流感病毒NP蛋白与宿主蛋白CAP1相互作用的研究

流感病毒核衣壳蛋白(NP)是病毒核糖核蛋白复合体(v RNP)的主要组成部分,主要调控病毒基因组的转录和复制。为筛选与流感病毒NP相互作用的宿主蛋白,本研究采用酵母双杂交技术(Y2H)在A549细胞c DNA文库中筛选到与其相互作用的宿主蛋白Adenylate cyclase-associated protein 1(CAP1),并通过酵母回交验证、免疫共沉淀技术(Co-IP)和GST pull down试验进一步证实NP与CAP1之间存在特异性的直接相互作用。此外,利用激光共聚焦试验证实NP与CAP1共定位于A549细胞的细胞质内。在HEK293T细胞中过表达CAP1蛋白后,流感病毒的复制能力下降,表明CAP1对流感病毒复制具有抑制作用。本研究对该蛋白的鉴定为流感病毒的复制机理研究奠定了基础。     

猪宿主蛋白G3BP1对猪圆环病毒2型在PK15细胞上复制的影响

猪宿主蛋白G3BP1在宿主抵抗病毒感染过程中起着重要作用,然而还未有相关报道G3BP1与猪圆环病毒2型(PCV2)感染过程中的联系。本文构建了G3BP1真核表达载体和干扰RNA,实现在PK15细胞上G3BP1的过表达及敲低。结果发现,PCV2感染细胞24 h后病毒复制出现显著差异,感染48 h相比于感染24 h病毒复制差异更显著,表明G3BP1能显著促进PCV2复制。过表达或敲低G3BP1后,用干扰素刺激DNA(ISD)刺激12 h,G3BP1能促进ISD诱导IFN-βmRNA的表达;感染PCV212 h后,G3BP1也能正调控IFN-βmRNA的转录。本文揭示了宿主蛋白G3BP1显著促进PCV2的复制,很可能是通过上调IFN-β表达引起的。     

猪圆环病毒2型复制与转录模式研究进展

猪圆环病毒是一种单股负链环状DNA病毒,有PCV1和PCV2两种血清型,PCV1一般认为无致病性,而PCV2是导致断奶仔猪多系统衰竭综合症的重要病原,给养猪业造成了巨大的经济损失。PCV复制模式为坩埚滚环复制,须依赖于Rep和Rep,蛋白的共表达、病毒复制起始区和双链DNA片段的形成,三者缺一不可;其转录模式也较为复杂,可双向进行且通过选择性剪切从而产生不同的RNA。主要对PCV2复制模式和转录模式等方面进行综述,以期为PCV的相关研究提供借鉴和参考。     

猪圆环病毒2型Cap蛋白互作宿主核蛋白的筛选与鉴定

为探究在猪圆环病毒2型(PCV2)入核复制过程中发挥生物学功能的宿主核相关蛋白,本研究借助免疫沉淀技术联合蛋白质谱鉴定技术,从宿主细胞核膜提取物中筛选到了可与PCV2 Cap蛋白发生潜在互作的2个蛋白质：高迁移率族蛋白1(HMGB1)和精氨酸酶1(ARG1)。通过进一步的免疫共沉淀试验及激光共聚焦试验,证明了HMGB1、ARG1与PCV2 Cap蛋白的相互作用,且互作与Cap蛋白的核定位信号(NLS)无关。该研究为进一步探索PCV2入核复制的机制解析提供了重要信息。     

流感病毒NP蛋白与宿主蛋白SNRPA的相互作用研究

流感病毒核蛋白(NP)是由病毒RNA节段5编码的主要结构蛋白,与病毒基因组RNA及聚合酶(PB1、PB2、PA)构成核糖核蛋白(vRNP)复合体,是病毒基因组的转录和复制的基本功能单位。本实验室采用酵母双杂交技术从人细胞系cDNA文库中筛选与流感病毒NP蛋白相互作用的宿主蛋白U1 small nuclear ribonucleoprotein A(SNRPA)。本研究通过酵母回交验证、免疫共沉淀(Co-IP)和GST pull down试验进一步证实NP与SNRPA之间存在直接相互作用。利用siRNA干扰基因的表达后,流感病毒的复制滴度在24 h和48 h分别下降2.7倍和1.75倍,表明SNRPA蛋白表达对流感病毒的复制发挥正调控作用。本研究丰富了流感病毒蛋白与宿主蛋白之间的蛋白质调控网络,为进一步研究流感病毒复制调控的机制奠定了基础。     

宿主蛋白TRIB2与流感病毒NP蛋白相互作用的研究

流感病毒的核衣壳蛋白(NP)是病毒粒子中重要的结构蛋白,参与流感病毒生命周期的多个过程。为筛选与流感病毒NP相互作用的宿主蛋白,本研究应用酵母双杂交技术(Y2H)技术从A549细胞cDNA文库中筛选到与其相互作用的宿主蛋白Tribble 2(TRIB2),并通过免疫共沉淀技术(Co-IP)试验和GST pull down试验进一步证实NP与TRIB2之间存在特异性的直接相互作用。此外,利用激光共聚焦试验证实NP与TRIB2共定位于A549细胞的细胞质内。在A549细胞中过表达TRIB2蛋白后,能够降低流感病毒的滴度,表明TRIB2具有抑制流感病毒的复制功能。本实验为研究流感病毒的复制机理奠定了基础,完善了病毒与宿主相互作用网络。     

应用酵母双杂交系统筛选与流感病毒PB2蛋白相互作用的宿主蛋白

流感病毒PB2蛋白可以作用于病毒mRNA转录起始阶段,辅助病毒mRNA引物的合成,并能够影响病毒的宿主范围,参与禽流感病毒在哺乳动物宿主体内的适应过程。本研究利用2009甲型H1N1流感病毒PB2蛋白作为"诱饵"蛋白,通过经典酵母双杂交系统筛选由Calu-3、A549、THP-1和U251 4种细胞构建的cDNA文库,筛选到多个与PB2蛋白相互作用的宿主蛋白。针对其中筛选到的一种宿主蛋白FHL2,进行免疫共沉淀(Co-IP)和激光共聚焦(Confocal)试验,证实了PB2蛋白和FHL2蛋白二者之间存在相互作用,并且转染瞬时过表达FHL2发现该蛋白可以负调控流感病毒复制,为进一步研究PB2蛋白与FHL2蛋白互作及影响病毒复制的分子机制奠定了基础。     

猪圆环病毒2型ORF1部分位点突变对病毒复制能力的影响

猪圆环病毒2型(PCV2)可引起断奶仔猪多系统衰竭综合征,导致仔猪逐渐消瘦。PCV2 ORF1表达的Rep蛋白及其剪切体Rep′是PCV2复制所需的重要蛋白。为了研究PCV2 ORF1部分位点对PCV2复制能力的影响,本试验通过构建PCV2 ORF1区域点突变双拷贝感染性克隆质粒,在无PCV2污染的PK-15细胞中进行病毒拯救,使用荧光定量PCR检测不同位点突变病毒培养不同代次上清液的Ct值。结果：将PCV2 Rep的17 aa、19 aa、20 aa和21 aa突变为丙氨酸后病毒无法成功拯救,2 aa突变后严重影响病毒的复制能力,3 aa、5 aa和18 aa突变后病毒的复制能力增强且细胞病毒载量高于PCV2原毒株,推测17 aa、19 aa、20 aa和21 aa是影响PCV2复制的关键作用位点。本研究结果为未来PCV2复制相关研究提供了试验依据。     

抑制伪狂犬病病毒复制的宿主蛋白的筛选与鉴定

本试验旨在利用TMT蛋白组学分析并筛选出抑制伪狂犬病病毒(pseudorabies virus, PRV)复制的关键宿主蛋白。前期通过TMT定量蛋白组学技术筛选出241个差异蛋白，Western blot和RT-qPCR验证4个差异表达的蛋白，结果与蛋白组学结果一致，表明蛋白组学结果真实可信。通过构建4个差异表达蛋白的真核表达载体，Western blot、RT-qPCR、病毒噬斑结果显示这4个宿主蛋白均可抑制PRV的复制；然后通过双荧光素报告基因检测IFN-β启动子活性，结果显示，MCCC1、STRAP促进IFN-β启动子活性效果更为显著；针对MCCC1和STRAP设计siRNA,Western blot和病毒噬斑结果显示，敲低STRAP显著促进PRV的复制。本试验最终筛选出显著影响PRV复制的宿主蛋白STRAP,为研究PRV与宿主互作的分子机制奠定基础。     

猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用

为了验证猪肾细胞系(PK-15)源3种蛋白质基因(AP2α2、Elf4和ISCU)表达水平对猪圆环病毒2型(PCV2)复制的调节作用,采用RT-PCR扩增这3种蛋白质编码基因,构建真核表达载体;同时设计这3种蛋白质的RNAi片段,分别插入到pGPU6-GFP载体构建shRNA干扰载体。以荧光定量PCR法检测干扰效率,选取干扰效率较高的干扰载体,利用G418筛选转染真核表达载体或干扰载体后的PK-15细胞。采用酶联免疫吸附试验(ELISA)和免疫过氧化物酶单层细胞试验(IPMA)法检测PCV2LG毒株在这些细胞中的复制效率。结果表明,Elf4蛋白和ISCU蛋白基因过表达能够显著增强PCV2的复制;而AP2α2蛋白基因过表达对PCV2的复制无显著影响。AP2α2、ISCU和Elf4这3种蛋白质基因被干扰表达后均可降低PCV2复制效率,表明这3种宿主细胞蛋白质对该病毒复制具有调节作用。     

猪圆环病毒2型复制及其影响因素

猪圆环病毒2型（PCV2）DNA的复制是滚环复制,Rep和Rep′蛋白是PCV复制所必需的蛋白质,核心元件A*TA*TAC为必需的结合位点。复制的影响因素包括病毒内部因素如病毒基因内的CpG寡核苷酸和干扰素反应元件、病毒黏附受体(硫酸乙酰肝素和硫酸软骨素B乙糖胺)和病毒内吞作用（小GTP酶和肌动蛋白）;外部因素如DL-硒蛋氨酸和刀豆素A。抑制PCV2复制的措施有猪营养的全面,干扰RNA技术,疫苗的使用等。     

Geminivirus DNA replication and cell cycle interactions

The Geminiviridae family includes a large number of viruses that infect plants and have a unique geminate virion particle, a single-stranded genome of approximately 2.6-3.0 kb, and replicate through a rolling-circle mechanism. Since they encode for just a few proteins (4-6 depending on the members that belong to four different genera), a rich variety of interactions has evolved between viral proteins and host factors to develop the virus replicative cycle. Among them, we have been particularly interested so far: (i). in the interference with cell cycle regulatory proteins of the retinoblastoma-related (RBR)/E2F pathway and (ii). in the interaction with host DNA replication factors necessary for the assembly of a functional replication complex at the viral origin of DNA replication during the rolling-circle stage. Yeast two-hybrid assays revealed that wheat dwarf virus RepA protein, but nor Rep protein, interacts with plant RBR protein. Interestingly, deletion of the C-terminal domain of Rep confers the truncated protein the ability to interact with RBR, suggesting that this domain may hinder the LXCXE RBR-binding motif. Secondary structure predictions support such a possibility.     

Nanoviruses: genome organisation and protein function

Nanoviruses, single-stranded DNA (ssDNA) plant viruses with a multipartite genome, share similarities with members of the Circoviridae family that infect mammals or birds as well as with the Geminiviridae, the only other plant virus family with circular ssDNA genomes. Although the virions of the latter are unique and different from that of the circoviruses, the mode of replication of viruses with monopartite or multipartite circular ssDNA genomes is strikingly similar. They multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyse initiation of ssDNA replication and resolution of replicative ssDNA into circular single-stranded virion DNA. All these ssDNA viruses exploit host polymerases for DNA synthesis and code for proteins that modulate the host's cell cycle favourably for virus multiplication. Recent three-dimensional structure analyses of a geminivirus and a parvovirus Rep protein have revealed an intriguing similarity between the catalytic domains of their respective Rep proteins. Furthermore, these structural data revealed that ssDNA virus replication initiator proteins might represent evolutionary intermediates between certain RNA-binding proteins and some multifunctional origin-binding proteins of papovaviruses.     

Identification and functional analysis of the novel ORF6 protein of porcine circovirus type 2 <Emphasis Type="Italic">in vitro</Emphasis>

In the present study, the function of a novel ORF6 gene in the PCV2 genome was determined and functionally analyzed in vitro. ORF6 expression was demonstrated by indirect immunofluorescence in PCV2-infected cells. The antibody against ORF6 was detected in PCV2-infected pigs. The start codon of ORF6 was mutated and an infectious clone was used to create an ORF6-deficient mutant virus. Viral DNA replication curves and immunofluorescence analysis indicated that ORF6 is unnecessary for viral replication and ORF6 deletion reduces viral DNA replication in PK-15 cells. The activities of caspases 3 and 8 in ORF6-deficient virus-infected cells were significantly different from those in wild-type virus-infected cells. The ORF6 protein can increase the expression of IFN-β, TNF-α, IL-1b, IL-10, and IL-12p40. These results demonstrated that the newly discovered ORF6 protein may be involved in caspases regulation and the expression of multiple cytokines in PCV2-infected cells. The functions of this gene in viral pathogenesis remain to be further elucidated.     

稳定表达IL-10的PK-15细胞的构建及其在PCV2增殖中的应用

【目的】 研究白细胞介素-10（IL-10）对猪圆环病毒2型（Porcine circovirus type 2,PCV2）复制的影响,筛选PCV2高感染性细胞系和提高PCV2病毒滴度,为后续疫苗的研发及IL-10在PCV2感染中的作用研究提供参考。【方法】 利用PCR技术扩增猪IL-10基因,将目的基因与慢病毒表达载体（pCDH-CMV-MCS-EF1-GFP+Puro）进行连接,获得重组质粒pCDH-CMV-IL-10,将其与包装质粒psPAX2和pMD2.G共转染293T细胞进行慢病毒包装。用收集的慢病毒液感染PK-15细胞,经嘌呤霉素筛选后得到细胞株PK-15-IL-10,对照组细胞分别命名为PK-15-pCDH和PK-15。PCV2感染PK-15-IL-10、PK-15-pCDH和PK-15细胞株后,在24、48和72 h分别收集细胞液,利用CCK-8检测细胞活力。利用实时荧光定量PCR和Western blotting检测IL-10基因的表达水平和PCV2的复制情况;利用间接免疫荧光试验（IFA）观察PCV2在细胞中的复制情况及测定PCV2的病毒滴度（TCID₅₀）。【结果】 试验成功构建了重组质粒pCDH-CMV-IL-10,将其与包装质粒psPAX2和pMD2.G共转染293T细胞后,48 h时细胞状态最好,荧光最强。分别收集共转染48和72 h的慢病毒液上清感染PK-15细胞,pCDH-CMV-IL-10组的荧光最强,将其在嘌呤霉素浓度为2.5 μg/mL的完全培养基中继续培养,获得仍有绿色荧光的稳转细胞株。实时荧光定量PCR和Western blotting检测发现,IL-10基因在pCDH-IL-10细胞株中的表达量明显高于对照组PK-15-pCDH和PK-15,PCV2的拷贝数增加了4倍,复制能力增强,且将病毒稀释连续传3代后,PK-15-IL-10细胞中的PCV2极显著高于PK-15细胞（P<0.01）。细胞增殖试验表明,猪IL-10基因在细胞中过表达对细胞活力无明显影响;IFA结果表明,PK-15-IL-10细胞中的荧光比PK-15细胞更强,PCV2在PK-15-IL-10细胞中的TCID₅₀在感染后48 h极显著高于PK-15细胞（P<0.01）。【结论】 本研究成功构建了pCDH-CMV-IL-10的慢病毒表达载体,并利用其感染PK-15细胞,继续培养后筛选出过表达IL-10的PK-15-IL-10细胞株,用PCV2感染该细胞株能促进PCV2在PK-15细胞中的复制。本试验结果为后期疫苗研究提供了参考,为进一步研究IL-10对PCV2在PK-15细胞中复制的影响奠定了基础。     

猪圆环病毒2型10JS-2株的分离与全基因组序列分析

本试验从江苏某猪场采集猪血清,进行猪圆环病毒2型(PCV2)的检测和分离,根据GenBank中登录的PCV2全基因组序列,设计1对特异性引物,扩增PCV2全基因组,并且进行序列测定和分析。发现一株PCV2 10JS-2的基因组全长为1779 nt,在病毒复制起始区域含有11个碱基的插入。在GenBank中对含有11个碱基插入的毒株序列进行BLAST发现,有两株PCV2(AY321993和EF565360)也有11个碱基的插入,但是与10JS-2比较,插入的碱基和插入的位置不同。遗传进化分析显示10JS-2属于PCV2a基因型,AY321993和EF565360属于PCV2b基因型,因此推断10JS-2可能是由PCV2a基因型的毒株在复制起始区域发生碱基插入形成的。这是首次发现此类PCV2毒株。     

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.     

Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK,as promoted by reducing glutathione levels

Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.     

猪圆环病毒Ⅱ型Rep基因在PK15细胞中的表达及特性

为研究猪圆环病毒Ⅱ型(PCV2)Rep基因在PK15细胞中的表达特性，通过PCR方法克隆了PCV2杭州株(HZ0201)Rep基因全长945bp片段，与真核表达栽体pCI—neo构建为重组质粒pCI-PCV2-Rep。pCI-PCV2-Rep质粒转染PK15细胞后48h，通过RT-PCR可检测到PCV2 Rep mRNA的转录；用猪PCV2多抗血清作间接免疫荧光试验，可检测到Rep基因表达产物。在表达量低的细胞中，PCV2 Rep蛋白主要位于PK15的细胞浆，在表达量高的细胞中，细胞浆和细胞核中均含有大量的Rep蛋白，表明Rep对PK15细胞的细胞浆和细胞核的亲嗜性没有明显差别。