【作者】 孙佳增；

【导师】 刘维全；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 病毒与宿主的相互作用一直以来就是病毒学家们高度关注的重要科学问题。目前,虽然二者互作的一些机制得到阐明,但microRNA (miRNA)参与的相互作用网络还亟待研究和发现。MiRNA是一类内源的长度约为18-23个核苷酸的单链非编码小分子RNA,通过抑制靶基因mRNA的翻译过程或者降解靶基因的mRNA分子,介导转录后基因表达的调控。MiRNA不但可以参与调控细胞增殖、分化、造血等一系列的生理过程,还可以参与调控病毒与宿主细胞的相互作用。目前,在泡沫病毒、丙型肝炎病毒、猪繁殖和呼吸综合征病毒及流感病毒中均存在宿主miRNA调节病毒感染的现象。为了便于进行miRNA参与病毒感染过程相应的探索,我们选取了一种简单的细小病毒—水貂肠炎病毒(Mink enteritis virus, MEV)作为模型进行研究。MEV是细小病毒科的一种,普遍认为是猫细小病毒的一个变种。MEV有强大的抵抗力并且传播速度很快,能够造成养貂业的巨大损失。MEV是一种单链DNA病毒,其基因组主要包含两个ORF,一个ORF编码功能性非结构蛋白(None-structural protein, NS),另一个ORF主要编码衣壳蛋白。为了探究miRNA在MEV感染过程中的作用,本文利用高通量测序技术和生物信息学手段对F81细胞感染MEV前后miRNA表达谱进行分析研究,从而筛选出可能参与到MEV感染过程中的miRNA,最终通过实验对miRNA的功能进行鉴定。我们首先得到F81细胞系感染MEV前后的两个小RNA数据库。经过比对发现,两个数据库有196个成熟体miRNA和97个miRNA*与哺乳动物的已知miRNA是同源的,这些miRNA分属于152个miRNA家族；在F81细胞系感染MEV前后的两个数据库中,分别预测得到384和398个新的miRNA前体。采用实时荧光定量PCR (qPCR)的方法对F81细胞感染MEV前后miRNA的表达差异进行了鉴定,在随机挑选的16个miRNA中,大部分miRNA的差异水平(12/16)与高通量测序结果是相符的。利用miRNA靶标预测软件从数据库中筛选出可能直接作用于MEV mRNA的8个miRNA和可能作用于MEV受体-转铁蛋白受体(Transferrin receptor,TfR)的6个miRNA,这些miRNA可能与MEV感染相关。在预测得到的8个有可能直接作用于MEV mRNA的miRNA中,高通量测序结果显示miR-181b的表达量较高,并且预测得到miR-181b的假定靶标为MEV的NS1mRNA,该靶标序列在不同的细小病毒中是高度保守的。分别转染合成的miRNA模拟物mimics和抑制剂inhibitors,测定不同感染时间MEV病毒滴度及其基因组DNA水平,并采用流式细胞术对细胞感染MEV的水平进行测定,然后对MEV造成细胞病变水平及MEV间接免疫荧光进行观察,结果证实miR-181b mimics能够抑制病毒增殖并且miR-181b inhibitors在病毒感染后期能够促进病毒增殖。双荧光素酶报告基因检测实验证明了miR-181b假定靶标的真实性。MiR-181b通过靶向NS1mRNA编码区导致NS1蛋白的翻译抑制。通过检测miR-181b对MEV假定靶标突变株的作用,验证了miR-181b是通过靶向预测靶标抑制MEV增殖的。Argonaute2(Ago2)蛋白的免疫共沉淀实验证明miR-181b能够在RNA诱导沉默复合物(RNA-induced silencing complex, RISC)中与NS1mRNA相互结合。qPCR结果显示MEV感染也会导致宿主F81细胞miRNA表达量发生变化,miR-181b随MEV感染时间的延长其表达量逐渐降低。这些表明F81细胞的miR-181b能够通过靶向MEV NS1mRNA的编码区抑制NS1表达,从而抑制MEV的增殖。MEV感染需要转铁蛋白受体TfR介导。通过同源比对,克隆出了猫的TfR mRNA3’非翻译区Untranslated region, UTR),并预测得到6个miRNA可能作用于其上。qPCR和western blot实验鉴定出miR-320a和miR-140能够抑制TfR的表达,流式细胞术测定实验也证明2个miRNA能够降低细胞TfR的表达。MEV感染能够导致2个niRNA表达逐渐上调,而TfR随之逐渐下调。双荧光素酶报告基因检测实验证明了2个miRNA假定靶标的真实性。Ago2蛋白的免疫共沉淀实验证明2个miRNA能够在RISC中与TfR mRNA相互结合。通过对不同病毒感染时间病毒基因组DNA的定量,验证了2个miRNA能够通过阻止病毒进入宿主细胞而控制MEV感染。共转染2个miRNA后对TfR表达水平及病毒基因组DNA水平测定,结果发现二者对TfR表达及MEV感染细胞能起到协同抑制作用。这些结果表明F81细胞的miR-320a和miR-140能够靶向MEV受体TfR mRNA的3’UTR区抑制TfR的表达,从而减少MEV侵入细胞。本研究构建了F81细胞感染MEV前后的miRNA数据库；并证实miR-181b能够通过抑制MEV NS1的表达抑制病毒增殖；而miR-320a和miR-140又能够通过抑制MEV受体TfR的表达控制MEV的感染。上述研究结果在miRNA层面上进一步阐明了MEV感染机制,为防治MEV提供了新的思路。更多还原

【Abstract】 Interaction between virus and host, as a scientific issue, has caused great attention of virologist. Currently, although some mechanisms about their interaction have been elucidated, little is known about a number of interaction networks involving microRNA (miRNA). MiRNAs are endogenous small noncoding RNAs of length18-23nucleotides (nt), which mediate post-transcriptional regulation of target genes through mRNA degradation or translational repression and play critical roles in many biological processes including cell proliferation, differentiation and haematopoiesis. Recent studies have also noted the role of miRNAs as modulators in host-pathogen interaction networks, such as foaming virus, hepatitis C virus, porcine reproductive and respiratory syndrome virus and influenza virus.Therefore, a simple parvovirus-mink enteritis virus (MEV) was selected as a model to explore the interaction networks. MEV, a subspecies of the feline parvovirus (FPV) and belonging to the family Parvoviridae, is one of the most important viral pathogens in the mink industry, which causes a highly infectious acute disease and has a high rate of morbidity and mortality in mink, resulting in huge economic losses in the worldwide. MEV is a single-stranded negative sense DNA virus with a genome comprised mainly of2ORFs, one coding for none-structural protein (NS) and another coding for capsid protein.To study the involvement of miRNAs in the MEV infection process, we used Illumina’s ultra high throughput approach to sequencing miRNA libraries from the feline kidney (F81) cell line before and after infection with MEV and identified miRNAs involved in MEV infection.Using this bioinformatics approach we identified196known mammalian miRNA orthologs belonging to152miRNA families in F81cells. Additionally,97miRNA*s of these miRNAs were also detected. Besides known miRNAs,384and398novel miRNA precursor candidates were identified from uninfected F81cells and MEV-infected cells respectively that have not been reported in other mammals. The majority (12of16) of randomly selected miRNAs expression profiles by qRT-PCR were consistent with those by deep sequencing. After target prediction,8miRNAs appeared to target the MEV mRNA coding region; and6to target the3’untranslated region (UTR) of MEV-specific receptor transferring receptor (TfR) mRNAs.Among the8miRNAs predicted to target MEV mRNA coding region, miR-181b showing comparatively higher expression level was selected. The potential target of miR-181b in NS1mRNA was highly conserved in different parvoviruses. After transfection with synthetic miRNA mimics and inhibitors, detection the level of viral titer, viral genomic DNA, the quantity of infected cells, cytopathic effect and immunostaining were carried out. The results showed that miR-181b mimics inhibited MEV production and miR-181b inhibitors increased it in the late virus infection. Dual-luciferase reporter assay validated the authenticity of miR-181b potential target. Western blot assay of NS1protein showed the inhibiton of miR-181b in NS1expression. Through detection the effect of miR-181b on the potential target mutated MEV, we confirmed that miR-181b inhibited MEV production through the potential target. Argonaute2(Ago2) co-immunoprecipitation demonstrated that miR-181b bound NS1mRNA in RNA-induced silencing complex (RISC). In the meantime, MEV infection led to the change of miRNA expression including miR-181b. Experiment results presented here show that cellular miR-181b in F81cells inhibits replication of MEV by targeting its NS1mRNA coding region resulting in NS1translational repression.MEV infection is dependent on induction of transferrin receptor (TfR). By homologous alignment, TfR mRNA3’UTR was cloned and sequenced. From the6predicted miRNAs, qPCR and western blot assay revealed that miR-320a and miR-140inhibited TfR expression and flow cytometric assay also verified the results. MEV infection led to gradually increase of the2miRNAs and decrease of TfR. Dual-luciferase reporter assay validated the authenticity of the2miRNA potential targets. Ago2co-immunoprecipitation demonstrated that the2miRNAs bound TfR mRNA in RISC. Quantification of MEV genomic DNA level at the indicated times validated the inhibition of the2miRNAs in MEV infection. Additionally, the2miRNAs played the inhibiton roles on TfR and MEV in a synergistic manner. Experiment results also show that miR-320a and miR-140inhibit MEV entry into F81cells by down-regulating its receptor TfR through targeting the3’UTR of TfR mRNA.This is the first time to construct the miRNA base of F81cells before and after MEV infection and to describe that cellular miR-181b inhibits replication of MEV by targeting its NS1mRNA coding region resulting in NS1translational repression; and miR-320a and miR-140inhibit MEV entry into F81cells by down-regulating its receptor TfR through targeting the3’UTR of TfR mRNA. These results provide further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.更多还原