CRL4在增殖的肝細(xì)胞系中上調(diào)乙型肝炎病毒抗原的分泌表達(dá)

朱園飛，魚康康，鄧強(qiáng)，高月求

1. 上海中醫(yī)藥大學(xué)附屬曙光醫(yī)院細(xì)胞免疫研究室，上海 201203； 2. 中國科學(xué)院上海巴斯德研究所分子病毒與免疫重點(diǎn)實(shí)驗(yàn)室，上海 200032

?

·論著·

CRL4在增殖的肝細(xì)胞系中上調(diào)乙型肝炎病毒抗原的分泌表達(dá)

朱園飛1,2，魚康康2，鄧強(qiáng)1,2，高月求1

1. 上海中醫(yī)藥大學(xué)附屬曙光醫(yī)院細(xì)胞免疫研究室，上海 201203； 2. 中國科學(xué)院上海巴斯德研究所分子病毒與免疫重點(diǎn)實(shí)驗(yàn)室，上海 200032

乙型肝炎病毒(hepatitis B virus，HBV)編碼的X蛋白(hepatitis B virus X protein，HBx)對(duì)HBV感染的啟始和維持至關(guān)重要。HBx可能作為病毒來源的接頭分子，介導(dǎo)Cullin-RING E3泛素連接酶4(Cullin-RING ubiquitin E3 ligase 4，CRL4)復(fù)合物對(duì)染色體外DNA限制因子SMC5/6的降解。最近研究發(fā)現(xiàn)，CRL4接頭分子DNA損傷結(jié)合蛋白1(DNA damage-binding protein 1，DDB1)可不依賴與HBx的相互作用而直接上調(diào)病毒的表達(dá)和復(fù)制。本研究基于HBx基因刪除(X-null)的HBV重組共價(jià)閉合環(huán)狀DNA(recombinant covalently closed circular DNA，rcccDNA)模型系統(tǒng)，在多種體外培養(yǎng)肝細(xì)胞系中證實(shí)上述發(fā)現(xiàn)。有意思的是，CRL4刺激rcccDNAX-null轉(zhuǎn)染細(xì)胞抗原分泌表達(dá)的效應(yīng)能被血清饑餓實(shí)驗(yàn)抵消。應(yīng)用尾靜脈高壓注射小鼠模型，同樣發(fā)現(xiàn)CRL4并不上調(diào)rcccDNAX-null在非增殖小鼠肝臟細(xì)胞中的表達(dá)。以上結(jié)果提示，細(xì)胞增殖特征與CRL4不依賴HBx上調(diào)病毒抗原分泌表達(dá)的效應(yīng)密切相關(guān)，有助于HBx生物學(xué)意義的準(zhǔn)確分析和理解。

乙型肝炎病毒；乙型肝炎病毒X蛋白；共價(jià)閉合環(huán)狀DNA；Cullin-RING E3泛素連接酶4；細(xì)胞增殖

很多病毒能利用宿主泛素-蛋白酶體系統(tǒng)調(diào)控其生命周期[1]。CUL4屬Cullin蛋白家族，包括兩個(gè)同源的CUL4A和CUL4B蛋白，與RING結(jié)構(gòu)域蛋白R(shí)OC1及DNA損傷結(jié)合蛋白1(DNA damage-binding protein 1，DDB1)組成Cullin-RING E3泛素連接酶4(Cullin-RING ubiquitin E3 ligase 4，CRL4)復(fù)合體。其中，DDB1作為接頭蛋白連接不同的底物識(shí)別亞基DCAF(DDB1-CUL4 associated factor)，降解特異性底物[2]。CRL4復(fù)合物參與機(jī)體內(nèi)廣泛的生理過程，包括細(xì)胞周期調(diào)控、染色質(zhì)表觀遺傳修飾、DNA復(fù)制和修復(fù)等[2]。

乙型肝炎病毒(hepatitis B virus，HBV)屬嗜肝DNA病毒科。在病毒感染肝細(xì)胞核內(nèi)，HBV修復(fù)為共價(jià)閉合環(huán)狀DNA(covalently closed circular DNA，cccDNA)，是病毒轉(zhuǎn)錄復(fù)制的模板和中心環(huán)節(jié)[3]。cccDNA非常穩(wěn)定，被認(rèn)為是HBV慢性感染的分子基礎(chǔ)[4-5]。在早先研究中，本課題組基于Cre/loxP 介導(dǎo)的位點(diǎn)特異性重組策略，由前體質(zhì)粒誘導(dǎo)重組cccDNA(recombinant cccDNA，rcccDNA)產(chǎn)生，首次建立了HBV cccDNA的體外培養(yǎng)細(xì)胞及小鼠實(shí)驗(yàn)?zāi)Ｐ?，獲得廣泛關(guān)注[6]。

HBV X蛋白(HBV X protein，HBx)能顯著上調(diào)HBV的轉(zhuǎn)錄和復(fù)制[7]。有研究認(rèn)為，HBV感染后宿主細(xì)胞在表觀遺傳學(xué)水平形成對(duì)cccDNA微小染色體的抑制[8]。最近報(bào)道顯示，HBx通過結(jié)合DDB1挾持CRL4，對(duì)一種宿主蛋白復(fù)合體SMC5/6[9]實(shí)現(xiàn)泛素化降解[10-11]，后者可能涉及宿主細(xì)胞針對(duì)染色體外病毒DNA的天然免疫機(jī)制。這一研究在HBV領(lǐng)域引起廣泛興趣，也引發(fā)了一定爭(zhēng)議。例如，Kim等基于HBx的突變分析發(fā)現(xiàn)，DDB1可上調(diào)HBV在宿主細(xì)胞的轉(zhuǎn)錄和復(fù)制，但并不依賴HBx與DDB1的相互作用[12]。

本研究應(yīng)用HBx基因刪除(X-null)的rcccDNA模型系統(tǒng)，對(duì)CRL4調(diào)控的HBV抗原分泌表達(dá)進(jìn)行分析，在多種體外培養(yǎng)肝細(xì)胞系中證實(shí)了Kim等[12]的發(fā)現(xiàn)。有意思的是，CRL4不依賴HBx上調(diào)病毒抗原分泌表達(dá)的效應(yīng)能被血清饑餓實(shí)驗(yàn)抵消，提示可能與體外培養(yǎng)細(xì)胞的增殖特征相關(guān)。與之一致的是，尾靜脈高壓注射小鼠模型顯示，CRL4并不上調(diào)rcccDNAX-null在非增殖小鼠肝臟細(xì)胞中的表達(dá)。

1 材料與方法

1.1 材料

prcccDNA和pCMV-Cre為本實(shí)驗(yàn)室早期構(gòu)建[6]。人肝癌細(xì)胞株Huh-7、HepG2和小鼠胚胎肝細(xì)胞BNL CL.2為本實(shí)驗(yàn)室保存。噻唑藍(lán)(methylthiazolyldiphenyl-tetrazolium，MTT)細(xì)胞增殖及細(xì)胞毒性檢測(cè)試劑盒購自碧云天生物技術(shù)研究所。FuGENE?HD 轉(zhuǎn)染試劑和海腎熒光素酶報(bào)告載體pRL-TK購自Promega公司。C57BL/6雄性小鼠購自上海靈暢生物科技有限公司。

1.2 方法

1.2.1 質(zhì)粒構(gòu)建 通過聚合酶鏈反應(yīng)(polymerase chain reaction，PCR)，在HBx基因ATG起始碼下游第22位引入C→T突變(CAA→TAA)，構(gòu)建HBx基因刪除的突變質(zhì)粒prcccDNAX-null。pCMV-CUL4A、pCMV-CUL4B及pCMV-DDB1基于真核表達(dá)載體pcDNA3.1構(gòu)建，分別表達(dá)人CUL4A、CUL4B和DDB1蛋白。短發(fā)夾RNA(short hairpin RNA，shRNA)編碼質(zhì)?；趐LKO.1載體構(gòu)建，插入序列如表1所示。根據(jù)pLKO.1 TRC Cloning Vector Protocol(Addgene)操作流程，在pLKO.1載體中插入相應(yīng)shRNA編碼序列，構(gòu)建質(zhì)粒由DNA測(cè)序鑒定(上海博尚生物技術(shù)有限公司)。

表1 shRNA編碼質(zhì)粒插入序列

Tab.1 Oligonucleotides used for construction of shRNA-encoding plasmids

NameSequence(5'-3')shCUL4A-1CCGGACTGTTTAGAACCCATATTATCTCGAGATAATATGGGTTCTAAACAGTTTTTTGshCUL4A-2CCGGGCAGAACTGATCGCAAAGCATCTCGAGATGCTTTGCGATCAGTTCTGCTTTTTGshCUL4B-1CCGGGCCATGAAAGAAGCATTTGAACTCGAGTTCAAATGCTTCTTTCATGGCTTTTTGshCUL4B-2CCGGGCAATTCTTCAGAAAGGTTTACTCGAGTAAACCTTTCTGAAGAATTGCTTTTTGshDDB1-1CCGGCAGCATTGACTTACCAGGCATCTCGAGATGCCTGGTAAGTCAATGCTGTTTTTGshDDB1-2CCGGCGTGTACTCTATGGTGGAATTCTCGAGAATTCCACCATAGAGTACACGTTTTTG

1.2.2 細(xì)胞培養(yǎng)和細(xì)胞轉(zhuǎn)染 Huh-7、HepG2和BNL CL.2細(xì)胞常規(guī)培養(yǎng)于含10%胎牛血清、100 U/mL青/鏈霉素的DMEM培養(yǎng)基(Gibco BRL)。血清饑餓實(shí)驗(yàn)中，胎牛血清濃度降至0.2%，每24 h更換培養(yǎng)液。DNA轉(zhuǎn)染采用FuGENE?HD 轉(zhuǎn)染試劑，按廠商提供的指導(dǎo)手冊(cè)進(jìn)行。用pRL-TK共轉(zhuǎn)染細(xì)胞，轉(zhuǎn)染后3 d收集細(xì)胞，檢測(cè)熒光素酶活性以校正轉(zhuǎn)染效率。

1.2.3 MTT檢測(cè) HepG2細(xì)胞在正?；蜓屦囸I條件下培養(yǎng)3 d，胰酶消化后以每孔10 000個(gè)細(xì)胞的密度接種于96孔板。貼壁后每孔加入10 μL MTT試劑，孵育4 h，加入100 μL甲瓚溶解液，孵育3～4 h至光鏡下觀察結(jié)晶紫全部溶解，在570 nm處測(cè)定光密度(optical density，OD)。

1.2.4 酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay，ELISA) 采用上?？迫A生物工程股份有限公司的ELISA試劑盒，檢測(cè)細(xì)胞培養(yǎng)上清液中HBV e抗原(HBV e antigen，HBeAg)和表面抗原(HBV surface antigen，HBsAg)的表達(dá)。用Bench Mark ELISA酶標(biāo)儀(Bio-Rad)讀取450 nm處OD。調(diào)整樣品稀釋度，使OD450為0.4～1.6(線性檢測(cè)范圍)，以信噪比(signal/noise)計(jì)算相對(duì)的抗原濃度。

1.2.5 小鼠尾靜脈高壓注射 小鼠尾靜脈高壓注射按文獻(xiàn)[6]進(jìn)行。在4～5周齡C57BL/6雄性小鼠中，共注射4 μg prcccDNAX-null，4 μg pCMV-Cre，以及8 μg真核表達(dá)質(zhì)粒pCMV-CUL4A、pCMV-CUL4B或pCMV-DDB1，4 d后眼眶后靜脈叢采血，檢測(cè)血清HBsAg和HBeAg表達(dá)。

2 結(jié)果

2.1 CRL4在Huh-7細(xì)胞中上調(diào)基于rcccDNAX-null的病毒抗原分泌表達(dá)

用prcccDNA/pCMV-Cre共轉(zhuǎn)染肝腫瘤細(xì)胞系，在細(xì)胞核中誘導(dǎo)大量rcccDNA產(chǎn)生，后者含有一段較短的loxP雜合內(nèi)含子編碼序列，能在病毒轉(zhuǎn)錄過程被高效移除[6]。通過過表達(dá)CRL4組成蛋白，評(píng)價(jià)CRL4對(duì)rcccDNA抗原表達(dá)的影響。應(yīng)用真核表達(dá)質(zhì)粒pCMV-CUL4A、pCMV-CUL4B及pCMV-DDB1，分別與prcccDNA/pCMV-Cre共轉(zhuǎn)染Huh-7細(xì)胞，在培養(yǎng)上清液中檢測(cè)到顯著升高的HBeAg和HBsAg(圖1A)。進(jìn)一步通過點(diǎn)突變方法構(gòu)建prcccDNAX-null突變體，誘導(dǎo)HBx基因刪除的rcccDNAX-null。過表達(dá)CUL4A或DDB1同樣觀察到基于rcccDNAX-null病毒抗原表達(dá)的上升，但過表達(dá)CUL4B未出現(xiàn)相似的結(jié)果(圖1B)。結(jié)果提示，在培養(yǎng)的Huh-7細(xì)胞中，CRL4能通過某種HBx不相關(guān)機(jī)制直接刺激HBV抗原的分泌表達(dá)。

2.2 CRL4在HepG2和BNL CL.2細(xì)胞中上調(diào)基于rcccDNAX-null的病毒抗原分泌表達(dá)

以前有研究顯示Huh-7細(xì)胞不適用于研究HBx相關(guān)生物學(xué)功能。本研究采用HepG2及BNL CL.2細(xì)胞，進(jìn)一步檢驗(yàn)CRL4對(duì)rcccDNAX-null抗原表達(dá)的影響。與圖1B相似，過表達(dá)CUL4A或DDB1，而不是CUL4B，能上調(diào)基于rcccDNAX-null的HBV抗原表達(dá)(圖2)。進(jìn)一步構(gòu)建shRNA編碼質(zhì)粒，能特異性敲低轉(zhuǎn)染細(xì)胞中內(nèi)源表達(dá)的CUL4A、CUL4B或DDB1(數(shù)據(jù)未列出)；分別與prcccDNAX-null/pCMV-Cre共轉(zhuǎn)染HepG2細(xì)胞，其中共轉(zhuǎn)染pLKO.1-shCUL4A或pLKO.1-shDDB1能顯著下調(diào)rcccDNAX-null的抗原表達(dá)。 以上結(jié)果顯示， CRL4 能在多種體外培養(yǎng)細(xì)胞系中通過HBx不相關(guān)機(jī)制調(diào)控HBV抗原的分泌表達(dá)。

A: Plasmids encoding the core components of CRL4 (pCMV-CUL4A, pCMV-CUL4B, and pCMV-DDB1) were cotransfected, respectively, with prcccDNA and pCMV-Cre, at a 2∶1∶1 ratio. Secretions of HBeAg and HBsAg in the cell culture medium were determined on day 3 after transfection. Results (normalized to luciferase activity) are expressed as fold change over that in the medium of pcDNA3.1-transfected cells. B: An HBx-mutant of prcccDNA was constructed to induce rcccDNAX-null. The effect of CRL4 on rcccDNAX-null-based viral expression was analyzed as described in A.*P<0.05,**P<0.01,***P<0.001. n.s., not significant.

圖1 CRL4不依賴HBx刺激Huh-7細(xì)胞中基于rcccDNA的HBV抗原分泌表達(dá)

Fig.1 CRL4 stimulated rcccDNA-based HBV antigen expression in Huh-7 cells in an HBx-independent manner

A: rcccDNAX-null-based HBV antigen expression in HepG2 and BNL CL.2 cells was determined as described in Fig.1. B: In HepG2 cells, shRNA-encoding plasmids targeting the expressions of human CUL4A, CUL4B, and DDB1 were transfected, respectively, with prcccDNA and pCMV-Cre at a 2∶1∶1 ratio. HBV antigen expression was determined on day 6 after transfection.*P<0.05,**P<0.01. n.s., not significant.

圖2 CRL4在HepG2和BNL CL.2細(xì)胞中上調(diào)基于rcccDNAX-null的HBV抗原分泌表達(dá)

Fig.2 CRL4 upregulated rcccDNAX-null-based HBV antigen expression in HepG2 and BNL CL.2 cells

2.3 CRL4不依賴HBx上調(diào)HBV抗原分泌表達(dá)可能與細(xì)胞增殖相關(guān)

大量證據(jù)表明CRL4調(diào)控細(xì)胞周期，在DNA復(fù)制和修復(fù)過程中起重要作用。本研究采用血清饑餓法抑制體外培養(yǎng)細(xì)胞的增殖[13]，對(duì)CRL4調(diào)控的rcccDNAX-null抗原分泌表達(dá)進(jìn)行分析。連續(xù)應(yīng)用0.2%胎牛血清體外培養(yǎng)HepG2，直接細(xì)胞計(jì)數(shù)和MTT實(shí)驗(yàn)顯示能明顯延緩細(xì)胞增殖(圖3A，左圖)，但仍保持相對(duì)健康的細(xì)胞活性(圖3A，右圖)。有意思的是，在此培養(yǎng)條件下，共轉(zhuǎn)染pCMV-CUL4A或pCMV-DDB1均不能顯著上調(diào)rcccDNAX-null的抗原分泌表達(dá)；共轉(zhuǎn)染pCMV-CUL4B明顯抑制rcccDNAX-null的抗原分泌表達(dá)(圖3B)。

生理?xiàng)l件下，體內(nèi)肝臟細(xì)胞的更迭頻率極低。鑒于CRL4在人與小鼠之間高度保守，本研究采用尾靜脈DNA高壓注射小鼠模型，對(duì)CRL4調(diào)控的rcccDNAX-null抗原表達(dá)進(jìn)行分析。對(duì)小鼠血清中HBV分泌抗原的分析表明，共注射pCMV-CUL4A、pCMV-CUL4B或pCMV-DDB1表達(dá)質(zhì)粒， 并不明顯影響 rcccDNAX-null在小鼠肝臟中的抗原表達(dá)(圖3C)。上述培養(yǎng)細(xì)胞和小鼠模型實(shí)驗(yàn)提示，CRL4不依賴HBx上調(diào)HBV抗原分泌表達(dá)可能與細(xì)胞增殖密切相關(guān)。

A: HepG2 cells were cultured in the presence of 0.2% or 10% fetal bovine serum (FBS). The cell proliferation and viability were estimated by direct cell counting (left) and MTT assay (right), at indicated time points. B: In the setting of serum starvation (0.2% FBS), the effects of CRL4 on rcccDNAX-nulll-based HBV antigen expression were analyzed as described in Fig.2A. C: pCMV-CUL4A, pCMV-CUL4B or pCMV-DDB1, was hydrodynamically coinjected with prcccDNA and pCMV-Cre at a 2∶1∶1 ratio to C57BL/6 mice (n=8 for each group). pcDNA3.1 was coinjected with prcccDNA and pCMV-Cre as control. HBV antigen production in mouse serum was determined by ELISA on day 4 after injection.**P<0.01. n.s., not significant.

圖3 CRL4基于rcccDNAX-null上調(diào)HBV抗原表達(dá)與快速細(xì)胞增殖相關(guān)

Fig.3 The upregulation of CRL4 on rcccDNAX-nulll-based HBV antigen expression was related to rapid cell proliferation

3 討論

HBV編碼的非結(jié)構(gòu)蛋白HBx被認(rèn)為具有多種復(fù)雜的調(diào)控功能，相關(guān)研究在近期獲得了一系列重要進(jìn)展。Lucifora等應(yīng)用刪除HBx的HBV突變株體外感染人原代肝臟細(xì)胞或分化的HepaRG細(xì)胞，發(fā)現(xiàn)HBx對(duì)有效HBV感染的啟始和維持至關(guān)重要，且與cccDNA微小染色體的H3組蛋白乙?；Ｊ矫芮邢嚓P(guān)[14]?；谶@一模型，Neuveut研究組報(bào)道，刪除HBx的病毒突變體通過H3K9二甲基化和三甲基化招募HP1因子，抑制cccDNA微小染色體的轉(zhuǎn)錄[8]。來自瑞士的Strubin研究組則報(bào)道，HBx可能作為一種病毒來源的DCAF分子，介導(dǎo)CRL4對(duì)染色體外DNA限制因子SMC5/6的泛素化降解[10]。

在眾多HBx候選結(jié)合分子中，DDB1與HBx的相互作用研究得較為確切，最早于1995年由Lee等[15]報(bào)道，并被許多實(shí)驗(yàn)室證實(shí)。DDB1是CRL4復(fù)合物中的接頭分子，在CUL4家族介導(dǎo)的蛋白泛素化降解過程中扮演重要角色。與Kim等的報(bào)道一致，本研究也觀察到過表達(dá)DDB1能不依賴HBx而直接上調(diào)HBV cccDNA的表達(dá)。為解釋這一現(xiàn)象，本課題組曾試圖將CRL4與HBV轉(zhuǎn)錄因子(如具有顯著轉(zhuǎn)錄抑制活性的Prox1[16]等)進(jìn)行關(guān)聯(lián)，但未獲得特別值得關(guān)注的發(fā)現(xiàn)。另一方面，CRL4的生物學(xué)功能主要涉及細(xì)胞增殖、DNA復(fù)制和修復(fù)等方面的精細(xì)調(diào)控，而HBV自然感染通常發(fā)生于靜止期的肝臟細(xì)胞。本研究設(shè)計(jì)了血清饑餓體外培養(yǎng)細(xì)胞實(shí)驗(yàn)及基于體內(nèi)肝細(xì)胞的小鼠尾靜脈高壓注射實(shí)驗(yàn)，均提示CRL4不依賴HBx上調(diào)HBV抗原分泌表達(dá)與細(xì)胞增殖相關(guān)。與之相一致的是，尾靜脈高壓注射小鼠模型中過表達(dá)CRL4相關(guān)蛋白并不上調(diào)野生(未刪除HBx)rcccDNA的轉(zhuǎn)錄。而生理?xiàng)l件下，小鼠模型中肝細(xì)胞基本處于G0期靜息狀態(tài)。

HBx作為重要的轉(zhuǎn)錄激活分子，對(duì)細(xì)胞的影響較為復(fù)雜。本研究在HBx氨基酸序列第7位引入終止密碼子時(shí)參考了很多實(shí)驗(yàn)室的通用做法[17-18]。值得注意的是，引入終止密碼子并不能終止mRNA的轉(zhuǎn)錄，有研究認(rèn)為HBV mRNA拼接產(chǎn)物能上調(diào)病毒的復(fù)制[19]，而HBV轉(zhuǎn)錄產(chǎn)物是否參與CRL4相關(guān)機(jī)制仍有待研究。

應(yīng)指出的是，HBV研究領(lǐng)域始終缺乏理想的細(xì)胞培養(yǎng)和體內(nèi)感染實(shí)驗(yàn)?zāi)Ｐ?。顯然，HBV在非增殖肝細(xì)胞和持續(xù)增殖的腫瘤細(xì)胞系中可能具有不同的病毒學(xué)特征。CRL4相關(guān)HBx調(diào)控機(jī)制研究是該領(lǐng)域的前沿和熱點(diǎn)，本研究的發(fā)現(xiàn)代表了一些不同的認(rèn)識(shí)，有助于對(duì)HBV病毒學(xué)進(jìn)行正確分析和理解。

[1] Minor MM, Slagle BL. Hepatitis B virus HBx protein interactions with the ubiquitin proteasome system [J]. Viruses, 2014, 6(11): 4683-4702.

[2] Jackson S, Xiong Y. CRL4s: the CUL4-RING E3 ubiquitin ligases [J]. Trends Biochem Sci, 2009, 34(11): 562-570.

[3] Seeger C, Mason WS. Hepatitis B virus biology [J]. Microbiol Mol Biol Rev, 2000, 64(1): 51-68.

[4] Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B [J]. Gut, 2015, 64(12):1972-1984.

[5] Zoulim F, Lebossé F, Levrero M. Current treatments for chronic hepatitis B virus infections [J]. Curr Opin Virol, 2016, 18: 109-116.

[6] Qi Z, Li G, Hu H, Yang C, Zhang X, Leng Q, Xie Y, Yu D, Zhang X, Gao Y, Lan K, Deng Q. Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice [J]. J Virol, 2014, 88(14): 8045-8056.

[7] Slagle BL, Bouchard MJ. Hepatitis B virus X and regulation of viral gene expression [J]. Cold Spring Harb Perspect Med, 2016, 6(3): a021402.

[8] Rivière L, Gerossier L, Ducroux A, Dion S, Deng Q, Michel ML, Buendia MA, Hantz O, Neuveut C. HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase [J]. J Hepatol, 2015, 63 (5): 1093-1102.

[9] Fernandez-Capetillo O. The (elusive) role of the SMC5/6 complex [J]. Cell Cycle, 2016, 15(6): 775-776.

[10] Decorsière A, Mueller H, van Breugel PC, Abdul F, Gerossier L, Beran RK, Livingston CM, Niu C, Fletcher SP, Hantz O, Strubin M. Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor [J]. Nature, 2016, 531(7594): 386-389.

[11] Murphy CM, Xu Y, Li F, Nio K, Reszka-Blanco N, Li X, Wu Y, Yu Y, Xiong Y, Su L. Hepatitis B virus X protein promotes degradation of SMC5/6 to enhance HBV replication [J]. Cell Rep, 2016, 16(11): 2846-2854.

[12] Kim W, Lee S, Son Y, Ko C, Ryu WS. DDB1 stimulates viral transcription of hepatitis B virus via HBx-independent mechanisms [J]. J Virol, 2016, 90(21): 9644-9653.

[13] Pirkmajer S, Chibalin AV. Serum starvation: caveat emptor [J]. Am J Physiol Cell Physiol, 2011, 301(2): C272-C279.

[14] Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, Zoulim F, Hantz O, Protzer U. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection [J]. J Hepatol, 2011, 55(5): 996-1003.

[15] Lee TH, Elledge SJ, Butel JS. Hepatitis B virus X protein interacts with a probable cellular DNA repair protein [J]. J Virol, 1995, 69(2): 1107-1114.

[16] Qin J, Zhai J, Hong R, Shan S, Kong Y, Wen Y, Wang Y, Liu J, Xie Y. Prospero-related homeobox protein (Prox1) inhibits hepatitis B virus replication through repressing multiple cis regulatory elements [J]. J Gen Virol, 2009, 90(Pt 5): 1246-1255.

[17] Melegari M, Scaglioni PP, Wands JR. Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication [J]. J Virol, 1998, 72 (3): 1737-1743.

[18] Gearhart TL, Bouchard MJ. The hepatitis B virus X protein modulates hepatocyte proliferation pathways to stimulate viral replication [J]. J Virol, 2010, 84(6): 2675-2686.

[19] Chen WN, Chen JY, Lin WS, Lin JY, Lin X. Hepatitis B doubly spliced protein, generated by a 2.2 kb doubly spliced hepatitis B virus RNA, is a pleiotropic activator protein mediating its effects via activator protein-1- and CCAAT/enhancer-binding protein-binding sites [J]. J Gen Virol, 2010, 91(Pt 10): 2592-2600.

《微生物與感染》征訂啟事

《微生物與感染》重點(diǎn)介紹國內(nèi)外微生物學(xué)基礎(chǔ)研究與臨床相結(jié)合的研究成果和新進(jìn)展，內(nèi)容涉及與人類、動(dòng)物和植物感染有關(guān)的微生物，如病毒、細(xì)菌、真菌、立克次體、螺旋體、支原體等的生物學(xué)及分子生物學(xué)特性、抗感染免疫、實(shí)驗(yàn)室診斷技術(shù)以及臨床感染等方面的研究。主要欄目有特約專稿、論著、病例分析、綜述等?？晒氖挛⑸锱c感染的教學(xué)、科研、醫(yī)療等工作者參考。歡迎廣大讀者到當(dāng)?shù)剜]局訂閱。

地址：上海市醫(yī)學(xué)院路138號(hào) 郵編：200032 電話：021-54237633

電子郵箱：jmi@fudan.edu.cn 網(wǎng)站：http://jmi.fudan.edu.cn

雙月25日出版 統(tǒng)一刊號(hào)：ISSN 1673-6184 CN31-1966/R 郵發(fā)代號(hào)：4-341

定價(jià)：12.00元/冊(cè) 全年定價(jià)：72.00元/冊(cè)

《微生物與感染》編輯部

s. GAO Yueqiu, E-mail: gaoyueqiu@hotmail.com；DENG Qiang, E-mail:dengqiang@sibs.ac.cn

CRL4 upregulates secretion of hepatitis B virus antigens in proliferating liver cell lines

ZHU Yuanfei1,2, YU Kangkang2, DENG Qiang1,2, GAO Yueqiu1

1．Laboratory of Cellular Immunity, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; 2. Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200032, China

Hepatitis B virus (HBV) regulatory protein HBx is required to initiate and maintain productive virus replication. HBx is thought to achieve these functions by hijacking Cullin-RING ubiquitin E3 ligase 4 (CRL4) to target the SMC5/6 complex, a restriction factor for extrachromosomal viral DNA degradation. Studies also indicated that DNA damage-binding protein 1 (DDB1), an adaptor protein within CRL4 complex, may stimulate HBV transcription via a mechanism that does not involve an interaction with HBx. In the present study, using a model system of recombinant covalently closed circular DNA (rcccDNA) of HBV, it was confirmed that CRL4 could upregulate the secretion of HBV antigens in the absence of HBx (rcccDNAX-null) in cultured hepatoma cell lines. However, this effect of CRL4 was abolished by serum deprivation in the cell culture. In addition, it was found that CRL4 failed to stimulate the expression of rcccDNAX-nullin non-proliferating hepatocytes in a mouse model with DNA hydrodynamic injection. Thus, the HBx-independent, CRL4-upregulated HBV expression is likely to be closely related to rapid proliferation of cultured hepatoma cell lines. The study thus revealed a previously unappreciated role of CRL4 in HBV virology, and would be helpful for better understanding of the biological significance of HBx.

Hepatitis B virus; Hepatitis B virus X protein; Covalently closed circular DNA; Cullin-RING ubiquitin E3 ligase 4; Cell proliferation

國家自然科學(xué)基金(81471950、81672034)，上海市科學(xué)技術(shù)委員會(huì)科研計(jì)劃項(xiàng)目(16401970600)

高月求，鄧強(qiáng)

2017-02-28)