CD81的分子生物学研究进展

CD81是一种分子量为26kD的四跨膜蛋白分子。在细胞膜上可与CD4、CD8、CD19、CD21、CD82、Leu13、HLA-DR和α3β1整合素等结合,调节跨膜信号转导。最近又证实CD81可与丙型肝炎病毒(HCV)包膜糖蛋白E2结合,可能是HCV感染靶细胞的受体蛋白。     

CD81分子的生物学功能

CD81分子又名抗增生抗体识别的靶抗原－1（TAPA－1）或分子量为38KD的蛋白,即M38抗原,是四跨膜蛋白（TM4SF）超家族的成员之一,CD81分子蛋白质的氨基和羧基末端皆位于细胞的膜内。从其氨基末端开始的第一、二段跨膜区和第三、四段跨膜区,分别围成膜外较小和较大的环状结构区,是细胞膜上CD81分子感知细胞外部信号的关键结构位点。CD81的特异性抗体可以导致表达CD81分子的T淋巴瘤细胞系的增殖反应,抑制人T 巴细胞白血病病毒－1（HTLV－1）感染引起的靶细胞合胞体的形成。最近的研究表明,CD81分子可能是丙型肝炎病毒（HCV）感染靶细胞的受体分子。     

CD81外显子6、7、8在中国人丙型肝炎病毒感染中单核苷酸多态性的研究

目的 探讨CD81在中国人丙型肝炎病毒（hepatitis C virus,HCV)感染中是否存在遗传易感性。方法 针对人和非洲绿猴的CD81有4个氨基酸位点（163、186、188、196）不同，而非洲绿猴不感染HCV，以正常人群和HCV感染阳性患者的基因组DNA为研究对象，用PCR结合DNA测序的方法对以上4个氨基酸对应的CD81基因位点进行分析。结果 在CD81外显子6、7、8没有发现单核苷酸多态性；尽管在一部分内含子6和3′端调控区发现了3个多态位点：11028G／T、11860C／T、11960G／A，但它们与HCV的感染无关（P＞0．05）。结论 未见CD81外显子6、7、8与HCV感染的易感性相关；由于CD81蛋白的其它位点在种间是高度保守的，因此在中国人群中CD81蛋白的多态性稀少或不存在。     

CD81分子的生物学功能

CD8 1分子又名抗增生抗体识别的靶抗原 - 1(TAPA - 1)或分子量为 38KD的蛋白 ,即M38抗原 ,是四跨膜蛋白 (TM4SF)超家族的成员之一。CD81分子蛋白质的氨基和羧基末端皆位于细胞的膜内。从其氨基末端开始的第一、二段跨膜区和第三、四段跨膜区 ,分别围成膜外较小和较大的环状结构区 ,是细胞膜上CD81分子感知细胞外部信号的关键结构位点。CD81的特异性抗体可以导致表达CD81分子的T淋巴瘤细胞系的增殖反应 ,抑制人T淋巴细胞白血病病毒 - 1(HTLV - 1)感染引起的靶细胞合胞体的形成。最近的研究表明 ,CD81分子可能是丙型肝炎病毒 (HCV)感染靶细胞的受体分子     

CD81分子与丙型肝炎病毒感染

CD8 1(TAPA 1)是一种非糖基化膜蛋白和细胞膜表面粘附分子 ,在体内分布广泛 ,是四跨膜蛋白 (TM4SF)超家族成员之一。具有影响细胞的粘附激活、形态改变、增殖分化及信号传导等生物学功能。新近研究表明 ,CD81能与HCV包膜糖蛋白E2结合 ,它可能是丙型肝炎病毒 (HCV)感染靶细胞的受体分子。     

介导HCV入胞相关分子的研究进展

丙型肝炎病毒(HCV)受体一直是HCV研究的热点之一,除了已经提出的可能受体:CD81、低密度脂蛋白受体、粘多糖、清道夫受体及C型(钙离子依赖型)凝集素DC/L-SIGN等,目前研究者又发现了新的受体claudin、occludin等.本文就上述HCV受体相关分子的研究进展作一综述.     

人CD81胞外区EC2基因的克隆和原核表达

CD８１是一种跨膜的非糖基化蛋白。在人类和黑猩猩中,CD８１胞外区EC２区氨基酸序列高度保守。CD８１可影响细胞的粘附、激活、增殖和分化,改变细胞形态及抑制合胞体形成等生物学功能〔１〕。近年研究发现,CD８１可能作为HCV的受体引导病毒进入细胞内〔２〕。进一步研究发现,CD８１胞外区EC２是CD８１与HCVE２相互作用的部位,是HCV感染的关键因素〔３〕。目前,CD８１的功能及作用机制尚有待进一步研究,我们采用分子生物学技术,构建了CD８１胞外区EC２原核表达载体,为今后进一步研究CD８１与HCV感染的关系奠定了基础…     

人CD81的克隆及在COS-7细胞中的表达

目的 从人外周血淋巴细胞中克隆出CD81基因，构建真核表达质粒，并在COS－7细胞中进行表达。方法 分离外周血淋巴细胞，提取细胞总RNA，采用RT－PCR扩增CD81基因。将CD81基因克隆至载体pcDNA3.1( )中，进行酶切及测序鉴定。以质粒pcDNA3.1-CD81转染COS－7细胞进行瞬时表达，并用免疫细胞化学染色法和流式细胞术检测蛋白的表达。结果 RT－PCR产物已插入载体pcDNA3.1( )构建成真核表达质粒pcDNA3.1-CD81。经双酶切和测序鉴定表明，克隆出的人CD81全长编码序列同GenBank收录的序列一致，并且真核表达质粒的构建正确。以脂质体转染COS－7细胞后，用免疫细胞化学染色法和流式细胞术检测表明，细胞可表达人CD81。结论 成功地构建真核表达载体pcDNA3.1-CD81，为进一步研究HCV和CD81的相互作用，以及建立可能的HCV细胞感染模型打下了基础。     

CD81与免疫应答

CD8 1(TAPA 1)是一种非糖基化膜蛋白 ,属于四跨膜区蛋白超家族成员 ,具有影响细胞的粘附、激活、增殖和分化等生物学功能。本文主要就CD81对免疫应答的影响作一综述     

054 CD81与免疫应答

CD81(TAPA-1)是一种非糖基化膜蛋白,属于四跨膜区蛋白超家族成员,具有影响细胞的粘附、激活、增殖和分化等生物学功能.本文主要就CD81对免疫应答的影响作一综述.     

Variation of hepatitis C virus load,hypervariable region 1 quasispecies and CD81 hepatocyte expression in hepatocellular carcinoma and adjacent non-cancerous liver

Hepatitis C virus (HCV) infection is etiologically associated with the development of hepatocellular carcinoma (HCC) worldwide. HCV has been reported to exist and replicate in both HCC and adjacent non-cancerous liver tissue, but limited information was available on HCV viral load and quasispecies composition in HCC relative to adjacent non-cancerous hepatocytes. Previous study has also suggested CD81, a surface hepatocyte protein, as a receptor for HCV. To clarify the above, HCV-RNA and CD81-RNA titers in 20 paired hepatectomized liver and serum were quantitatively measured by chemiluminescent RT-cPCR. Hypervariable region 1 (HVR-1) variations of parallel specimens were analyzed after subcloning in 6 patients. HCV-RNA levels in serum and non-cancerous liver were markedly higher for HCV genotype 1 than genotype non-1. HCV levels were markedly higher in non-cancerous liver than in HCC (P = 0.001) in a genotype-independent manner, with a mean ratio of 56:1 for non-cancerous tissue to HCC. Both non-cancerous and HCC tissues had the same level of CD81-RNA expression, which was not linked to HCV load. HCV-RNA quantity in both HCC and non-cancerous liver correlated with the number of HVR-1 quasispecies in the tissue, and distinct HVR-1 subclones existed.     

The roles of CD81 and glycosaminoglycans in the adsorption and uptake of infectious HCV particles

Because appropriate cell-culture systems or small-animal models have been lacking, the early steps in the HCV life cycle have been difficult to study. A cell culture system was developed recently that allows production of infectious HCV. In this study, infectious HCV particles produced in cultured cells were used. To clarify the role of CD81 in HCV attachment and entry, the effect of anti-CD81 antibody was examined. The antibody blocked HCV virion entry but not particle attachment. Only the fraction bound to a heparin affinity column and eluted with 0.3 M NaCl productively infected Huh7 cells, indicating that infectious HCV particles bind to heparin. Both heparin treatment of the virus particles and heparinase treatment of the Huh7 cells reduced virus-cell binding without substantially inhibiting HCV infectivity. Finally, to confirm the role of both heparin sulfate proteoglycan (HSPG) and CD81 in HCV entry, the effects of heparinase I and anti-CD81 antibody were analyzed. No productive RNA replication was detected in the Huh7 cells in the presence of both heparinase I and anti-CD81 antibody. In conclusion, these data suggested that both HSPG and CD81 are important for HCV entry. HSPG may play a role in the initial cell surface binding of infectious HCV particles and CD81 is conceivably correlated with HCV entry after viral attachment.     

The small extracellular loop of CD81 is necessary for optimal surface expression of the large loop, a putative HCV receptor

Human tetraspanin CD81 is a putative receptor for hepatitis C virus (HCV), because it has been shown to bind ‘bona fide’ HCV particles. CD81, as all tetraspanins, spans the membrane four times forming two extracellular loops: a small (SEL) and a large one (LEL). We have shown previously that a recombinant form of LEL is sufficient for binding HCV through the major envelope glycoprotein E2. The role of SEL in the CD81–HCV interaction was questioned. We found that transfectants expressing LEL alone bind the recombinant HCV-E2 protein at much lower levels than cells expressing the wild type CD81. And therefore whether SEL contributes to the CD81–HCV interaction or whether it influences the expression of LEL was examined. We have found that in the absence of SEL, LEL is expressed at significantly reduced levels on the cell surface because it is retained intracellularly, while HCV-E2 still binds LEL. Our data suggest that SEL of CD81 does not mediate interaction with HCV, but contributes to optimal cell surface expression of LEL by mediating translocation of the whole CD81 molecule to the cell surface.     

Small molecule inhibition of hepatitis C virus E2 binding to CD81

The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.     

Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry

CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.     

CD81 engineered with endocytotic signals mediates HCV cell entry: implications for receptor usage by HCV in vivo

Although CD81 has been shown to bind HCV E2 protein, its role as a receptor for HCV remains controversial. In this study, we constructed two CD81 chimeras by linking the cytoplasmic domains of recycling surface receptors, low-density lipoprotein receptor (LDLR), and transferrin receptor (TfR), respectively, to CD81 and compared their internalization properties to wild-type CD81. Binding experiments with anti-hCD81 antibody showed that cell-surface CD81 chimeric receptors were internalized much more efficiently than wild-type CD81. In addition, CD81 chimeras, but not wild-type CD81, could internalize recombinant E2 protein and E2-enveloped viral particles from the serum of HCV-infected patients into Huh7 liver cells. The latter resulted in persistent positive-strand viral RNA and accumulation of replication intermediates, negative-strand viral RNA, in the infected cells, suggesting that the internalized viruses have undergone replication. Therefore, it appeared that CD81, possibly in association with a liver-specific endocytotic protein(s), represents one of the pathways by which HCV can infect hepatocytes.     

HBeAg与CD81分子结合的研究

目的 :研究HBeAg与CD81分子在细胞内、外的相互作用。方法 :应用RT PCR技术 ,从HepG2细胞中扩增CD81全基因 ,并构建重组真核表达载体。将其与pGBKT7 eAg共转染营养缺陷型酵母细胞 ,观察生长情况。应用网织红细胞裂解体外翻译及免疫共沉淀试验 ,进一步验证CD81分子与HBeAg的结合。结果 :经EcoRI和BamHI酶切和DNA序列测定鉴定表明 ,构建的CD81基因的重组表达载体正确。共转染后的酵母细胞在营养缺陷的培养基中生长良好。体外免疫共沉淀试验证实 ,CD81与HBeAg出现沉淀带。结论 :HBeAg与CD81分子在细胞内、外均可结合 ,推测CD81在HBV的致病过程中起着重要的作用