BB細胞株NNV持續性感染的機制中扮演一個角色，以NNV專一性兔子抗血清處理BB細胞五代後，得到一株無病毒的負對照組細胞，在治療的BB細胞 (cured BB, cBB) 中皆測不到NNV的力價、RNA及capsid protein。第一型IFN可誘發Mx抗病毒 蛋白，在BB細胞及NNV感染的cBB細胞中發現有Mx基因的表現，但在無NNV感染的cBB細胞中則否。以單源抗體中和過的BB細胞上清液，處理cBB細胞後，可誘發Mx基因的表現，及抗NNV之抗病毒活性。以低病毒感染劑量 (multiplicity of infection, MOI) (≦1) 感染cBB細胞後，可再誘發cBB細胞形成NNV持續性感染。從以上這些實驗結果得知，BB細胞上清液存在類IFN的細胞激素；又IFN誘發的抗病毒反應，在保護大部分細胞免於病毒爆破性感染，及調節BB細胞株NNV持續性感染機制中扮演了一個重要的角色。 以RACE (rapid amplification of cDNA ends) PCR方法放大金目鱸腦細胞株cBB的Mx mRNA，得到金目鱸Mx基因的全長cDNA (2.2 kb)包含一個長度為1875個核苷酸的open reading frame (ORF)，可轉譯出一個624個氨基酸之Mx蛋白，預測分子量為71.4 kDa，且在氨基端含有一tripartite guanosinetriphosphate (GTP)-binding motif，以及在羧基端含有一leucine zipper，具有全部已知的Mx蛋白之特性。在cBB細胞中，poly I:C的轉染會誘發Mx基因的表現；在28 ℃的誘發量會高於20 ℃的誘發量；且魚類結病毒、雙股RNA病毒及虹彩病毒的感染也會誘發Mx基因的表現，但結病毒比其他兩種病毒誘發Mx基因表現量高。利用抗病毒活性測試，發現poly I:C轉染的cBB細胞，對魚類結病毒及雙股RNA病毒具有抗病毒活性，但對虹彩病毒則沒有抗病毒活性；以siRNA抑制Mx基因的表現後，可恢復結病毒及雙股RNA病毒的複製量，這些結果顯示，金目鱸Mx基因的表現具有抑制魚類結病毒及雙股RNA病毒增殖的功能。 在NNV感染cBB細胞24小時內，NNV RNA-dependent RNA polymerase (RdRp)的表現量會先增加，當金目鱸Mx蛋白在感染後24小時表現時，NNV RdRp的量即減少；以siRNA抑制cBB細胞中金目鱸Mx蛋白的表現後，NNV RdRp、capsid protein、RNA2以及子代病毒力價皆會上升；以免疫螢光染色法在NNV感染的cBB細胞中，發現金目鱸Mx蛋白會與NNV RdRp共同座落在細胞核周圍的區域；在共同免疫沈澱法分析中，發現金目鱸Mx蛋白不僅會與NNV RdRp共同沈澱下來，也會與NNV capsid protein共同沈澱，且金目鱸Mx蛋白與NNV RdRp之複合物會與lysosomes共同座落在一起，隨後NNV RdRp會被lysosome系統所分解，因此顯示金目鱸Mx蛋白是藉由隔離NNV RdRp於細胞核周圍的區域，接著在lysosome中被分解的機制來抑制NNV RNA的合成。

The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (≦1). These results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced antiviral response played an important role in protecting the majority of cells from virus lytic infection and mediating NNV persistence in the BB cell line. We obtained a full-length cDNA clone for the Mx gene of barramundi (Lates calcarifer), using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from a barramundi brain cell line cBB. The Mx cDNA of 2.2 kb contains an open reading frame (ORF) of 1875 nucleotides encoding a protein of 624 amino acids. The predicted barramundi Mx protein is 71.4 kDa and contains a tripartite guanosinetriphosphate (GTP)-binding motif at the amino terminal and a leucine zipper at the carboxyl terminal which are characteristics of all known Mx proteins. Poly I:C-transfection induced the expression of Mx gene in cBB cells, and the induction level at 28 ℃ was higher than that at 20 ℃. Moreover, Mx gene expression was also induced by viral infection, including fish nodavirus, birnavirus, and iridovirus. Among these, nodavirus was a stronger inducer than the other two viruses. Using an antiviral activity assay, we revealed that poly I:C-transfected cBB cells had antiviral activity against fish nodavirus and birnavirus, but not iridovirus. Furthermore, the replication of nodavirus and birnavirus could be restored after the expression of Mx gene was down-regulated by Mx-specific siRNA. These results indicated that the expression of barramundi Mx gene was able to inhibit the proliferation of fish nodavirus and birnavirus. The expression of NNV RNA-dependent RNA polymerase (RdRp) increased in NNV-infected cBB cells within 24 h post-infection (hpi), and later decreased as soon as the expression of barramundi Mx protein at 24 hpi. Moreover, NNV RdRp, capsid protein, RNA2, and viral titer of progeny NNV all elevated in NNV-infected cBB cells when the expression of barramundi Mx protein was down-regulated by siRNA. Barramundi Mx protein was observed to colocalize with NNV RdRp at the perinuclear area in NNV-infected cBB cells by immunofluorescence staining. In the coimmunoprecipitation assay, barramundi Mx protein was coprecipitated with NNV RdRp and capsid protein. Furthermore, the complexes of barramundi Mx protein and NNV RdRp could colocalize with lysosomes, and then NNV RdRp would be degragded by the lysosome system. Therefore, it was suggested that barramundi Mx protein suppressed NNV RNA synthesis by sequestration of NNV RdRp to the perinuclear area for the following degradation in the lysosomes.