人呼吸道合胞病毒感染的SPC-A1细胞相关基因 zzz171con的电子克隆与分析

采用mRNA差异显示RT-PCR方法克隆到人呼吸道合胞病毒感染的SPC-A1细胞中相关的50个表达序列标签,其中一个片段g17-1在HRSV感染的细胞中高表达。利用生物信息学的方法对g17-1进行分析鉴定,推测是一个新基因。利用电子克隆,得到一全长2101bp的cDNA,对其ORF、启动子、电子表达谱、染色体定位以及所编码的蛋白进行分析,结果表明,该cDNA含有1个ORF,与人类假定蛋白XP_097121同源性达90%,定位于人17号染色体,在肿瘤细胞、腺癌细胞、扁桃体初级B细胞中表达,其编码的蛋白预测定位于细胞核内,推测其与细胞抗病毒感染有一定的关系。     

人呼吸道合胞病毒感染的SPC-A1细胞相关基因 zzz171con的电子克隆与分析

采用mRNA差异显示RT-PCR方法克隆到人呼吸道合胞病毒感染的SPC-A1细胞中相关的50个表达序列标签,其中一个片段g17-1在HRSV感染的细胞中高表达。利用生物信息学的方法对g17-1进行分析鉴定,推测是一个新基因。利用电子克隆,得到一全长2101bp的cDNA,对其ORF、启动子、电子表达谱、染色体定位以及所编码的蛋白进行分析,结果表明,该cDNA含有1个ORF,与人类假定蛋白XP_097121同源性达90%,定位于人17号染色体,在肿瘤细胞、腺癌细胞、扁桃体初级B细胞中表达,其编码的蛋白预测定位于细胞核内,推测其与细胞抗病毒感染有一定的关系。     

Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein

Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of < or =22 degrees C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.     

Surface display of rice stripe virus NSvc2 and analysis of its membrane fusion activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper.How RSV enters an insect cell to initiate the infection cycle is poorly understood.Sequenc...     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

日本血吸虫新基因Sj-MA的克隆、表达及保护性免疫

为发现新基因 ,寻找日本血吸虫病新疫苗候选分子 ,采用Sj雄虫免疫血清筛选Sj成虫cDNA文库。经测序发现新基因Sj MA含有一个完整的阅读框 ,推测其由 2 4 9个氨基酸组成 ,编码分子量为 2 8.8kD的可溶性蛋白质 ,并带有多个能被磷酸化激活的位点 ,提示其可能为一重要的信息传递分子。将Sj MA的cDNA亚克隆至原核表达载体pGEX 5X ,获得Sj MA原核表达的重组体rSj MA/GST ,并在E .coli中高效表达为谷胱甘肽S 转移酶 (GST)融合蛋白 ,分子量为 5 4 .8kD ,Western印迹显示融合蛋白质能被抗雄虫和抗GST血清识别。融合蛋白质免疫小鼠可诱导 34.2 9%的减虫率 ,与对照组有显著性差异 (P <0 .0 0 1 )。表明新基因Sj MA表达的蛋白质能诱导小鼠的抗日本血吸虫的保护性免疫 ,提示其作为日本血吸虫疫苗候选分子的潜在价值     

M phase-specific association of human topoisomerase IIIbeta with chromosomes

Two isoforms, 1 and 2, of human DNA topoisomerase IIIbeta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform 1 of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform 1 is in the C-terminal part that is different between the two isoforms.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

OX40 ligand and programmed cell death 1 ligand 2 expression on inflammatory dendritic cells regulates CD4 T cell cytokine production in the lung during viral disease

CD4 Th differentiation is influenced by costimulatory molecules expressed on conventional dendritic cells (DCs) in regional lymph nodes and results in specific patterns of cytokine production. However, the function of costimulatory molecules on inflammatory (CD11b(+)) DCs in the lung during recall responses is not fully understood, but it is important for development of novel interventions to limit immunopathological responses to infection. Using a mouse model in which vaccination with vaccinia virus vectors expressing the respiratory syncytial virus (RSV) fusion protein (rVVF) or attachment protein (rVVG) leads to type 1- or type 2-biased cytokine responses, respectively, upon RSV challenge, we found expression of CD40 and OX40 ligand (OX40L) on lung inflammatory DCs was higher in rVVF-primed mice than in rVVG-primed mice early after RSV challenge, whereas the reverse was observed later in the response. Conversely, programmed cell death 1 ligand 2 (PD-L2) was higher in rVVG-primed mice throughout. Inflammatory DCs isolated at the resolution of inflammation revealed that OX40L on type 1-biased DCs promoted IL-5, whereas OX40L on type 2-biased DCs enhanced IFN-γ production by Ag-reactive Th cells. In contrast, PD-L2 promoted IFN-γ production, irrespective of conditions, suppressing IL-5 only if expressed on type 1-biased DCs. Thus, OX40L and PD-L2 expressed on DCs differentially regulate cytokine production during recall responses in the lung. Manipulation of these costimulatory pathways may provide a novel approach to controlling pulmonary inflammatory responses.     

The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1

The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.     

新城疫病毒HN基因诱导人肺癌细胞SPC-A1凋亡的作用机制

为了探索新城疫病毒血凝素神经氨酸酶(HN)基因对人肺癌细胞SPC-A1的作用及机制,将含有HN基因的重组质粒pVHN经脂质体介导转染人肺癌细胞SPC-A1,通过MTT方法检测细胞活力;采用吖啶橙/溴化乙锭（AO/EB）染色分析肿瘤细胞凋亡;罗丹明123和DCFA染色测定线粒体跨膜电位（ΔΨm）和活性氧水平变化;流式细胞仪分析MHCⅠ分子表达; 底物染色反应检测caspase-3活性结果重组质粒pVHN转染人肺癌细胞SPCA1 48 h后,细胞活力明显降低; AO/EB染色可见明显的细胞凋亡形态学变化;与空质粒对照相比,线粒体ΔΨm下降(Ｐ<0.01),活性氧水平升高(Ｐ<0.05);细胞表面MHC-Ⅰ分子表达上调(P>0.05);caspase-3活性增强(Ｐ<0.01). 以上结果提示,新城疫病毒HN基因能够上调SPC-A1细胞表面MHC-Ⅰ分子表达,并通过上调ROS水平,下调线粒体ΔΨm,进而激活caspase-3,最终诱导人肺癌细胞凋亡.     

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

Five proteins of Laodelphax striatellus are potentially involved in the interactions between rice stripe virus and vector

Rice stripe virus (RSV) is the type member of the genus Tenuivirus, which relies on the small brown planthopper (Laodelphax striatellus Fallén) for its transmission in a persistent, circulative-propagative manner. To be transmitted, virus must cross the midgut and salivary glands epithelial barriers in a transcytosis mechanism where vector receptors interact with virions, and as propagative virus, RSV need utilize host components to complete viral propagation in vector cells. At present, these mechanisms remain unknown. In this paper, we screened L. striatellus proteins, separated by two-dimensional electrophoresis (2-DE), as potential RSV binding molecules using a virus overlay assay of protein blots. The results, five L. striatellus proteins that bound to purified RSV particles in vitro were resolved and identified using mass spectrometry. The virus-binding capacities of five proteins were further elucidated in yeast two-hybrid screen (YTHS) and virus-binding experiments of expressed proteins. Among five proteins, the receptor for activated protein kinase C (RACK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH3) did not interact with RSV nucleocapsid protein (NCP) in YTHS and in far-Western blot, and three ribosomal proteins (RPL5, RPL7a and RPL8) had specific interactions with RSV. In dot immunobinding assay (DIBA), all five proteins were able to bind to RSV particles. The five proteins' potential contributions to the interactions between RSV and L. striatellus were discussed. We proposed that RACK and GAPDH3 might be involved in the epithelial transcytosis of virus particles, and three ribosomal proteins probably played potential crucial roles in the infection and propagation of RSV in vector cells.     

Incorporation of chimeric gag protein into retroviral particles.

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.     

在大肠杆菌体内有效地表达以组蛋白为骨架的融合蛋白HNHG

研究了在大肠杆菌体内表达了以组蛋白C 末端为骨架的融合蛋白HNHG[H(HA2 0 )N(NLS)H(组蛋白H10 的C末端 97个氨基酸 )G(GE7) ],并发现质粒DNA与其形成复合体后 ,在电镜下出现与体内染色体相同的凝聚、螺旋现象。由于HNHG的这种有效地结合质粒、并使之凝聚的特性 ,有望使之成为新兴的能将外源基因导入体内的载体系统。     

Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.     

Involvement of ADAM9 in multinucleated giant cell formation of blood monocytes

Monocytes-macrophages are converted to multinucleated giant cells by stimulation with various cytokines, and osteoclasts are the multinucleated giant cells derived from a monocyte-macrophage lineage. However, at present, the fusion peptides have not been clearly identified in monocytes-macrophages. The ADAM are a family of transmembrane glycoproteins that have a role in various biological functions. Interestingly, fertilin-alpha, ADAM9, and ADAM11 have potential fusion peptides. In this study, which ADAM was specifically expressed in monocytes stimulated with anti-CD98 antibody or RANKL and which factor(s) was functioning in monocytes as a fusion protein were investigated. ADAM1, 8, 10, 12, 15, 17, 20, and 21 mRNAs are expressed in blood monocytes incubated with control antibody, anti-FRP-1/CD98 antibody, or RANKL + M-CSF, while ADAM2, 7, 11, 13, 19, 23, 29, and 30 mRNAs could not be detected in these blood monocytes. Expression of ADAM9 and ADAM10 mRNAs are enhanced by either RANKL + M-CSF or anti-CD98 antibody. The expression of ADAM9 and ADAM10 is also induced in blood monocytes by anti-CD98 mAb. An anti-ADAM9 antibody enhances CD98-mediated cell aggregation, while it blocks CD98-mediated and RANKL-mediated multinucleated giant cell formation. A hydroxamate-based metalloprotease inhibitor, SI-27, which is found to suppress ADAM9 activity, suppresses multinucleated giant cell formation. New protein synthesis is necessary for the expression of ADAM9 mRNA and genistein suppresses induction of ADAM9 mRNA. This is the first report that ADAM9 is involved in monocyte fusion, such as CD98-mediated and RANKL-mediated cell fusion of blood monocytes. Furthermore, AMAM9 is one candidate for a fusion peptide in blood monocytes.