Structure of the dodecahedral penton particle from human adenovirus type 3

The sub-viral dodecahedral particle of human adenovirus type 3, composed of the viral penton base and fiber proteins, shares an important characteristic of the entire virus: it can attach to cells and penetrate them. Structure determination of the fiberless dodecahedron by cryo-electron microscopy to 9 Angstroms resolution reveals tightly bound pentamer subunits, with only minimal interfaces between penton bases stabilizing the fragile dodecahedron. The internal cavity of the dodecahedron is approximately 80 Angstroms in diameter, and the interior surface is accessible to solvent through perforations of approximately 20 Angstroms diameter between the pentamer towers. We observe weak density beneath pentamers that we attribute to a penton base peptide including residues 38-48. The intact amino-terminal domain appears to interfere with pentamer-pentamer interactions and its absence by mutation or proteolysis is essential for dodecamer assembly. Differences between the 9 Angstroms dodecahedron structure and the adenovirus serotype 2 (Ad2) crystallographic model correlate closely with differences in sequence. The 3D structure of the dodecahedron including fibers at 16 Angstroms resolution reveals extra density on the top of the penton base that can be attributed to the fiber N terminus. The fiber itself exhibits striations that correlate with features of the atomic structure of the partial Ad2 fiber and that represent a repeat motif present in the amino acid sequence. These new observations offer important insights into particle assembly and stability, as well as the practicality of using the dodecahedron in targeted drug delivery. The structural work provides a sound basis for manipulating the properties of this particle and thereby enhancing its value for such therapeutic use.     

Co‐chaperone BAG3 and adenovirus penton base protein partnership

The BAG family of Hsp70/Hsc70 co‐chaperones is characterised by the presence of a conserved BAG domain at the carboxyl‐terminus. BAG3 protein is the only member of this family containing also the N‐terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N‐terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co‐migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co‐chaperone activity, suggesting that this interaction is not related to the classical BAG3 co‐chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad‐infected cells, suggesting that the interaction between virus penton base protein and cellular co‐chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host–pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co‐chaperone. J. Cell. Biochem. 111: 699–708, 2010. © 2010 Wiley‐Liss, Inc.     

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100-containing inclusions during adenovirus infection

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.     

Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants.

The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII.     

Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli

Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.     

Structure of gamma-secretase and its trimeric pre-activation intermediate by single-particle electron microscopy

The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active γ-secretase using single-particle cryo-electron microscopy. γ-Secretase has a cup-like shape with a lateral belt of ∼40–50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.     

The nucleotide sequence of the polypeptide IX gene of human adenovirus type 3

The nucleotide sequence of the DNA segment encompassing the polypeptide IX gene of class B human adeno-virus serotype 3 (Ad3) has been determined using cloned restriction fragments. There is only a single, open translational reading frame capable of specifying a protein of 138 amino acids, comparable to the M_r 12000–13000 of protein IX detected in virions (Wadell, 1980). The corresponding region of a closely related class B virus, Ad7, is virtually identical (Dijkema et al., 1981), but the comparable segments of class C viruses Ad2 or Ad5 are much less homologous (Aleström et al., 1980; Maat et al., 1980). There are 150 single bp changes and 19 deletion-insertions, at least one frameshift, together affecting 210 nucleotides within the 455 bp comparison positions of the protein-coding regions of Ad2 (423 bp) and Ad3 (417 bp). Each of the 19 deletion-insertions involves an integral multiple of 3 bp in phase with the open translation frame. There is no “TATA” promoter box in Ad3 DNA at the position comparable to that of Ad2. The deduced protein sequences near the amino-terminus are extensively conserved between the two classes of viruses, but the carboxy-terminal portion and the nucleotide sequences flanking the gene are much more diverged. In both classes, these N- and C-terminal regions of the inferred proteins are linked by an alanine-rich chain, an arrangement suggestive of two functional domains.     

Expression of the adenovirus receptor and its interaction with the fiber knob

The coxsackievirus group B (CVB) and adenovirus (Ad) receptor (HCVADR, formerly HCAR) is a cell surface protein with two immunoglobulin-like regions (IG1 and IG2) that serves as a receptor for two structurally unrelated viruses. We have established the tissue distribution of the receptor in the rodent by immunohistochemistry and show that the receptor is broadly expressed during embryonic development in the central and peripheral nervous systems and in several types of epithelial cells. The tissue distribution is more restricted in the adult but remains high mainly in epithelial cells. Using site-directed mutagenesis, based on computer modeling of the IG1 region, Ad5 binding could be inhibited but CVB attachment was unaffected. A double amino acid substitution in a three-stranded anti-parallel beta sheet that may form a face of the receptor completely inhibited Ad5 binding. Therefore, we conclude that the molecular interactions critical for Ad5 binding to HCVADR do not overlap with those of CVB3. In fact a specific antibody interfering with only CVB binding recognizes the IG2 domain in the receptor, suggesting that the CVB interacts with this region or an overlap between the IG1 and the IG2 regions.     

人成纤维细胞生长因子受体2IIIc及其突变型重组腺病毒的获得和在乳腺癌细胞中的表达

获得人成纤维细胞生长因子受体2Ⅲc(FGFR2Ⅲc)及其S252W突变型重组腺病毒,感染乳腺癌细胞MDA-MB-231,为下一步研究FGFR2Ⅲc基因的功能和作用机制奠定基础。以本实验室保存的含FGFR2Ⅲc基因的质粒为模板,PCR扩增得到FGFR2Ⅲc基因,重叠延伸法PCR获得FGFR2ⅢcS252W突变型基因;分别将上述野生型和突变型基因克隆至腺病毒穿梭质粒pAdTrack-CMV上,得到重组穿梭质粒pAdTrack-FGFR2Ⅲc和pAdTrack-FGFR2ⅢcS252W,DNA测序证实。Pme I酶切后分别与腺病毒骨架质粒pAdEasy-1共转化BJ-5183感受态细菌同源重组,得到的重组表达质粒Ad-FGFR2Ⅲc和Ad-FGFR2ⅢcS252W Pac I酶切线性化后转染HEK293A细胞进行重组腺病毒的包装和扩增,通过GFP报告基因观察病毒表达情况。收集重组病毒颗粒并测定滴度,进一步感染乳腺癌细胞MDA-MB-231,RT-PCR和Western blotting方法检测目的基因的表达,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法和流式细胞术分析细胞增殖情况。结果表明,成功构建了人FGFR2Ⅲc及其S252W突变型基因的重组腺病毒表达载体,获得的重组腺病毒颗粒能高效感染MDA-MB-231细胞,并表达目的基因。MTT结果显示FGFR2Ⅲc和S252W均能抑制MDA-MB-231细胞增殖,S252W抑制效果更加明显。流式细胞术表明FGFR2Ⅲc和S252W均能使MDA-MB-231细胞周期停滞于G0/G1期,抑制细胞增殖。     

针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体的构建及其对肝癌细胞的抑制作用

转录因子Oct-4和Survivin是细胞增殖的关键调控因子,构建针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体Ad5-Dual-shRNA,并研究其对肝癌细胞及移植瘤的生长抑制作用。合成Oct-4和Survivin基因的shRNA序列,插入腺病毒穿梭载体pDC312,含有shRNA的穿梭载体与腺病毒骨架载体pBHGloxdeltaE13Cre共转染HEK293细胞,经Cre/LoxP位点特异性重组获得重组腺病毒Ad5-Dual-shRNA;腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1,经Western blotting检测Oct-4和Survivin基因的表达情况,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐染色法(MTT实验)和裸鼠荷瘤实验检测对肿瘤细胞生长的影响。研究结果显示,双靶向重组腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1能够有效沉默Oct-4与Survivin基因的表达,并且在MTT实验和裸鼠荷瘤试验中都显示出较单一靶向的shRNA腺病毒载体Ad5-Surv-shRNA、Ad5-Oct4-shRNA具有更为明显的肿瘤细胞生长抑制作用。实验结果表明,特异性双靶向shRNA腺病毒载体Ad5-Dual-shRNA是一种更为高效的靶向肿瘤基因治疗载体。     

人CXCL9重组腺病毒的制备及其表达产物生物活性检测

目的构建携带人CXCL9基因的重组腺病毒,分析其表达产物的生物学活性。方法采用PCR法从pBLAST2-hCXCL9质粒上扩增出hCXCL9基因,再将其克隆至pENTR11载体上,构建pENTR11-hCXCL9质粒,通过同源重组将hCXCL9基因片段重组至pAd/CMV/V5-DEST上,最后在293A细胞内进行包装、扩增后得到携带hCX-CL9基因的重组腺病毒。用包装好的病毒感染Hela细胞,分析目的基因的表达及其生物学活性。结果成功地将hCXCL9基因片段克隆至重组腺病毒载体上,并经293A细胞包装出病毒颗粒。该病毒感染Hela细胞后,可在培养上清液中检测到显著表达的hCXCL9。通过趋化活性分析发现,该表达产物对T淋巴细胞具有明显的趋化活性。结论成功构建了hCXCL9重组腺病毒,并可在体外高效表达具有生物活性的目的产物。     

重组腺病毒Ad-HGF体外感染成纤维细胞后转染效率及表达的检测

押检测携带人肝细胞生长因子基因的重组腺病毒Ad-HGF在体外对成纤维细胞的感染效率以及感染细胞对目的蛋白的表达。以不同感染复数(m.o.i.)(25,50,100,200)的Ad-GFP感染NIH3T3细胞,48h时用流式细胞仪检测转染效率;以50m.o.i.感染NIH3T3细胞后48h,用ELISA和Western印迹杂交法分别检测感染上清中HGF的表达。分别以50m.o.i.的Ad-GFP和Ad-HGF感染原代培养人瘢痕成纤维细胞,以检测重组腺病毒对原代培养人瘢痕成纤维细胞的转染效率和其对HGF的表达。结果表明,当m.o.i.为50时,重组腺病毒对NIH3T3细胞的转染效率已达95%以上;HGF的表达量可达每2×106细胞249ng;并可检测到HGF蛋白的一特异杂交带。以50m.o.i.的Ad-GFP感染原代培养人瘢痕成纤维细胞,72h时GFP表达达高峰,此时转染效率可高达36.75%。Ad-HGF感染原代培养人瘢痕成纤维细胞后HGF的表达在72h时达高峰,表达量可达每3.3×105细胞66ng。初步认为重组腺病毒可有效地介导HGF基因转染正常或瘢痕成纤维细胞,且感染细胞可有效表达目的蛋白。     

eIF-4E腺病毒载体构建及对乳腺癌MCF-7细胞转移能力的影响

摘要 目的：构建乳腺癌真核细胞起始因子一4E(eukaryotic initiation factor 4E,eIF-4E)基因重组腺病毒载体,并观察其对乳腺癌细胞MCF-7转移能力的影响。方法：应用基因重组技术将eIF-4E基因构建于腺病毒载体pAD-X,转染293包装细胞得到高滴度重组腺病毒,并用real-time PCR进行eIF-4E基因表达的验证。将重组腺病毒pAD-eIF-4E感染MCF-7细胞后,应用transwell小室法观察其对细胞侵袭和运动能力的影响。结果：酶切结果与预期相符,real-time PCR可检测到转染后MCF-7细胞有eIF-4E基因表达。且病毒转染后transwell小室可见,MCF-7细胞的侵袭和运动能力均受到显著的抑制（均为p<0.01）。结论：重组eIF-4E腺病毒载体正确构建,其对乳腺癌细胞MCF-7的侵袭和运动都有抑制作用。     

Role of dietary fiber in the recovery of the human gut microbiome and its metabolome

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Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.