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        DC-SIGN與mCEACAM1a分子相互作用調(diào)控鼠冠狀病毒復(fù)制

        2018-06-12 07:27:36張浩旸郭佳慧趙蘭娟姚茜茜文榮徐婭佳王玉燕葉榮
        微生物與感染 2018年3期
        關(guān)鍵詞:病毒感染孵育磷酸化

        張浩旸,郭佳慧,趙蘭娟,姚茜茜,文榮,徐婭佳,王玉燕,葉榮

        1. 復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)系, 上海 200032; 2. 第二軍醫(yī)大學(xué)微生物學(xué)教研室, 上海 200433

        C型凝集素受體(C-type lectin receptor,CLR)調(diào)節(jié)病毒感染與病毒種類(lèi)和易感細(xì)胞存在的其他病毒特異性受體密切相關(guān)。來(lái)源不同的CLR如樹(shù)突細(xì)胞特異性細(xì)胞間黏附分子-3結(jié)合非整合素分子(dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin,DC-SIGN)、肝/淋巴結(jié)特異性細(xì)胞間黏附分子-3結(jié)合非整合素分子(liver/lymph node-specific intercellular adhesion molecules-3-grabbing non-integrin, L-SIGN)和LSECtin(liver and lymph node sinusoidal endothelial cell C-type lectin)均能與病毒表面糖蛋白相互作用,協(xié)助多種病毒感染和擴(kuò)散,包括皰疹病毒、反轉(zhuǎn)錄病毒、正黏和副黏病毒、冠狀病毒及大多數(shù)出血熱病毒和腦炎病毒等[1]。病毒對(duì)CLR的依賴程度也不同。如DC-SIGN是決定登革熱病毒感染DC并在其中復(fù)制的受體[2],但對(duì)丙型肝炎病毒(hepatitis C virus,HCV)而言只是攜帶和轉(zhuǎn)運(yùn)的輔助性受體[3]。對(duì)大多數(shù)病原體而言,DC-SIGN主要表現(xiàn)為模式識(shí)別受體(pattern recognition receptor,PRR)功能,如結(jié)核分枝桿菌和人類(lèi)免疫缺陷病毒(human immunodeficiency virus,HIV)與DC-SIGN相互作用后,可通過(guò)激活核因子κB(nuclear factor κB,NF-κB)產(chǎn)生免疫應(yīng)答分子白細(xì)胞介素10(interleukin 10,IL-10)[4]。病毒進(jìn)入細(xì)胞復(fù)制是誘發(fā)細(xì)胞應(yīng)答的關(guān)鍵,NF-κB激活還需病原體同時(shí)激活Toll樣受體(Toll-like receptor,TLR)[4-5]。

        鼠肝炎病毒(mouse hepatitis virus,MHV)是乙型冠狀病毒(beta coronavirus)原型種,可導(dǎo)致小鼠肝炎和中樞神經(jīng)性病變,是研究冠狀病毒分子生物學(xué)和致病性的經(jīng)典模式病毒[6-7]。鼠癌胚抗原相關(guān)細(xì)胞黏附分子1(mouse carcinoembryonic antigen-related cell adhesion molecule 1,mCEACAM1)是MHV的主要受體[8]。mCEACAM1是典型的細(xì)胞黏附分子,其膜內(nèi)區(qū)包括蛋白激酶C(protein kinase C,PKC)和免疫受體酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif,ITIM)等,ITIM通過(guò)激活SHP1/2信號(hào)通路參與免疫耐受機(jī)制[9]。

        人DC-SIGN能促進(jìn)動(dòng)物冠狀病毒感染,如貓傳染性腹膜炎病毒(feline infectious peritonitis virus,F(xiàn)IPV)和禽傳染性支氣管炎病毒(infectious bronchitis virus,IBV)感染[10-11]。但沒(méi)有證據(jù)表明人DC-SIGN能介導(dǎo)鼠冠狀病毒感染異源細(xì)胞[6-7]。本研究比較了穩(wěn)定表達(dá)DC-SIGN與L-SIGN的不同類(lèi)型細(xì)胞中鼠冠狀病毒的復(fù)制水平,發(fā)現(xiàn)L-SIGN對(duì)鼠冠狀病毒復(fù)制的抑制作用較DC-SIGN更明顯,這主要與兩者胞內(nèi)區(qū)序列差異有關(guān)。DC-SIGN調(diào)節(jié)鼠冠狀病毒復(fù)制的作用依賴病毒受體mCEACAM1a的存在,不能介導(dǎo)病毒感染無(wú)受體表達(dá)的細(xì)胞。其調(diào)控機(jī)制可能是以三聚體的形式與病毒受體相互作用,改變了病毒受體和細(xì)胞的功能,包括阻止鼠冠狀病毒感染導(dǎo)致的細(xì)胞外信號(hào)調(diào)節(jié)激酶(extracellular signal-regulated kinase,ERK)途徑信號(hào)轉(zhuǎn)導(dǎo)下調(diào)等。

        1 材料與方法

        1.1 材料

        小鼠腦神經(jīng)瘤細(xì)胞Neuro-2a購(gòu)自中國(guó)科學(xué)院典型培養(yǎng)物保藏委員會(huì)細(xì)胞庫(kù);小鼠成纖維細(xì)胞NIH/3T3購(gòu)自美國(guó)典型微生物菌種保藏中心(American Type Culture Collection,ATCC);人神經(jīng)瘤細(xì)胞SH-SY5Y來(lái)自復(fù)旦大學(xué)腦科學(xué)研究院黃芳博士實(shí)驗(yàn)室;大鼠胚胎肺細(xì)胞L2來(lái)自美國(guó)紐約州衛(wèi)生部沃茲沃思研究中心Dr. Paul Masters實(shí)驗(yàn)室。鼠肝炎病毒(MHV-A59)來(lái)自Dr. Paul Masters實(shí)驗(yàn)室[12],豬傳染性胃腸炎病毒(porcine transmissible gastroenteritis virus,TGEV)(Purdue)購(gòu)自ATCC。真核表達(dá)質(zhì)粒pcDNA3.1/myc-HisA(Invitrogen V385-20)由本實(shí)驗(yàn)室保存,含人DC-SIGN、L-SIGN基因的cDNA質(zhì)粒來(lái)自美國(guó)馬里蘭州弗雷德里克國(guó)家衛(wèi)生研究院Dr. Wu實(shí)驗(yàn)室[13]。感受態(tài)DH5α和Top10細(xì)胞購(gòu)自天根生化科技(北京)有限公司??故蠊跔畈《臼蠖嗫寺】贵w(pAb-MHV)由本實(shí)驗(yàn)室制備[14];鼠抗人DC-SIGN(CD209)、L-SIGN(CD209L)抗體購(gòu)自BD PharMingen;兔抗Raf-1、絲裂原活化的細(xì)胞外信號(hào)調(diào)節(jié)激酶(mitogen-activated extracellular signal-regulated kinase,MEK)、ERK及磷酸化抗體購(gòu)自Cell Signaling Technology公司;β-actin和GADPH購(gòu)自BD公司;抗鼠和抗兔二抗購(gòu)自GE醫(yī)療生命科學(xué)。

        1.2 方法

        1.2.1細(xì)胞培養(yǎng)與病毒感染Neuro-2a、SH-SY5Y、NIH/3T3及NIH/3T3_vR細(xì)胞用含10%胎牛血清(fetal bovine serum,F(xiàn)BS)(Gibco公司)的高糖DMEM(Hyclone公司)于37 ℃、5% CO2傳代培養(yǎng),傳代間隔3~4 d。將病毒感染用細(xì)胞培養(yǎng)36~48 h,密度達(dá)60%~80%,根據(jù)不同實(shí)驗(yàn)需要,調(diào)整病毒感染復(fù)數(shù)(multiplicity of infection,MOI)為1~10,感染1 h后更換為新鮮完全培養(yǎng)基,感染計(jì)時(shí)從加入病毒開(kāi)始。

        1.2.2流式細(xì)胞術(shù)細(xì)胞于T25培養(yǎng)瓶中培養(yǎng) 48 h,密度約為80%,用 0.25% 胰酶消化細(xì)胞,完全培養(yǎng)基終止消化。轉(zhuǎn)移細(xì)胞懸液至15 mL離心管,1 000 r/min離心8 min,用磷酸鹽緩沖液(phosphate buffered saline,PBS)洗細(xì)胞2次。250 μL PBS〔含1%牛血清白蛋白(bovine serum albumin,BSA)〕重懸細(xì)胞,分裝至 1.5 mL管中(每管100 μL約含1×106個(gè)細(xì)胞),2 500 r/min離心5 min,棄上清液。加入4 μg/mL CD209 mAb或CD209L mAb 100 μL,室溫孵育1 h,800 μL PBS洗細(xì)胞2次。加入100 μL稀釋的異硫氰酸熒光素(fluorescein isothiocyanate,F(xiàn)ITC)-羊抗鼠IgG,4 ℃避光孵育30 min,800 μL PBS洗細(xì)胞2次。用400 μL 1%多聚甲醛重懸細(xì)胞,4 ℃避光保存。用FACSCalibur流式細(xì)胞儀(BD公司)檢測(cè),CELLQuest軟件獲取和分析結(jié)果。

        1.2.3病毒復(fù)制曲線測(cè)定與噬斑實(shí)驗(yàn)病毒感染后定時(shí)收集培養(yǎng)上清液和細(xì)胞裂解物,間隔時(shí)間前期為4 h,后期為8~12 h。采用噬斑實(shí)驗(yàn)測(cè)定上清液病毒滴度,蛋白免疫印跡法檢測(cè)細(xì)胞中病毒S或N蛋白。將鼠L2細(xì)胞鋪至D60培養(yǎng)皿中,37 ℃、5% CO2培養(yǎng)36~48 h,使密度達(dá)80%。樣品用含2% FBS的1×DMEM進(jìn)行10倍系列稀釋至10-6。每個(gè)培養(yǎng)皿加入1 mL稀釋上清液,37 ℃吸附2 h;等量混合60 ℃ 1.95% 瓊脂和37 ℃含10% FBS的2×DMEM,并維持50 ℃左右備用;吸去培養(yǎng)皿中的病毒液,加7 mL上述混合瓊脂,凝固后置37 ℃、5% CO2培養(yǎng)40 h。將中性紅染液3 mL加至培養(yǎng)皿,37 ℃染色8 h后計(jì)數(shù)。

        1.2.4人DC-SIGN和L-SIGN及融合突變體重組真核表達(dá)質(zhì)粒構(gòu)建分子克隆按常規(guī)方法進(jìn)行[15],試劑來(lái)自NEB和TaKaRa公司。pcDNA3_DC-SIGN和pcDNA3_L-SIGN通過(guò)聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)擴(kuò)增基因片段(引物T7:5′-AATACGACTCACTATAG-3′/BGH-R: 5′-TAGAAGGCACAGTCGAGG-3′),分別經(jīng)BamH Ⅰ和EcoR Ⅴ位點(diǎn)插入pcDNA3.1/myc-HisA。pcDNA3_LYdc通過(guò)二輪PCR將DC-SIGN N端膜內(nèi)區(qū)片段(引物T7/SIGN-2R:5′-CCATGGCCAAGACACCCTG-3′)和L-SIGN C端跨膜區(qū)-頸區(qū)-糖類(lèi)識(shí)別區(qū)(carbohydrate recognition domain,CRD)片段(引物SIGN-1F:5′-CAGGGTGTCTTGGCCATGG-3′/BGH-R)融合(引物T7/BGH-R);pcDNA3_LCdc通過(guò)引物將DC-SIGN C端糖類(lèi)結(jié)合區(qū)末端16個(gè)氨基酸加至L-SIGN C端(引物T7/SIGN-4R:5′-TGGATA-TCTGCAGAATTCTACGCAGGAGGGGGGTT-TGGGGTGGCAGGGGCTGGAGAAAGAAAC-TGTTCTTCGTCTCTGAAGCAGGC-3′),融合片段經(jīng)BamH Ⅰ和EcoR Ⅴ位點(diǎn)插入pcDNA3.1/myc-HisA。

        1.2.5穩(wěn)定表達(dá)細(xì)胞系建立細(xì)胞培養(yǎng)于6孔板,達(dá)70%~80%密度時(shí)更換為無(wú)血清Opti MEM(Gibco公司),繼續(xù)培養(yǎng)2 h。取5 μL(2.5~5 μg)質(zhì)粒加至100 μL無(wú)血清Opti MEM,另取5 μL Lipofectamine 2000(Invitrogen公司)加至100 μL無(wú)血清Opti MEM,將兩者輕輕混勻,室溫靜置5 min后加至培養(yǎng)板,37 ℃、5% CO2培養(yǎng)6 h,培養(yǎng)液換為10% FBS-DMEM。每個(gè)樣品轉(zhuǎn)染兩個(gè)培養(yǎng)孔,一孔于48 h收集細(xì)胞裂解液檢測(cè)蛋白表達(dá)情況,另一孔于72 h更換為含500~1 000 ng/μL G418(Invitrogen公司)的10% FBS-DMEM維持培養(yǎng)。1周后轉(zhuǎn)入T25培養(yǎng)瓶,每周更換2~3次培養(yǎng)基和1次培養(yǎng)瓶,3~4周后轉(zhuǎn)入T75培養(yǎng)瓶擴(kuò)大培養(yǎng)并凍存。

        1.2.6RNA提取及反轉(zhuǎn)錄PCR吸去D60培養(yǎng)皿或T25培養(yǎng)瓶中培養(yǎng)基,加入1 mL TRIzol。室溫靜置5 min后轉(zhuǎn)移至離心管中,加0.2 mL氯仿并充分混勻,4 ℃、12 000 r/min離心5 min。將上層水相轉(zhuǎn)移至新離心管中,加入0.5 mL異丙醇,充分混勻于 -20 ℃ 靜置2 h以上。4 ℃、12 000 r/min離心10 min,棄上清液,沉淀用70%乙醇洗2次,室溫晾干。用25~50 μL無(wú)菌水(含1 U/μL RNase Inhibitor)溶解RNA,-80 ℃保存。cDNA合成:取10 μL RNA與2 μL Oligo-dT(10 mmol/L)混勻,80 ℃ 孵育10 min,冷卻至室溫;加入4 μL 5×M-MLV Buffer、2 μL 10 mmol/L dNTP、1 μL RNase Inhibitor(40 U/μL)、1 μL M-MLV(200 U/μL),42 ℃ 孵育1 h,70 ℃孵育15 min,冰上冷卻。PCR:混合32 μL ddH2O、10 μL 5×PCR Buffer、1 μL dNTP(10 mmol/L)、0.5 μLTaq酶,加入2 μL特異性引物和5 μL cDNA,94 ℃變性5 min,30個(gè)循環(huán)(94 ℃ 30 s、52 ℃ 45 s、72 ℃ 60 s),72 ℃延伸5 min。產(chǎn)物用1%瓊脂糖電泳檢測(cè)。

        1.2.7蛋白免疫印跡法蛋白電泳及轉(zhuǎn)印實(shí)驗(yàn)用Bio-Rad Mini裝置完成。樣本緩沖液(Tris/Glycine/SDS)購(gòu)自CalBioChem公司。細(xì)胞裂解液樣品加等體積2×樣品緩沖液,100 ℃、5 min變性處理。配制丙烯酰胺(Amresco公司)凝膠(濃縮膠5%,分離膠8%~12%),15 mA恒流電泳60~90 min。蛋白相對(duì)分子質(zhì)量采用預(yù)染PageRuler標(biāo)準(zhǔn)。分離膠中蛋白在4 ℃、250 mA條件下經(jīng)1~3 h轉(zhuǎn)移至聚偏氟乙烯(polyvinylidene fluoride,PVDF)膜(Bio-Rad公司)。PVDF膜用5%脫脂奶粉封閉,與一定稀釋度的一抗4 ℃孵育過(guò)夜,洗滌后與二抗室溫孵育1 h,采用 增強(qiáng)化學(xué)發(fā)光法(enhanced chemiluminescence,ECL)(Bio-Rad公司)發(fā)光,X線片顯影。

        1.2.8免疫熒光染色將傳代細(xì)胞鋪至玻璃底小型細(xì)胞培養(yǎng)皿(In Vitro Scientific公司),培養(yǎng)24~48 h至細(xì)胞密度為60%~80%,用MOI=10.0 的病毒感染至特定時(shí)間,收集培養(yǎng)液。單層細(xì)胞用冷PBS洗3次,4%多聚甲醛(Sigma公司)室溫固定30 min,5% BSA封閉1 h,4 ℃保存。一抗4 ℃孵育過(guò)夜,洗滌后二抗室溫孵育1 h,與4′,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)孵育30 min,晾干封片。用Leica TCS SP8激光共聚焦顯微鏡觀察結(jié)果。

        2 結(jié)果

        2.1 DC-SIGN 和 L-SIGN過(guò)表達(dá)抑制鼠冠狀病毒復(fù)制

        為觀察DC-SIGN和L-SIGN對(duì)鼠冠狀病毒復(fù)制的影響,將構(gòu)建的真核重組質(zhì)粒轉(zhuǎn)染至NIH/3T3細(xì)胞,經(jīng)篩選、傳代培養(yǎng)、流式細(xì)胞檢測(cè)分析,獲得能穩(wěn)定表達(dá)人DC-SIGN和L-SIGN的NIH/3T3細(xì)胞系(圖1A)。鼠冠狀病毒(MHV-A59)感染穩(wěn)定表達(dá)DC-SIGN或L-SIGN的細(xì)胞系。病毒測(cè)定結(jié)果表明,DC-SIGN和L-SIGN過(guò)表達(dá)均可降低MHV-A59滴度(圖1B);病毒S和N蛋白表達(dá)水平降低并滯后(圖1C)。L-SIGN對(duì)MHV-A59復(fù)制和蛋白表達(dá)的抑制作用比DC-SIGN更顯著,可能與DC-SIGN和L-SIGN之間的差異有關(guān)(圖1C)。

        2.2 DC-SIGN和L-SIGN調(diào)節(jié)鼠冠狀病毒復(fù)制主要與膜內(nèi)區(qū)序列有關(guān)

        DC-SIGN與L-SIGN的CRD同源,但兩者分布在不同類(lèi)型細(xì)胞對(duì)病毒感染的調(diào)節(jié)存在差異[16-17]。比較一級(jí)結(jié)構(gòu)可看出,兩者N端胞內(nèi)區(qū)均包含雙亮氨酸和三酸性氨基酸簇(LL/EED),胞外區(qū)C端的CRD同源性為84%,但L-SIGN胞內(nèi)區(qū)序列較長(zhǎng),無(wú)YxxL酪氨酸激酶信號(hào),末端缺少16個(gè)富含脯氨酸的序列(圖2A)。為此,分別將DC-SIGN膜內(nèi)區(qū)序列和膜外區(qū)C端16個(gè)氨基酸同源替換或添加至L-SIGN,構(gòu)建兩個(gè)嵌合突變體LYdc和LCdc(圖2A)。

        將pcDNA3_DC-SIGN、pcDNA3_L-SIGN、pcDNA3_LYdc、pcDNA3_LCdc質(zhì)粒分別轉(zhuǎn)染鼠腦神經(jīng)瘤細(xì)胞Neuro-2a,建立穩(wěn)定表達(dá)相應(yīng)蛋白的細(xì)胞系(圖2B)。在這些Neuro-2a細(xì)胞系中,DC-SIGN和L-SIGN均能抑制MHV-A59復(fù)制, L-SIGN的抑制作用較DC-SIGN更顯著(圖2C)。而同源替換DC-SIGN的功能域顯著減弱了L-SIGN對(duì)病毒復(fù)制的抑制作用,LYdc比LCdc顯著。在過(guò)表達(dá)LYdc的細(xì)胞系中,病毒滴度恢復(fù)至對(duì)照細(xì)胞水平(圖2C)。病毒S蛋白的表達(dá)水平與病毒滴度變化趨勢(shì)基本一致(圖2D)。

        鼠冠狀病毒感染導(dǎo)致Neuro-2a細(xì)胞信號(hào)通路分子MEK和ERK磷酸化水平顯著下降,Raf-1磷酸化水平無(wú)明顯影響(圖2E)。鼠冠狀病毒感染過(guò)表達(dá)DC-SIGN、L-SIGN及其嵌合蛋白LYdc和LCdc的Neuro-2a細(xì)胞系中,ERK和Raf-1磷酸化水平不受影響,MEK磷酸化水平顯著下降,但下降程度低于未轉(zhuǎn)染細(xì)胞(圖2E)。

        2.3 DC-SIGN 和 L-SIGN抑制鼠冠狀病毒復(fù)制依賴病毒受體的存在

        鼠冠狀病毒可感染表達(dá)病毒受體mCEACAM1a的NIH/3T3和Neuro-2a細(xì)胞(圖1~2)。為研究DC-SIGN調(diào)節(jié)鼠冠狀病毒復(fù)制與受體mCEACAM1a的關(guān)系,將NIH/3T3于39 ℃反復(fù)傳代,獲得不能轉(zhuǎn)錄mCEACAM1a mRNA的缺陷細(xì)胞株(NIH/3T3_vR),MHV-A59在其內(nèi)的復(fù)制水平顯著降低(圖3A)。

        將pcDNA3_DC-SIGN、pcDNA3_L-SIGN、pcDNA3_LYdc、pcDNA3_LCdc分別轉(zhuǎn)染缺陷細(xì)胞株NIH/3T3_vR,能穩(wěn)定表達(dá)4種蛋白(圖3B);MHV-A59在4種轉(zhuǎn)染細(xì)胞中及不表達(dá)重組蛋白的NIH/3T3_vR細(xì)胞中病毒滴度無(wú)顯著差異(圖3B)。MHV-A59在穩(wěn)定表達(dá)DC-SIGN但不表達(dá)病毒受體mCEACAM1a的人神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y中不能復(fù)制,感染細(xì)胞中檢測(cè)不到病毒N蛋白;而表達(dá)DC-SIGN的鼠Neuro-2a細(xì)胞中N蛋白表達(dá)水平顯著下降(圖3C)。結(jié)果提示,DC-SIGN和L-SIGN對(duì)病毒復(fù)制的影響依賴病毒受體mCEACAM1a的表達(dá)。

        2.4 DC-SIGN與病毒受體相互作用

        為分析細(xì)胞表面DC-SIGN與mCEACAM1a可能存在的相互作用,用MHV-A59感染穩(wěn)定表達(dá)DC-SIGN的NIH/3T3細(xì)胞,免疫熒光法檢測(cè)細(xì)胞表面的DC-SIGN和mCEACAM1a。結(jié)果顯示,隨MHV-A59孵育時(shí)間延長(zhǎng),DC-SIGN和mCEACAM1a共定位更明顯,而在無(wú)病毒感染細(xì)胞中兩者共定位不明顯(圖4A)。蛋白免疫印跡檢測(cè)結(jié)果表明,MHV-A59感染穩(wěn)定表達(dá)DC-SIGN的Neuro-2a細(xì)胞可導(dǎo)致受體mCEACAM1a減少,蛋白交聯(lián)劑DTSSP處理后mCEACAM1a L亞型增加(圖4B),而同樣處理的NIH/3T3細(xì)胞無(wú)增加現(xiàn)象(圖4B),與兩種細(xì)胞的mCEACAM1a異型分布差異一致。

        A: DC-SIGN or L-SIGN expressed on the membrane of murine NIH/3T3 cells stably transfected with eukaryotic expression plasmids were detected by flow cytometry. B: Replication of murine coronavirus in murine NIH/3T3 cells stably transfected with DC-SIGN or L-SIGN plasmids. Viruses in supernatants were titrated at 4 to 48 h postinfection (hpi) by plaque assay (titer shown in lg PFU/mL). C: Murine coronavirus N and S proteins in murine NIH/3T3 cells stably transfected with DC-SIGN or L-SIGN plasmids were analyzed by Western blotting.

        圖1DC-SIGN和L-SIGN對(duì)鼠冠狀病毒在NIH/3T3細(xì)胞中復(fù)制的影響

        Fig.1EffectsofDC-SIGNandL-SIGNonreplicationofmurinecoronavirusinNIH/3T3cells

        A: Diagram shows partial amino acid sequences and functional domains of DC-SIGN and L-SIGN. Signal motifs in N-terminal endodomain and C-terminal 21 or 5 residues of carbohydrate recognition domain (CRD) are shown. The transmembrane (TM) and neck regions (NRs) are the representive of rectangles. Chimeric L-SIGN_Ydc and _Cdc were constructed by grafting YxxL and C-terminal 16-peptide from DC-SIGN into L-SIGN respectively. B: Expression levels were analyzed by Western blotting in murine Neuro-2a cells stably transfected with DC-SIGN, L-SIGN, chimeric L-SIGN_Ydc or _Cdc plasmids. C: Titers of murine coronavirus in murine Neuro-2a cells stably transfected with above plasmids were titrated (lg PFU/mL of supernatant) at 4, 8, 12, 16, 20, 24 hpi. D: Viral S protein in the cell lysates was measured by Western blotting at 12,16,20,24 hpi. E: Regulation of MAPK pathway at early infection of murine coronavirus. Neuro-2a cells stably expressing DC-SIGN, L-SIGN, chimeric L-SIGN_Ydc or _Cdc were infected with 2.0 MOI of murine coronavirus (MHV-A59) for 1 h. Total and phosphorylated (p-) Raf, MEK, and ERK were analyzed by Western blotting. β-actin is shown as a loading control.

        圖2DC-SIGNN端和C端氨基酸在鼠冠狀病毒復(fù)制調(diào)控中的作用

        Fig.2RolesofN-andC-terminalresiduesofDC-SIGNinreplicationofmurinecoronavirus

        A: RT-PCR of mCEACAM1a and murine coronavirus replication in receptor-deficient NIH/3T3 (NIH/3T3_vR), wild-type NIH/3T3 (NIH/3T3_wt), Raw264.7 and Neuro-2a cells. Diagram shows the functional domains of polypeptide and mRNA of mCEACAM1a. Amplification primers and product size were marked. B: DC-SIGN, L-SIGN, chimeric L-SIGN_Ydc and _Cdc plasmids were transfected in receptor-deficient NIH/3T3 cells. N protein and titres of murine coronavirus at 24 hpi were analyzed by Western blotting and plaque assay respectively.**P<0.01. NS: no significant difference. C: N protein of murine coronavirus was detected in Neuro-2a cells expressing DC-SIGN, SH-SY5Y cells expressing mCEACAM1a-2S or DC-SIGN at 24 hpi by Western blotting.

        圖3DC-SIGN對(duì)鼠冠狀病毒復(fù)制的調(diào)控依賴病毒受體分子mCEACAM1a

        Fig.3RegulationofDC-SIGNonmurinecoronavirusreplicationisdependentonviralreceptormCEACAM1a

        蛋白交聯(lián)劑處理后,DC-SIGN形成明顯的三聚體和四聚體(圖4B)。與非同源TGEV感染細(xì)胞和無(wú)病毒感染細(xì)胞比較,MHV-A59感染導(dǎo)致DC-SIGN三聚體減少,而DC-SIGN四聚體及單體與mCEACAM1a異源二聚體均輕度增加(圖4B)。結(jié)果提示,鼠冠狀病毒既可能直接激活DC-SIGN,也可能同時(shí)結(jié)合DC-SIGN與mCEACAM1a,促使兩者相互作用,協(xié)同調(diào)控病毒在細(xì)胞中的復(fù)制。

        A: Co-location of DC-SIGN and mCEACAM1a in NIH/3T3 cells infected with murine coronavirus. DC-SIGN is stained with AF-488 (green) and mCEACAM1a is stained with PE (red). Confocal photography was conducted at 15 and 60 min after virus infection, at 30 min after mock infection. Bar is 50 μm. The insets show an enlarged cell. B:Co-expression of DC-SIGN and mCEACAM1a in Neuro-2a and NIH/3T3 cells. Cells stably transfected with DC-SIGN plasmid were infected with 10 MOI of murine coronavirus MHV-A59 or porcine coronavirus TGEV for 30 min and then cells were treated with 10 μmol DTSSP for 15 min.

        圖4DC-SIGN與鼠冠狀病毒受體mCEACAM1a相互作用

        Fig.4InteractionofDC-SIGNwithmurinecoronavirusreceptormCEACAM1a

        3 討論

        人源CLR在冠狀病毒感染過(guò)程中的作用多樣而復(fù)雜,可直接促進(jìn)病毒進(jìn)入細(xì)胞提高復(fù)制水平,也能誘導(dǎo)細(xì)胞產(chǎn)生抗病毒應(yīng)答。如L-SIGN可作為嚴(yán)重急性呼吸綜合征冠狀病毒(severe acute respiratory syndrome coronavirus,SARS-CoV)的受體介導(dǎo)病毒感染,也可表現(xiàn)為免疫保護(hù)作用[18-20]。人DC-SIGN和L-SIGN能促進(jìn)人及異源的貓和禽冠狀病毒感染[10-11]。本研究發(fā)現(xiàn),在表達(dá)人DC-SIGN和L-SIGN的鼠細(xì)胞中,鼠冠狀病毒的復(fù)制受到抑制(圖1)。在不表達(dá)mCEACAM1a的人神經(jīng)細(xì)胞中,即使表達(dá)DC-SIGN也不能介導(dǎo)病毒感染(圖3)。DC-SIGN可能獨(dú)立或通過(guò)與病毒受體相互作用,拮抗病毒感染導(dǎo)致的ERK通路下調(diào),引起鼠冠狀病毒復(fù)制降低(圖2、圖4)。

        DC-SIGN主要由DC表達(dá),L-SIGN主要由肝竇或淋巴結(jié)內(nèi)皮細(xì)胞表達(dá),前者通過(guò)內(nèi)體形成促進(jìn)病毒進(jìn)入細(xì)胞,而后者主要表現(xiàn)為免疫調(diào)節(jié)作用,兩者在功能上的差異與來(lái)源細(xì)胞和氨基酸序列的不同有關(guān)[5,16,21]。兩者較短的N端膜內(nèi)區(qū)中存在的保守雙亮氨酸和三酸性氨基酸簇(LL/DDD)為內(nèi)化所必需,有助于介導(dǎo)病毒進(jìn)入細(xì)胞[17]。DC-SIGN膜內(nèi)區(qū)含有YxxL基序,而L-SIGN無(wú)此基序,膜內(nèi)區(qū)更長(zhǎng)(圖2A)。同源序列替換證明,DC-SIGN胞內(nèi)區(qū)YxxL信號(hào)有消除L-SIGN抑制病毒復(fù)制的作用,但CRD末端16個(gè)氨基酸不明顯(圖2)。膜內(nèi)YxxL基序?qū)ψR(shí)別真菌和寄生蟲(chóng)抗原CLR Dectin-1和CLEC-2信號(hào)是必需的,但在DC-SIGN中作用不明顯[17,22]。本研究發(fā)現(xiàn),鼠冠狀病毒感染后,Raf-1磷酸化(S338)變化不明顯,但MEK和ERK磷酸化水平明顯下調(diào),DC-SIGN、L-SIGN及其N(xiāo)端和C端序列嵌合體均能拮抗這種下調(diào)作用(圖2E)。大部分病毒及其膜蛋白通過(guò)DC-SIGN和L-SIGN激活絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路信號(hào)分子[23-24],而病毒受體本身具有的信號(hào)激活功能作用可能相反。

        病原體本身通過(guò)DC-SIGN激活Raf-1磷酸化依賴激酶Src和Pak,進(jìn)入細(xì)胞后通過(guò)其他組分激活TLR,最終激活NF-κB產(chǎn)生免疫調(diào)節(jié)作用[4-5]。但與DC不同,鼠Neuro-2a和NIH/3T3細(xì)胞不具有免疫細(xì)胞功能,導(dǎo)致DC-SIGN作用不明顯,而上皮細(xì)胞來(lái)源的L-SIGN對(duì)病毒復(fù)制發(fā)揮主導(dǎo)調(diào)控作用。

        鼠冠狀病毒特異性受體mCEACAM1是典型黏附分子,通過(guò)識(shí)別細(xì)胞自身的糖蛋白如癌胚抗原(carcinoembryonic antigen,CEA)、IgFc、細(xì)胞間黏附分子2(intercellular adhesion molecule 2,ICAM-2)和ICAM-3等參與免疫調(diào)節(jié)與腫瘤發(fā)生[9]。不同組織來(lái)源細(xì)胞的mCEACAM1有不同RNA拼接異型,N端膜外區(qū)結(jié)構(gòu)域異型2[1,4]和4[1-4]可能導(dǎo)致不同鼠細(xì)胞對(duì)病毒的易感性不同(圖3A)。mCEACAM1a C端膜內(nèi)區(qū)結(jié)構(gòu)域異型S和L所含的信號(hào)激活序列不同,L和S型均有PKC激活信號(hào),但只有L型含有重復(fù)的ITIM信號(hào)[9]。Neuro-2a細(xì)胞同時(shí)表達(dá)S和L兩種胞內(nèi)區(qū)亞型,病毒感染導(dǎo)致PKC磷酸化上調(diào),而有DC-SIGN存在時(shí)PKC下調(diào)(數(shù)據(jù)未列)。鼠冠狀病毒感染后PKC如何下調(diào)MAPK通路信號(hào)分子,以及ITIM信號(hào)中YxxL與DC-SIGN膜內(nèi)區(qū)YxxL在激活受體酪氨酸激酶(receptor tyrosine kinase,RTK)信號(hào)通路中的差異仍有待進(jìn)一步闡明。此外,NIH/3T3于39 ℃反復(fù)傳代后產(chǎn)生的mCEACAM1a 表達(dá)缺陷細(xì)胞株(NIH/3T3_vR)仍然支持MHV-A59在其內(nèi)復(fù)制,但病毒滴度和N蛋白表達(dá)明顯降低(圖3A、圖3B)。推測(cè)當(dāng)mCEACAM1a表達(dá)減少或缺陷時(shí),細(xì)胞中其他黏附分子代償性增加,而這些糖蛋白結(jié)構(gòu)功能域與鼠冠狀病毒結(jié)合的能力增強(qiáng),也不排除NIH/3T3細(xì)胞存在鼠冠狀病毒的輔助受體。

        DC-SIGN和L-SIGN的CRD識(shí)別含甘露聚糖或果糖寡聚糖結(jié)構(gòu)的配體分子,能直接與SARS-CoV S蛋白相互作用介導(dǎo)病毒進(jìn)入細(xì)胞[25-26]。本研究發(fā)現(xiàn),DC-SIGN不能介導(dǎo)鼠冠狀病毒進(jìn)入細(xì)胞,病毒粒子更有可能發(fā)揮配體的作用,直接與CLR結(jié)合并激活MAPK信號(hào)分子(圖3)。此外,鼠冠狀病毒可能同時(shí)結(jié)合DC-SIGN和mCEACAM1a,促進(jìn)兩者靠近發(fā)生相互作用,從而抑制mCEACAM1a介導(dǎo)病毒進(jìn)入或改變其誘導(dǎo)的信號(hào)通路(圖4)。

        鼠冠狀病毒感染可引發(fā)肝臟和中樞神經(jīng)系統(tǒng)免疫病理[6-7]。在小鼠中存在人DC-SIGN和L-SIGN同源基因簇,表達(dá)7~8種同源分子,稱(chēng)為mSIGNR1~8[27]。但這些分子在小鼠組織細(xì)胞中的表達(dá)不恒定,且呈功能多樣性,導(dǎo)致建立DC-SIGN敲除小鼠模型困難,從而限制了DC-SIGN的深入研究[28]。本研究構(gòu)建了穩(wěn)定表達(dá)人DC-SIGN和L-SIGN的NIH/3T3和Neuro-2a細(xì)胞系,探討了鼠冠狀病毒復(fù)制調(diào)控的相關(guān)機(jī)制,有助于加深對(duì)冠狀病毒感染與致病機(jī)制的理解。

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