劉晴晴 常章梅 白金金 王 艷 龍健兒

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)系-衛(wèi)生部、教育部醫(yī)學(xué)分子病毒學(xué)重點(diǎn)實(shí)驗(yàn)室 上海 200032)

RNA編輯酶ADAR1對(duì)EV71感染及變異的影響

劉晴晴 常章梅 白金金 王 艷 龍健兒△

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)系-衛(wèi)生部、教育部醫(yī)學(xué)分子病毒學(xué)重點(diǎn)實(shí)驗(yàn)室 上海 200032)

目的 研究RNA編輯酶腺苷酸脫氨酶 (adenosine deaminase acting on RNA,ADAR1)對(duì)新型腸道病毒71型(enterovirus 71,EV71)感染及變異的影響。方法 運(yùn)用RNAi技術(shù)篩選ADAR1基因沉默穩(wěn)定細(xì)胞株,通過(guò)MTT分析病毒感染后細(xì)胞活力變化、噬斑形成分析病毒滴度及細(xì)胞對(duì)病毒的敏感性,以及Western blot測(cè)定病毒蛋白表達(dá)水平等,分析ADAR1對(duì)EV71感染的影響。由于ADAR1介導(dǎo)的RNA編輯可使基因形成A-G或T-C突變,為確定ADAR1影響EV71感染是否與其編輯EV71基因組導(dǎo)致病毒變異有關(guān),對(duì)EV71流行區(qū)EV71的突變特征進(jìn)行分析;并利用EV71感染ADAR1基因沉默細(xì)胞,通過(guò)對(duì)病毒基因組測(cè)序分析,研究ADAR1是否直接編輯EV71基因組。結(jié)果ADAR1基因沉默后,與對(duì)照細(xì)胞相比,病毒感染細(xì)胞的存活率下降更快,并形成更多、更大的噬斑。病毒感染細(xì)胞中的VP1蛋白和細(xì)胞培養(yǎng)基上清中的病毒滴度均明顯增加。EV71突變特征分析表明,雖然A-G和T-C之間的變異是病毒突變的主要類(lèi)型,但EV71感染實(shí)驗(yàn)初步證明,ADAR1并不直接編輯EV71病毒基因組。結(jié)論 ADAR1可能具有抗EV71感染的作用,但ADAR1可能并不直接編輯EV71基因組。

EV71; ADAR1; RNA編輯; 病毒突變

新型腸道病毒71型(enterovirus 71,EV71)是嬰幼兒手足口病(hand,food and mouth disease,HFMD)的主要病原體。HFMD臨床癥狀主要表現(xiàn)為手、足、口等部位的皰疹,重癥病例可引起神經(jīng)系統(tǒng)相關(guān)并發(fā)癥,病情進(jìn)展快,死亡率高[1-2]。雖然EV71預(yù)防性滅活疫苗已獲批生產(chǎn),但尚無(wú)特異性抗病毒藥物[3-5]。因此對(duì)EV71的致病機(jī)制以及影響EV71感染的宿主因子等進(jìn)行研究仍十分必要。

EV71為單股正鏈RNA病毒,在細(xì)胞質(zhì)中復(fù)制,復(fù)制過(guò)程中形成雙鏈RNA(dsRNA)復(fù)制中間體。而dsRNA依賴(lài)的腺苷酸脫氨酶(adenosine deaminase acting on RNA 1,ADAR1)是一類(lèi)dsRNA編輯酶,不但能編輯宿主細(xì)胞內(nèi)的dsRNA分子,而且還可編輯部分病毒dsRNA[6]。近來(lái)還發(fā)現(xiàn)ADAR1是MDA5-MAVS誘生干擾素生成途徑的負(fù)調(diào)控因子[7-8],并且ADAR1本身也是干擾素刺激基因(interferon stimulated gene,ISG)之一[6,9],提示ADAR1在抗病毒感染和免疫應(yīng)答過(guò)程中可能有重要作用。研究表明,ADAR1可影響多種病毒的感染和復(fù)制,如水皰性口炎病毒(vesicular stomatitis virus,VSV)[10]、人類(lèi)免疫缺陷病毒(human immunodeficiency virus,HIV)[11-13]、麻疹病毒(measles virus,MV)[14-15]、丙型肝炎病毒(hepatitis C virus,HCV)[16]和丁型肝炎病毒(hepatitis delta virus,HDV)[17]等。ADAR1對(duì)EV71感染是否有影響尚不清楚。

ADAR1的N-端具有雙鏈RNA結(jié)合結(jié)構(gòu)域(dsRNA-binding domain,dsRBD),C-端具有RNA編輯酶催化結(jié)構(gòu)域。此外,N-端還具有兩個(gè)Z-DNA結(jié)合域:Zα和Zβ[18-19]。ADAR1可催化dsRNA部分腺苷(A)脫氨基,形成次黃嘌呤(I),次黃嘌呤在轉(zhuǎn)錄和翻譯時(shí)被識(shí)別為鳥(niǎo)嘌呤(G),因此可能導(dǎo)致被編輯RNA的基因形成A-G或T-C突變,并影響其表達(dá)[20]。因此,本研究擬通過(guò)構(gòu)建ADAR1基因沉默穩(wěn)定細(xì)胞株來(lái)研究ADAR1對(duì)EV71感染和復(fù)制的影響,并探討ADAR1影響EV71感染是否通過(guò)直接編輯EV71基因組而產(chǎn)生影響及是否因此增加EV71的突變率。

材 料 和 方 法

材料和試劑 EV71病毒株(Genbank Access No.HQ891927,064-Shanghai)由本實(shí)驗(yàn)室分離并保存。橫紋肌肉瘤(rhabdomysarcoma,RD)細(xì)胞、子宮頸癌(Henrietta Lacks,HeLa)細(xì)胞、人胚腎細(xì)胞(human embryonic kidney cell,293FT)購(gòu)自中科院細(xì)胞庫(kù)?；虺聊d體pLKO.1-TRC克隆載體來(lái)源于Addgene(美國(guó))。

ADAR1基因沉默HeLa細(xì)胞、RD細(xì)胞的構(gòu)建與篩選 以HeLa細(xì)胞為例,RD細(xì)胞的構(gòu)建與此類(lèi)似。為構(gòu)建ADAR1基因沉默穩(wěn)定細(xì)胞,設(shè)計(jì)與人類(lèi)ADAR1 mRNA(NM_001111) 1398-1418以及4976-4996位點(diǎn)靶向結(jié)合的短發(fā)夾RNA(short hairpin,shRNA)。將shRNA克隆入慢病毒載體pLKO.1,與病毒包裝質(zhì)粒psPAX2和pMD2.G共轉(zhuǎn)染293FT細(xì)胞,包裝獲得有感染性的慢病毒。然后將慢病毒感染HeLa細(xì)胞,通過(guò)嘌呤霉素(2 mg/mL)篩選穩(wěn)定整合慢病毒的細(xì)胞株HeLa-pLKO.1-shADAR1。同時(shí)構(gòu)建和篩選整合陰性對(duì)照慢病毒載體pLKO.1-SCR的細(xì)胞作為對(duì)照。

MTT法檢測(cè)細(xì)胞存活力 為確定ADAR1基因沉默后對(duì)病毒誘導(dǎo)細(xì)胞病變和裂解的影響,以HeLa細(xì)胞為例,將HeLa-pLKO.1-shADAR1和對(duì)照HeLa-pLKO.1-SCR細(xì)胞鋪96孔板(1×104/孔),37 ℃培養(yǎng)24 h后,EV71分別以不同的感染復(fù)數(shù)(multiplicity of infection,MOI)感染上述細(xì)胞(從MOI為100 PFU/細(xì)胞開(kāi)始2倍連續(xù)稀釋)。細(xì)胞分別培養(yǎng)72 h和96 h后,加入MTT (10 mL/孔),37 ℃孵育3 h,吸掉培養(yǎng)基,再加入DMSO(100 mL/孔),37 ℃孵育15 min,用酶標(biāo)儀測(cè)定570 nm處的吸光度值。病毒感染后細(xì)胞的存活率按以下公式計(jì)算:細(xì)胞存活率=感染組吸光度值/未感染組吸光度值×100%。

病毒噬斑分析病毒滴度及細(xì)胞對(duì)病毒的敏感性為測(cè)定病毒滴度,鋪RD細(xì)胞于六孔板(1×106/孔),培養(yǎng)24 h。EV71按梯度稀釋感染RD細(xì)胞,2 h后棄去病毒上清。然后用0.25%低熔點(diǎn)瓊脂膠2 mL覆蓋細(xì)胞并培養(yǎng)72 h,用0.1%中性紅染色3 h后檢測(cè)噬斑形成數(shù)量。為測(cè)定細(xì)胞對(duì)病毒的敏感性,以HeLa細(xì)胞為例,鋪HeLa-pLKO.1-shADAR1和HeLa-pLKO.1-SCR細(xì)胞于六孔板(1.2×106/孔)。培養(yǎng)8 h之后用相同EV71病毒量(4.8×107PFU/mL)按指定倍數(shù)稀釋分別感染ADAR1基因沉默細(xì)胞及對(duì)照細(xì)胞,2 h后棄去病毒上清,檢測(cè)EV71在不同細(xì)胞上形成的噬斑數(shù)量和大小。

Western blot檢測(cè)ADAR1及病毒VP1蛋白的表達(dá) 用含磷酸酶抑制劑、蛋白酶抑制劑、PMSF的細(xì)胞裂解液于冰上裂解細(xì)胞,用BCA法測(cè)定蛋白濃度。經(jīng)SDS-PAGE電泳分離后電轉(zhuǎn)到PVDF膜,膜封閉2 h后分別與下述單克隆抗體4 ℃搖床過(guò)夜,ADAR1抗體(英國(guó)Abcam公司,EPR7033,ab126745)稀釋5 000倍,EV71 VP1抗體(美國(guó)Abnova公司,3D7,MAB1255-M05)稀釋2 000倍,β-actin抗體(美國(guó)Cell Signaling Technology公司,8H10D10,#3700)稀釋3 000倍,洗膜后與HRP標(biāo)記羊抗兔IgG(1∶5 000稀釋)以及HRP標(biāo)記羊抗鼠IgG(1∶5 000稀釋)室溫孵育1 h;洗膜后用ECL發(fā)光顯色,暗室壓片,顯影定影,掃描后軟件定量分析。蛋白表達(dá)水平以目的蛋白/β-actin的相對(duì)倍數(shù)表示。

EV71的突變特征分析 由于ADAR1編輯dsRNA可能導(dǎo)致基因A-G或T-C的突變[20]。為進(jìn)一步確定ADAR1對(duì)EV71感染的影響是否與其編輯EV71基因組并導(dǎo)致病毒變異有關(guān),我們利用NCBI核酸數(shù)據(jù)庫(kù)中EV71全基因組序列,對(duì)EV71的突變特征進(jìn)行初步分析:選取曾經(jīng)EV71感染的5個(gè)高度流行區(qū)(中國(guó)的阜陽(yáng)、上海和臺(tái)灣地區(qū),以及馬來(lái)西亞和越南),每個(gè)流行區(qū)隨機(jī)挑選10株,進(jìn)行序列比對(duì),獲得各個(gè)流行區(qū)EV71病毒基因組保守序列作為參考序列,將各流行區(qū)靶基因序列與相應(yīng)流行區(qū)的保守序列進(jìn)行比較,統(tǒng)計(jì)分析其堿基變化情況,獲得各流行區(qū)EV71的主要堿基突變特征。

RNA編輯分析 EV71感染HeLa-pLKO.1-shADAR1和對(duì)照細(xì)胞,36 h后收集培養(yǎng)上清,用新增殖病毒再去感染重新鋪板的細(xì)胞,不斷感染和傳代,直至第15代(G15)。細(xì)胞培養(yǎng)上清中病毒RNA用Trizol試劑抽提,利用隨機(jī)引物進(jìn)行逆轉(zhuǎn)錄,隨后取5 mL cDNA進(jìn)行PCR,對(duì)PCR產(chǎn)物進(jìn)行測(cè)序。以PCR產(chǎn)物測(cè)序圖譜為分析對(duì)象,仔細(xì)比對(duì)RNA編輯熱點(diǎn)的測(cè)序峰值,以及測(cè)序主峰當(dāng)中可能含有其他堿基的測(cè)序峰,測(cè)序峰的積分值比例表明堿基在此位置的百分含量。如果在ADAR1編輯熱點(diǎn)出現(xiàn)A主峰中含G峰,或T主峰中含C峰,并隨病毒傳代該峰出現(xiàn)升高,則認(rèn)為出現(xiàn)ADAR1對(duì)EV71 RNA的A-G或T-C的定向編輯[11]。

統(tǒng)計(jì)學(xué)分析 不同組間的數(shù)據(jù)使用Student′st檢驗(yàn)進(jìn)行分析,所有數(shù)據(jù)均使用SPSS 16.0軟件進(jìn)行處理。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

ADAR1基因沉默細(xì)胞的構(gòu)建與篩選 為確定ADAR1基因沉默后對(duì)EV71感染和復(fù)制的影響,設(shè)計(jì)2對(duì)分別作用于ADAR1 mRNA的特異性shRNA(圖 1A),通過(guò)構(gòu)建慢病毒、篩選穩(wěn)定下調(diào)ADAR1的細(xì)胞株。首先構(gòu)建HeLa細(xì)胞ADAR1基因沉默穩(wěn)定細(xì)胞株ShADAR1-1398和ShADAR1-4976。Sh-ADAR1-4976穩(wěn)定細(xì)胞株下調(diào)ADAR1蛋白表達(dá)水平約90%,而Sh-ADAR1-1398穩(wěn)定細(xì)胞株下調(diào)ADAR1蛋白表達(dá)水平達(dá)96%,均有非常顯著的下調(diào)作用。故選擇Sh-ADAR1-1398穩(wěn)定細(xì)胞株完成后續(xù)的實(shí)驗(yàn)(圖 1B)。同時(shí)使用Sh-ADAR1-1398慢病毒感染并篩選RD細(xì)胞,并篩選ADAR1基因沉默穩(wěn)定細(xì)胞株RD-pLKO.1-shADAR1(圖 1C)。

A:Design of shRNA targetingADAR1 mRNA located at 1398-1418 and 4976-4996,respectively;B:ADAR1 expression levels were detected by immunoblotting and knocked down by shRNAs in HeLa cells;C:ADAR1 expression levels were detected in RD cells.The immunoblotting data were quantified and normalized with-actin.

圖1ADAR1基因沉默細(xì)胞的構(gòu)建與篩選

Fig 1 Construction and selection of stable cell lines withADAR1 knock-down

ADAR1基因沉默促進(jìn)EV71誘導(dǎo)細(xì)胞病變、裂解 EV71在不同MOI下感染HeLa-pLKO.1-shADAR1細(xì)胞和對(duì)照HeLa-pLKO.1-SCR細(xì)胞,隨著感染時(shí)間延長(zhǎng),HeLa-pLKO.1-shADAR1細(xì)胞和對(duì)照細(xì)胞的存活率都不斷降低,但在相同條件下,HeLa-pLKO.1-shADAR1細(xì)胞的存活率均低于對(duì)照細(xì)胞(圖2A)。提示ADAR1基因沉默后促進(jìn)病毒誘導(dǎo)HeLa細(xì)胞病變和裂解。利用ADAR1基因沉默RD細(xì)胞也發(fā)現(xiàn)類(lèi)似結(jié)果,即ADAR1基因沉默后促進(jìn)病毒誘導(dǎo)RD細(xì)胞的病變和裂解,與對(duì)照細(xì)胞相比,細(xì)胞存活率下降更快(圖2B)。

ADAR1基因沉默提高細(xì)胞對(duì)EV71的敏感性分別用等量EV71病毒感染HeLa-pLKO.1-shADAR1細(xì)胞和對(duì)照細(xì)胞2 h,HeLa-pLKO.1-shADAR1細(xì)胞形成的噬斑數(shù)量約為對(duì)照細(xì)胞的3倍,且噬斑明顯較大(圖3A)。提示ADAR1基因沉默后可提高HeLa細(xì)胞對(duì)EV71的敏感性。觀察EV71感染RD-pLKO.1-shADAR1細(xì)胞和對(duì)照細(xì)胞后形成的噬斑情況,得到一致結(jié)果,即細(xì)胞ADAR1基因下調(diào)后,細(xì)胞更容易被病毒感染,形成更多、更大的噬斑(圖3B)。

ADAR1基因沉默促進(jìn)EV71的復(fù)制 觀察EV71感染HeLa-pLKO.1-shADAR1細(xì)胞和對(duì)照細(xì)胞后病毒的復(fù)制情況。在感染后12～48 h,HeLa-pLKO.1-shADAR1細(xì)胞中EV71 VP1蛋白表達(dá)水平明顯高于對(duì)照細(xì)胞(圖 4A);在感染后6～48 h,HeLa-pLKO.1-shADAR1細(xì)胞上清中病毒滴度較對(duì)照細(xì)胞明顯升高(P<0.05,圖4A)。提示ADAR1基因沉默后可促進(jìn)EV71在HeLa細(xì)胞中的復(fù)制。EV71感染RD-pLKO.1-shADAR1細(xì)胞和對(duì)照細(xì)胞后檢測(cè)病毒VP1含量以及上清中病毒滴度,得到類(lèi)似的結(jié)果(圖4B)。

A:Cell viability of HeLa cells withADAR1 knock-down and the control cells were analyzed after EV71 infection;B:Cell viability of RD cells withADAR1 knock-down and the control cells were analyzed.

圖2ADAR1基因沉默促進(jìn)EV71誘導(dǎo)細(xì)胞病變和裂解

Fig 2ADAR1 knock-down promoted EV71-induced cell lysis

Plaques of EV71 formed on HeLa cells (A) and RD cells (B) withADAR1 knock-down.Graph showed the relative folds of plaque number normalized by the control cells.

圖3ADAR1基因沉默提高細(xì)胞對(duì)EV71的敏感性

Fig 3ADAR1 knock-down increased the cellular susceptibility to EV71

EV71的突變特征及ADAR1對(duì)EV71基因組的編輯分析 由于ADAR1具有RNA編輯酶活性,編輯EV71復(fù)制中間體dsRNA可導(dǎo)致病毒A-G或T-C的突變[20]。分析5個(gè)EV71流行地區(qū)病毒的突變特征,結(jié)果顯示A-G和T-C之間突變確實(shí)為EV71的主要突變類(lèi)型(表1)。為確定ADAR1是否直接編輯EV71基因組并影響病毒感染和突變,根據(jù)預(yù)測(cè)分析的ADAR1編輯熱點(diǎn)(AAG、UAG、AAU、UAU和CTT、CTA、ATT、ATA)序列特征,選擇EV71基因組中5個(gè)編輯熱點(diǎn)聚集區(qū)進(jìn)行測(cè)序分析。結(jié)果顯示病毒感染ADAR1基因沉默細(xì)胞及其不斷傳代后(至G15),尚未發(fā)現(xiàn)EV71基因組中被ADAR1定向編輯的位點(diǎn)(圖5),提示ADAR1可能并不通過(guò)直接編輯EV71基因組而影響病毒的感染。

A:EV71 VP1 expressions (upper panel) in HeLa cells withADAR1 knock-down,and virus titer (lower panel) were detected after EV71 infection (MOI=50 PFU/cell).B:EV71 VP1 expressions (upper panel) in RD cells withADAR1 knock-down,and virus titer (lower panel) were detected after EV71 infection (MOI=10 PFU/cell).The data were normalized by the control cells.

圖4 ADAR1基因沉默對(duì)EV71復(fù)制的影響

EV71 full-length genome sequences from five epidemic regions were collected.The Genbank Acesss No.showed as the following:EU703812,EU703814,HQ188292,JX025561,EU703813,FJ439769,EU812515,GU459070,GU459071 and GU198371 in Fuyang;FJ713137,HQ891925,HQ891926,KC570452,JQ736684,HQ891929,HQ891927,HQ891923,HQ891924 and HQ891928 in Shanghai;GQ231925,GQ231942,GQ231934,GQ231941,GQ231933,KJ186973,KF134486,KF154354,KF154355 and GQ231943 in Taiwan;AB550334,AB550335,DQ341367,DQ341362,DQ341363,DQ341365,DQ341366,AB550340,AB550341 and DQ341358 in Malaysia;KJ686299,KJ686301,KJ686304,KJ686302,KJ686308,KJ686306,KJ686300,KJ686303,KJ686305 and KJ686307 in Vietnam.

Five fragments of EV71 genome (sequence locations indicated with red box) clustering with RNA-editing hot sites by ADAR1 were analyzed by sequencing from G0 to G15.Arrows indicated the potential editing-sites.

圖5 EV71基因組RNA編輯分析結(jié)果示例

Fig 5 Representative RNA-editing assay for EV71 genome

討 論

新型腸道病毒EV71感染較其他HFMD病原體更容易致神經(jīng)性重癥[21],但是其致病機(jī)制并不清楚。研究表明一些重要宿主因子與EV71感染相關(guān)[21]。EV71與其他RNA病毒一樣,基因組易產(chǎn)生突變,而且在宿主抗病毒免疫系統(tǒng)的壓力下,EV71也容易產(chǎn)生逃逸突變,而高突變率引起的新基因亞型與EV71流行密切相關(guān)[3]。

RNA編輯酶ADAR1既可編輯宿主細(xì)胞因子,也可編輯病毒dsRNA[6],因而影響多種病毒的感染。ADAR1對(duì)EV71感染有何影響,及是否對(duì)該病毒進(jìn)行編輯并不清楚。通過(guò)MTT分析及噬斑形成分析發(fā)現(xiàn),ADAR1基因沉默后可促進(jìn)EV71誘導(dǎo)細(xì)胞病變和裂解,增強(qiáng)細(xì)胞對(duì)EV71的敏感性。Toth等[14]用RNAi技術(shù)沉默HeLa細(xì)胞ADAR1后,發(fā)現(xiàn)MV引起的細(xì)胞病變?cè)鰪?qiáng);Ward等[15]通過(guò)敲除MEF細(xì)胞ADAR1 p150異構(gòu)體,發(fā)現(xiàn)細(xì)胞對(duì)MV感染的敏感性增強(qiáng)。我們通過(guò)Western blot檢測(cè)發(fā)現(xiàn)ADAR1基因沉默后可以促進(jìn)EV71的復(fù)制。敲除ADAR1 p150后MV增殖病毒滴度顯著增高[15]。Taylor等[16]運(yùn)用小分子RNA特異性抑制ADAR1,發(fā)現(xiàn)HCV的復(fù)制能力增加了40倍。我們還通過(guò)基因過(guò)表達(dá)研究了ADAR1對(duì)EV71感染的影響,獲得了基本一致的結(jié)果。但由于細(xì)胞中ADAR1的基礎(chǔ)表達(dá)水平非常高,與看家基因表達(dá)水平相近(圖1),ADAR1過(guò)表達(dá)所能提升的蛋白表達(dá)水平有限,遠(yuǎn)未及ADAR1基因沉默改變明顯(最多約20倍),因此ADAR1過(guò)表達(dá)對(duì)病毒感染的影響也相應(yīng)較小(結(jié)果未顯示)。

本研究初步發(fā)現(xiàn)ADAR1具有抗EV71感染的作用,但其分子機(jī)制并不清楚。有研究表明ADAR1基因沉默后,MV引起的細(xì)胞病變?cè)鰪?qiáng)與激活PKR和干擾素調(diào)節(jié)因子3 (IRF3)有關(guān)[14]。ADAR1也可以通過(guò)RNA編輯影響病毒感染,如ADAR1可能通過(guò)對(duì)病毒RNA的編輯來(lái)達(dá)到清除HCV RNA的目的[16]。在HDV感染中,ADAR1過(guò)表達(dá)使amber/W處被過(guò)度編輯,導(dǎo)致大HDV抗原(HDAg-L)的異常表達(dá)而使復(fù)制過(guò)早的終止[17]。盡管通過(guò)EV71突變特征分析發(fā)現(xiàn)A-G和T-C的突變?yōu)镋V71的主要突變類(lèi)型,但本研究并未發(fā)現(xiàn)ADAR1可以直接編輯EV71 RNA,所以ADAR1影響EV71感染可能不是ADAR1編輯EV71基因組的結(jié)果,而可能與ADAR1誘導(dǎo)或抑制某些重要宿主因子的表達(dá)或通過(guò)ADAR1編輯某些影響病毒感染的宿主因子有關(guān),具體分子機(jī)制尚需進(jìn)一步研究。由于ADAR1不直接編輯EV71 RNA,因此EV71易突變形成新的基因型與ADAR1的關(guān)系可能并不密切。病毒基因突變除與病毒RNA聚合酶有關(guān)外[22-24],還有哪些因素影響基因突變的頻率和方式,也是目前研究的難點(diǎn)和熱點(diǎn),相關(guān)研究進(jìn)展將對(duì)防控病毒的感染和流行具有重要的理論和實(shí)際意義。

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The roles of RNA-editing enzyme ADAR1 in EV71 infection and virus mutation

LIU Qing-qing, CHANG Zhang-mei, BAI Jin-jin, WANG Yan, LONG Jian-er△

(KeyLaboratoryofMedicalMolecularVirology,MinistriesofEducationandHealth-DepartmentofMedicalMicrobiologyandParasitology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China)

Objective To identify the role of RNA-editing enzyme ADAR1 (adenosine deaminase acting on RNA) in EV71 infection and virus mutation. Methods RNAi technology was applied to establish ADAR1 knock-down stable cell lines.Then the cells were served to evaluate the role of ADAR1 in EV71 infection by MTT assay for detecting virus-induced cell viability,virus plaque assay for quantification of the virus titer and the cellular susceptibility to the virus,and Western blot for virus protein expressions.ADAR1-mediated RNA editing can result in the genetic A-G and T-C mutations.To further determine whether the effects ofADAR1 on EV71 infection were correlated withADAR1-mediated EV71 RNA editing and therefore increased the viral mutations during the infection,the characteristics of EV71 mutation were analyzed based on the different full-length viral genomes from epidemic regions.The viral genome was also sequenced from the infected ADAR1 knock-down cells. Results AfterADAR1 knock-down,the cell viability decreased quickly after the virus infection,and formed much more and larger sizes of plaques than the control cells.The virus capsid protein VP1 expressions and virus titer in the cells culture media were both increased inADAR1 knock-down cells.Statistic analysis showed that A-G and T-C mutations were the major mutations of EV71,which were believed to be the hot sites for RNA-editing.However,the results of viral RNA genomic sequencing data indicated that ADAR1 did not edit EV71 genome directly. Conclusions ADAR1 was a restriction factor for controlling EV71.However,ADAR1 does not directly edit EV71 genome.

EV71; ADAR1; RNA-editing; virus mutation

國(guó)家自然科學(xué)基金(31570156);傳染病防治國(guó)家科技重大專(zhuān)項(xiàng)(2012ZX10004503-003)

Q343.1+3

A

10.3969/j.issn.1672-8467.2017.03.001

2016-06-27;編輯:段佳)

△Corresponding author E-mail:longjianer@fudan.edu.cn

*This work was supported by the National Natural Science Foundation of China (31570156) and the National Science and Technology Major Project on Infectious Diseases (2012ZX10004503-003).