探討第一型干擾素對於水痘疱疹病毒 感染人類胚胎纖維母細胞的病毒基因表現的影響

Abstract

Varicella-zoster virus (VZV) 是感染人類常見的α形疱疹病毒，初次感染時會形成水痘，並有機會進入感覺神經節而潛伏，當被重新活化後則會產生帶狀疱疹。干擾素的抗病毒機制在免疫系統中扮演極重要的角色。從感染VZV的病人血清中發現含有高量的IFN-α，顯示IFN-α是抵禦VZV感染的重要激素。過去利用移植人類皮膚至SCIDhu mice感染VZV模式實驗顯示在阻斷了IFN-α的訊號傳遞後，會增加VZV感染皮膚細胞的能力；IFN-α具有抑制VZV感染纖維母細胞能力的實驗結果均顯示IFN-α不論是在體內或體外條件都具有抑制VZV感染的功能。病毒從進入細胞、釋出核殼體、完成病毒基因的複製到最終離開細胞等包含多重步驟，其中哪些步驟可能是IFN-α抑制病毒感染標的則為本篇論文想要研究探討的重點。為達此一目的，本論文設計如下：將經過CellTracker標定過的VZV感染細胞分別與經過IFN-α處理或是不經過IFN-α處理的纖維母細胞共同培養，在經過2、4、6、9、12與24小時後，以免疫螢光染色方法觀察VZV核殼體蛋白ORF23與轉錄蛋白IE62在新感染細胞內的表現情形；其次，以RT-PCR的方法分析IFN-α對於VZV在新感染細胞內的基因轉錄的作用。實驗結果發現感染後2-6小時，不論細胞是否經過IFN-α處理，病毒核殼體蛋白ORF23表現並無顯著差異，顯示IFN-α並不影響病毒進入細胞；但是感染後12小時，IFN-α處理後的細胞表現ORF23的比率較未處理的細胞減少了20%，顯示有較少的細胞在這段時間內成功地合成ORF23，顯示IFN-α並不會抑制病毒進入細胞。觀察IE62在感染後4-12小時的細胞內分布型態發現沒有經過IFN-α處理的細胞，IE62在感染細胞核內的表現由點狀、球狀到散布在核中，呈現典型的感染狀態；在IFN-α處理過的狀況下，不但IE62陽性細胞比例大幅減少，在感染的細胞核內直到24小時都維持著點狀結構，顯見IFN-α抑制IE62及IE62所啟動的病毒基因轉錄。利用RT-PCR的方式觀察病毒複製不同時期(IE、E、L)的基因表現實驗結果顯示在處理的情況下，ORF4(IE)、IE62(IE)、ORF61(IE)、ORF63(IE)、ORF68(L)相較於不經過IFN-α處理的情況有顯著減少的情形，顯示IFN-α可能是透過影響病毒複製的早期（IE）過程達到抑制VZV 感染的效果。

Varicella-zoster virus (VZV) is a ubiquitous human alpha-herpesvirus that causes chickenpox during primary infection and may reactivate to cause herpes zoster from latent dorsal root ganglia. The elevated level of interferon (IFN-α) production in VZV patient serum has suggested that IFN-α is an important cytokine in response to VZV infection. Blocking of IFN-α signal transduction in human skin xenografts enhances VZV infectivity in SCIDhu mouse model as well as the results that IFN-α inhibits VZV infection in IFN-α pretreated human embryonic lung fibroblasts in vitro has suggested that IFN-α inhibits VZV infection both in vitro and in vivo. However, which steps following viral entry including uncoating, DNA nuclear transport, DNA replication and viral assembly is or are target by IFN-α remains unclear. In this study, we have used CellTracker dye to label VZV cell inoculums (input cells) and co-cultured them with un-infected cell monolayer (output cells) to investigate the expression of viral nucleocapsid protein ORF23 and major viral transactivator IE62 by immunofluorescence staining at different time points after VZV infection. The results showed that no significant difference in ORF23 expression between mock-treated and IFN-α treated HELFs at 2-6 hours after infection, indicating that IFN-α did not affect the viral penetration. However, the percentage of newly infected cells expressing ORF23 was reduced by 20% in IFN-α treated cells as compared with mock-treated cells at 9-12h after infection. The percentages of IE62-positive cells in IFN-α-pretreated HELFs was lower than that in mock-treated HELFs. Moreover, IE62 expression profile exhibited as punctate, globular and diffused patterns in the nuclei of VZV-infected cells at 6-12h, expression of IE62 in HELFs pretreated with IFN-α remained punctate from 9h to 24h after infection, indicating that IFN-α inhibited VZV DNA replication. To further determined which stage of viral replication can be affected by IFN-α, we investigated the transcription of genes that are expressed at IE, E or L genes after infection by RT-PCR. The results showed that expression of all IE genes including IE4, IE62, ORF61, ORF63 and L gene ORF68 were reduced in HELFs pretreated with IFN-α, indicating that IFN-α inhibits IE stage of viral replication. Our results suggested that IFN-α may affect IE stage of viral replication to inhibit virus infection.