干扰素刺激基因ISG15对猪流行性腹泻病毒复制的作用及机制分析

【目的】探究干扰素刺激基因ISG15在猪流行性腹泻病毒(PEDV)复制中所发挥的作用及机制。【方法】利用实时荧光定量PCR检测ISG15基因组织表达谱及肠道组织差异表达情况,同时以猪小肠上皮细胞(IPEC-J2)为研究模型,检测PEDV CV777毒株感染细胞不同时间点PEDV M基因mRNA和N蛋白表达量,并从RNA和蛋白水平检测ISG15表达变化;分别构建猪ISG15基因干扰和过表达细胞,通过实时荧光定量PCR、Western blotting及间接免疫荧光试验检测ISG15基因表达对PEDV复制水平的影响;对ISG15基因过表达前后进行转录组测序分析,筛选其下游调控基因及信号通路。【结果】ISG15基因在仔猪肠道组织中特异性高表达,其中空肠和回肠中表达量极显著高于其他组织(P<0.01);PEDV感染组十二指肠、空肠及回肠中ISG15基因表达量显著或极显著高于健康组(P<0.05;P<0.01);M基因mRNA和N蛋白表达量出现上升趋势,与0 h相比,ISG15基因表达水平在24 h出现极显著上调(P<0.01);ISG15基因过表达后PEDV复制出现显著或极显著下降(P<0.05;P<0.01),而ISG15基因干扰后PEDV复制出现极显著上调(P<0.01);转录组测序发现,过表达ISG15基因前后存在1 532个差异表达基因,且其主要富集在自噬、MAPK、内吞等信号通路中。【结论】本研究揭示了PEDV感染过程中ISG15基因的调控功能及作用机制,发现ISG15基因上调可显著抑制PEDV复制,增进了对PEDV与宿主细胞互作分子机制的认识。

Analysis of differentially expressed genes in Verruca vulgaris vs.adjacent normal skin by RNA-sequencing

Introduction:Verruca vulgaris is one of the most common low-risk HPV infections and is characterized by excessive proliferation of keratinocytes.Currently,very little genetic information is available regarding verruca vulgaris in the Chinese population.This study aimed to obtain comprehensive transcript information of verruca vulgaris by RNA sequencing.Methods:High-throughput sequencing was performed on three fresh verruca vulgaris samples and adjacent normal skin on the Illumina sequencing platform.The transcriptomes were analyzed using bioinformatics and the differentially expressed genes(DEGs)were verified by immunohistochemistry.Verruca vulgaris exhibited a unique molecular signature.Results:In total,1,643 DEGs were identified in verruca vulgaris compared to normal skin.The functions of the DEGs were studies by Gene Ontology(GO)enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,DEGs Reactome analysis,disease annotation function,and STRING protein-protein interaction(PPI)network analysis.The results revealed 595 GO terms associated with the cell cycle,signal transduction,immune system,signaling molecules,and interaction.The Reactome analysis revealed enrichment in reversible hydration of carbon dioxide and BMP signaling,while the disease annotation function revealed that the enriched DEGs are involved in keratosis disorders.The STRING PPI network showed that the edges with the highest density mainly included the 2′-5′oligoadenylate synthase(OAS)family-related proteins.Furthermore,the M-code analysis found ISG15,IRF7,and OASL were scored as significant modules and their high expression compared to the control was verified by immunohistochemistry.Conclusion:These findings contribute to the genetic information of verruca vulgaris in the Chinese population,revealing that interferon-stimulated genes may play essential roles in verruca vulgaris.

BmCH25H,a vertebrate interferon-stimulated gene(ISG)homolog,inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori

Cholesterol-25-hydroxylase(CH25H)has been identified as an interferon-stimulated gene(ISG)in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol(25HC).However,invertebrates lack an antiviral system homologous to vertebrate interferons(IFNs)because the genomes of invertebrates do not encode IFN-like cytokines.Nevertheless,CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates.In this study,the Bombyx mori CH25H(BmCH25H)gene,of which the encoded protein has high homology with other lepidopteran species,was identified and located on chromosome 9.Interestingly,we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus(BmNPV)-infected BmN cells and silkworm(B.mori)larvae at the early infection stage.The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC.More importantly,we demonstrated that during BmNPV infection,BmCH25H expression was increased through the Janus kinase–signal transducer and activator of transcription(JAK–STAT)pathway,similar to the induction of ISGs following virus infection in vertebrates.This is the first report that CH25H has antiviral effects in insects;the study also elucidates the regulation of its expression and its mechanism of action.

Advances on genetic and genomic studies of ALV resistance

Avian leukosis(AL)is a general term for a variety of neoplastic diseases in avian caused by avian leukosis virus(ALV).No vaccine or drug is currently available for the disease.Therefore,the disease can result in severe economic losses in poultry flocks.Increasing the resistance of poultry to ALV may be one effective strategy.In this review,we provide an overview of the roles of genes associated with ALV infection in the poultry genome,including endogenous retroviruses,virus receptors,interferon-stimulated genes,and other immune-related genes.Furthermore,some methods and techniques that can improve ALV resistance in poultry are discussed.The objectives are willing to provide some valuable references for disease resistance breeding in poultry.

Effects of hepatitis E virus infection on interferon production via ISG15

AIM To assess the effects of hepatitis E virus(HEV) on the production of type Ⅰ interferons(IFNs) and determine the underlying mechanisms.METHODS We measured the production of interferon(IFN)-alpha and-beta(-α/β) in genotype 3 HEV-infected C3 A cells at different time points(0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay(ELISA). The expression levels of IFN-stimulated gene(ISG)15 in HEVinfected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3(ORF3) or control plasmids(green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/β and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/β was also measured by ELISA.RESULTS We showed that genotype 3 HEV could enhance the production of IFN-α/β and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/β and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/β. Overexpression of ISG15 resulted in the reduction of IFN-α/β.CONCLUSION HEV may promote production of IFN-α/β and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/β.

干扰素刺激基因（ISGs）的研究进展

干扰素刺激基因（ＩＳＧｓ）是干扰素作用机制研究的核心内容。干扰素与受体结合后，通过细胞内信号转换，激活胞浆转录调控因子与ＩＳＧｓ调控序列上的ｃｉｓ元件结合而诱导基因表达。

Host Interferon-Stimulated Gene 20 Inhibits Pseudorabies Virus Proliferation

Host interferon-stimulated gene 20(ISG20)exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling.Here,we examined the role of ISG20 during pseudorabies virus(PRV)proliferation.We found that ISG20 modulates PRV replication by enhancing IFN signaling.Further,ISG20 expression was upregulated following PRV infection and poly(I:C)treatment.Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells,whereas knockdown of ISG20 promoted PRV proliferation.In addition,ISG20 expression upregulated IFN-βexpression and enhanced IFN downstream signaling during PRV infection.Notably,PRV UL24 suppressed the transcription of ISG20,thus antagonizing its antiviral effect.Further domain mapping analysis showed that the N terminus(amino acids 1-90)of UL24 was responsible for the inhibition of ISG20 transcription.Collectively,these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay.

猪ifitm基因克隆及其序列和组织表达分析

为了研究ifitm基因的功能,本研究从猪肾PK15细胞中克隆了猪的3个ifitm c DNA序列,分析了猪ifitm1、ifitm2和ifitm3基因的染色体定位及其与其他物种基因序列的同源关系,并对不同ifitm在不同组织的表达进行分析和检测。结果显示,猪ifitm和人、鼠ifitm具有相同的基因和蛋白结构,进化上与牛ifitm高度同源,ifitm1和ifitm3在脾、肾、心、肝等组织中大量表达,而ifitm2只在脾和肾中检测到表达,在其他组织中的表达量相对较小。猪ifitm基因的克隆、生物信息学及组织表达分析为进一步研究其在猪细胞中的功能奠定了基础。

HCV感染过程中的相关免疫反应

丙型肝炎病毒(Hepatitis C Virus,HCV)是慢性丙型病毒性肝炎的主要病因,也是引发肝硬化和肝癌的主要诱因。在HCV感染过程中,伴随着干扰素信号通路的激活和干扰素刺激基因(IFN-stimulated gene,ISG)的持续表达,且有HCV独特的免疫逃逸和免疫细胞的功能损伤。现就HCV感染过程中机体的固有免疫反应和适应性免疫反应的研究进展作一综述。

鸭肠炎病毒感染诱导鸭胚成纤维细胞γ干扰素上调表达及初步机制的研究

目的探讨鸭肠炎病毒(DEV)诱导鸭胚成纤维细胞(DEF)中γ干扰素(IFNγ)的表达情况及初步机制。方法分别提取感染DEV后24h、36h、48h、60h的DEF总RNA,反转录成cDNA,使用特异性引物,运用荧光定量PCR方法 ,检测IFNγ及几个相关干扰素刺激基因(ISGs)mRNA水平的变化。结果感染DEV的细胞相对于未感染的细胞,IFNγ的mRNA在24 h、36 h、48 h、60 h均显著上调,且相关ISGs包括抗黏液病毒基因(Mx)、DEAD框蛋白50(DDX50,是一种ATP依赖的RNA解旋酶)、2'-5'寡腺苷酸合成酶L(OASL)等基因的mRNA表达水平也均显著上调。结论 DEV感染DEF能够诱导IFNγ产生及相关ISGs表达的上调。

干扰素刺激基因15的研究进展

干扰素刺激基因(interferon-stimulated genes,ISGs)是一类快速的干扰素刺激反应基因,主要参与抵抗病毒、细菌入侵及免疫功能调节。干扰素刺激基因15(interferon-stimulated gene 15,ISG15)是ISGs家族成员之一,也是第一个被鉴定出来的泛素样蛋白(ubiquitin-like protein,UBL),它既可以以游离形式存在,也可以与底物结合,对底物进行ISG化(ISGylation)修饰。ISG化修饰能够竞争性阻断底物的泛素化降解,进而影响底物的生物学功能。目前确证的ISG15底物约一百余种,它们的功能涉及病毒复制、细胞增殖、细胞周期调控、凋亡、自噬、DNA损伤修复、蛋白质翻译等诸多方向。本文将对近10年ISG15在对抗病毒侵袭、免疫功能调节和参与肿瘤发生、发展等方向的研究进展进行总结综述。

Identification of new type I interferon- stimulated genes and investigation of their involvement in IFN-β activation

Virus infection induces the production of type I interferons （IFNs）. IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes （ISGs）. ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon （Con-IFN）, an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expres- sions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR （qRT-PCR） confirmed 91 genes. We screened 89 genes for those involved in Sendal virus （SeV）-induced IFN-13 promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and iden- tify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.

TRIM25:A central factor in breast cancer

TRIM25 is emerging as a central factor in breast cancer due to its regulation and function.In particular,it has been shown that:(1)Estrogens modulate TRIM25 gene expression;(2)TRIM25 has activity as an E3-ligase enzyme for ubiquitin;and(3)TRIM25 is also an E3 ligase for interferon-stimulated gene 15 protein in the ISGylation system.Consequently,the proteome of mammary tissue is affected by TRIM25-associated pathways,involved in tumor development and metastasis.Here,we discuss the findings on the mechanisms involved in regulating TRIM25 expression and its functional relevance in breast cancer progression.These studies suggest that TRIM25 may be a biomarker and a therapeutic target for breast cancer.

重组犬IFN-α的原核表达及抗病毒活性研究

本试验旨在建立犬α干扰素(CaIFN-α)原核表达及纯化方法,并对其抗病毒活性进行研究。通过构建pET-28a/CaIFN-α表达载体,高效表达和纯化rCaIFN-α;采用CCK-8法在犬肾细胞(MDCK)上测定rCaIFN-α的细胞毒性,在MDCK/VSV系统中测定其抗病毒活性效价;RT-qPCR检测rCaIFN-α作用MDCK细胞后下游ISGs的表达水平;进一步利用TCID 50和间接免疫荧光(IFA)测定rCaIFN-α抗犬副流感病毒(CPIV)和犬瘟热病毒(CDV)的感染能力。电泳和Western-blot结果显示,成功制备原核表达的特异性rCaIFN-α;CCK-8结果显示重组蛋白无明显细胞毒性,细胞病变抑制法测定抗VSV病毒活性效价为1.39×107 U/mL;RT-qPCR显示rCaIFN-α可显著上调ISG15、Mxl等ISGs的转录水平;IFA等结果显示,rCaIFN-α对CPIV和CDV具有良好的抗病毒作用。结果表明,本试验制备的rCaIFN-α具有优良的抗病毒感染活性,为进一步研发新型宠物抗病毒制剂奠定了基础。

ISG20 inhibits bluetongue virus replication

ISG20 is an interferon-inducible exonuclease that inhibits virus replication.Although ISG20 is thought to degrade viral RNA,the antiviral mechanism and specificity of ISG20 remain unclear.In this study,the antiviral role of ovine ISG20(o ISG20)in bluetongue virus(BTV)infection was investigated.It was found that BTV infection upregulated the transcription of ovine ISG20(o ISG20)in a time-and BTV multiplicity of infection(MOI)-dependent manner.Overexpression of o ISG20 suppressed the production of BTV genome,proteins,and virus titer,whereas the knockdown of o ISG20 increased viral replication.o ISG20 was found to co-localize with BTV proteins VP4,VP5,VP6,and NS2,but only directly interacted with VP4.Exonuclease defective o ISG20 significantly decreased the inhibitory effect on BTV replication.In addition,the interaction of mutant o ISG20 and VP4 was weakened,suggesting that binding to VP4 was associated with the inhibition of BTV replication.The present data characterized the anti-BTV effect of o ISG20,and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

Polymorphism Near the Interleukin-28B Gene and Anti-Hepatitis C Viral Response

In a recent genome-wide association study, single nucleotide polymorphisms (SNPs) located near the interleukin-28B gene (IL28B), which encodes type Ⅲ interferon (IFN) λ3, were shown to be strongly associated with a viral response to pegylated IFNα (PEG-IFNα) and ribavirin (RBV) combination therapy and spontaneous viral clearance in patients chroni-cally and acutely infected with hepatitis C virus (HCV), respectively. The global distribution of allele frequencies shows a remarkable pattern, in which a favorable allele is nearly fixed in East Asia, has an intermediate frequency in Europe, and is least frequent in Africa. Although the under-lying mechanisms responsible for viral responses associated with IL28B SNPs have not been completely elucidated, IFN-stimulated gene expression in patients with unfavorable IL28B genotypes tends to be high at baseline and is insufficiently induced by exogenous IFN administration, resulting in poor treatment outcomes. Clinically, triple therapy with PEG-IFNα/RBV together with direct-acting anti-viral agents (DAAs) is currently used to treat chronic hepatitis C as a first-line therapy. Although the predictive power of IL28B status may be attenuated, the IL28B genotype will remain relevant to the outcomes of DAA therapy when used in combination with PEG-IFNα as a backbone. Even with the introduction of IFN-free therapies with a new class of highly effective DAAs, IL28B SNPs are still useful predictors of treatment outcomes and can be used to individualize treat-ment strategies to maximize cost-effectiveness and identify patients at risk of being refractory to treatment. This review summarizes the current understanding of the clinical sig-nificance and role of IL28B in HCV infection and response to therapy.

黏病毒抗性蛋白A(MxA)通过增强干扰素刺激应答元件(ISRE)活性诱导HepG2细胞干扰素刺激基因表达

目的研究黏病毒抗性蛋白A(MxA)对HepG2细胞Janus激酶/信号转导子和转录激活子(JAK/STAT)途径的影响。方法采用pcDNA3.1-Flag-MxA转染HepG2细胞,免疫荧光细胞化学染色检测MxA蛋白在HepG2细胞的表达和定位,Western blot法检测MxA蛋白表达;采用MxA小干涉RNA(si-MxA)转染HepG2细胞并以α干扰素(IFN-α)处理细胞,实时荧光定量PCR检测HepG2细胞黏液病毒抗性蛋白A(MxA)、蛋白激酶R(PKR)和寡聚腺苷酸合成酶(OAS)的mRNA表达,Western blot法检测MxA、PKR、OAS、细胞信号转导子与转录激活子1(STAT1)、磷酸化的STAT1(p-STAT1)、STAT2、p-STAT2和干扰素调节因子9(IRF9)的蛋白表达。此外,pcDNA3.1-Flag-MxA与pISRE-TA-luc分别共转染至HepG2和HepG2.2.15细胞,荧光素酶活性检测干扰素刺激应答元件(ISRE)活性。结果MxA蛋白在HepG2细胞质和细胞核均有表达且细胞质表达量高于胞核。敲低HepG2细胞中MxA表达虽不影响IFN-α诱导的STAT1、p-STAT1、STAT2、p-STAT2和IRF9蛋白的表达,但显著降低抗病毒蛋白PKR和OAS的表达。过表达MxA的HepG2细胞ISRE活性增强且PKR和OAS蛋白表达增高,但这种效应却在HepG2.2.15细胞中被抑制。结论MxA通过增强JAK/STAT信号通路ISRE活性诱导抗病毒蛋白表达。

猪外周血淋巴细胞干扰素诱导跨膜蛋白的拓扑学分析及其在猪繁殖与呼吸综合征病毒感染后转录水平的变化

目的对猪外周血淋巴细胞(peripheral blood lymphocytes,PBMCs)干扰素诱导跨膜蛋白(IFN-induced transmembrane,IFITM)进行拓扑学分析,并检测PBMCs在体外感染猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)后IFITM基因mRNA转录水平的变化。方法无菌采集PRRSV、猪圆环病毒2(porcine circovirus 2,PCV2)和乙型脑炎病毒(Japanese encephalitis virus,JEV)阴性的仔猪抗凝血,分离PBMCs,PCR扩增猪IFITM CDS序列,测序并进行拓扑学分析。PBMCs体外感染PRRSV,分别于感染后12、24、36和48 h收集细胞样品,RT-PCR法检测PBMCs中PRRSV感染情况,qRT-PCR法检测IFITM1、IFITM2和IFITM3基因mRNA转录水平的变化。结果成功分离到猪PBMCs,并克隆获得了PBMCs源IFITM CDS全长序列,猪IFITM蛋白可能存在两种拓扑学结构。PBMCs接种PRRSV后24 h可产生明显的细胞病变。PRRSV可在PBMCs中复制。PRRSV感染早期可显著上调PBMCs中IFITM1、IFITM2和IFITM3基因mRNA的转录水平,且均在感染后第12 h达高峰,随后逐渐下降;IFITM1基因mRNA的转录水平在病毒感染后第36 h有所回升,然后又迅速回落。结论PRRSV体外感染PBMCs可显著上调猪IFITM基因mRNA的转录水平,表明IFITM参与PBMCs抗病毒免疫反应。本研究为揭示机体抗PRRSV天然免疫反应提供了参考。

Splicing factor SF3B3,a NS5-binding protein,restricts ZIKV infection by targeting GCH1

Zika virus(ZIKV),a positive-sense single-stranded RNA virus,causes congenital ZIKV syndrome in children and Guillain-Barre Syndrome(GBS)in adults.ZIKV expresses nonstructural protein 5(NS5),a large protein that is essential for viral replication.ZIKV NS5 confers the ability to evade interferon(IFN)signalling;however,the exact mechanism remains unclear.In this study,we employed affinity pull-down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analyses and found that splicing factor 3b subunit 3(SF3B3)is associated with the NS5-Flag pull-down complex through interaction with NS5.Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene(ISG)expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication.GTP cyclohydrolase I(GCH1)is the first and ratelimiting enzyme in tetrahydrobiopterin(BH4)biosynthesis.NS5 upregulates the expression of GCH1 during ZIKV infection.And GCH1 marginally promoted ZIKV replication via the IFN pathway.Additionally,GCH1 expression is related to the regulation of SF3B3.Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels,whereas SF3B3 knockdown increased its levels.These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.

Ⅰ型和Ⅲ型干扰素抗病毒作用机制的研究进展

干扰素(interferon,IFN)是一类重要的细胞因子,直接参与上皮细胞的抗病毒免疫反应,介导天然免疫应答诱导IFN刺激基因(interferon stimulated gene,ISG)的表达,从而使机体快速建立针对病毒侵染的天然免疫防线。目前认为Ⅰ型和Ⅲ型IFN在很多功能上重叠相似,均具有抗病毒、抗肿瘤增殖和免疫调节作用。同时,在很大程度上,介导的信号通路也很相似。但Ⅰ型IFN在病毒感染早期即发挥抗病毒作用并可发挥长期免疫功能,期间反应迅速且能够强烈诱导ISG的产生,而Ⅲ型IFN则相对迟缓,且诱导ISG的能力有限,主要在天然免疫反应的黏膜免疫中发挥重要作用。此外,相关基因的表达导致IFN系统在遗传进化上出现差异,反映了物种间的差异性。本文对Ⅰ型和Ⅲ型IFN在免疫调节以及抗病毒领域方面的研究进展作一综述,以期为今后IFN介导相关免疫的研究提供参考。