Crystal structure of enteric adenovirus serotype 41 short fiber head

Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding.     

Characterization of type 5 adenovirus fiber protein.

Type 5 adenovirus fiber protein was purified and subjected to chemical characterization. Equilibrium sedimentation ultracentrifugation analysis indicated that the intact fiber has a molecular weight of approximately 183,000. Denaturation and chemical analyses implied that the fiber consists of three polypeptide chains, each of about 61,000 mol wt. Mapping of tryptic peptides and electrophoretic separation of the constituent chains suggested that the intact fiber consists of two identical and one unique polypeptide chains.     

Human adenovirus 2 temperature-sensitive mutant 112 contains three mutations in the protein IIIa gene

The temperature-sensitive (ts) mutant 112 of human adenovirus 2 is defective in the late stage of virus maturation. The region of functional mutation has been localised by marker rescue. It was observed that the ts mutation can be rescued by the left-hand part of the wild-type gene (nucleotides 12,301-12,891). By nucleotide sequencing, two mutations, both C to T (at position 12,386 and 12,741), were found in this region. The first one, in the glycine 20 codon, is silent, whereas the second changes alanine 145 to valine. A third mutation, which changed C to A (nucleotide 13,613), was identified in the right-hand part of the gene, resulting in the replacement of alanine-436 by threonine.     

原核表达人41型腺病毒(Ad41) 蛋白 V及其抗血清的制备

摘要：目的 人肠腺病毒Ad41被称为难养腺病毒,其难以培养的特性可能与次要核心蛋白V（Ad41 protein V,pV）表达不充分有关。本研究拟表达纯化Ad41 pV抗原,免疫动物制备抗血清,为研究Ad41难养性机理打下基础。方法 以野生型Ad41基因组DNA为模板,PCR扩增pV,克隆到原核表达载体pET30a(+),测序后,转化大肠杆菌BL21(DE3)菌株,异丙基-β-D-硫代半乳糖苷（IPTG）诱导目的蛋白表达,固化金属亲和层析(IMAC)方法纯化,免疫BALB/c小鼠制备抗血清,将获得的抗血清用于Western blot检测Ad41感染各细胞系后pV的表达。结果 克隆得到包括完全编码区的pV基因,表达质粒转化BL21(DE3)菌株,使用1 m mol/L IPTG 37℃诱导4 h,pV以包涵体形式表达,或使用0.5 m mol/L IPTG 25℃诱导8 h获得可溶性表达。利用皮下多点注射包涵体的方法免疫小鼠,得到抗pV抗血清;使用纯化的可溶性pV作为抗原对抗血清进行了鉴定,表明该抗血清可用于Western blot检测。等量野生型Ad41感染293或293E12细胞（一株稳定表达Ad41 E1B55K基因的293细胞）后,pV在293E12细胞的表达明显高于293细胞。结论 成功克隆了Ad41 pV基因,表达纯化了重组蛋白,获得了可用于Western blot检测的抗血清,为进一步研究Ad41难养性机理打下了基础。     

Human adenovirus serotype 3 fiber protein. Comparison of native and recombinant proteins

We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.     

Specific properties of two enteric adenovirus 41 clones mapped within early region 1A.

Enteric adenovirus type 40 (Ad40) and Ad41 form the sixth subgenus of human adenoviruses. They are associated with infantile diarrhea but cannot be isolated in conventional cell cultures. The genome of the fastidious enteric Ad41 has been cloned, and the cleavage sites of the genome produced by restriction endonucleases BamHI, EcoRI, HpaI, NruI, PvuI, and SalI have been mapped. To develop useful hybridization methods for direct detection of adenoviruses, a restriction fragment library containing Ad41 DNA, with plasmid pBR322 as vector, has been constructed. Clones have been isolated which contain 8 of 10 possible BamHI fragments of Ad41, inserted into the BamHI cleavage site of the vector. Two of these clones are particularly useful for the detection of adenoviruses. One clone detects members of all human adenovirus subgenera, and the second clone is specific for enteric adenoviruses, in particular Ad41. A conspicuous absence of detectable homology was noted at 1.5 to 3.3 map units of the Ad41 genome in hybridizations against other serotypes of adenoviruses, including the closely related enteric Ad40. This sequence corresponds to the 5' portion of early region Ia.     

The mouse adenovirus type 1 contains an unusual E3 region.

Since the E3 region of human adenoviruses codes for a series of proteins that are probably involved in viral pathogenesis, the nucleotide sequence for a 3.6-kilobase DNA fragment in the corresponding region (map units 77 through 89) of the mouse adenovirus type 1 genome has been determined. Analysis of the sequence revealed that the genes for the fiber and for the precursor to the hexon-associated protein, pVIII, that usually flank the E3 region, are well conserved. However, many of the open reading frames contained in the E3 region of human adenoviruses between the pVIII and the fiber genes were absent from the mouse adenovirus type 1 genome.     

Human skeletal muscle fiber type specific protein content

The aim of this project was to develop a method to assess fiber type specific protein content across the continuum of human skeletal muscle fibers. Individual vastus lateralis muscle fibers (n = 264) were clipped into two portions: one for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fiber typing and one for Western blot protein identification. Following fiber type determination, fiber segments were combined into fiber type specific pools (～20 fibers/pool) and measured for total protein quantity, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), citrate synthase (CS), and total p38 content. GAPDH content was 64, 54, 160, and 138% more abundant in myosin heavy chain (MHC) I/IIa, MHC IIa, MHC IIa/IIx, and MHC IIx fibers, respectively, when compared with MHC I. Inversely, CS content was 528, 472, 242, and 47% more abundant in MHC I, MHC I/IIa, MHC IIa, and MHC IIa/IIx fibers, respectively, when compared with MHC IIx. Total p38 content was 87% greater in MHC IIa versus MHC I fibers. These data and this approach establish a reliable method for human skeletal muscle fiber type specific protein analysis. Initial results show that particular proteins exist in a hierarchal fashion throughout the continuum of human skeletal muscle fiber types, further highlighting the necessity of fiber type specific analysis.     

Sequence of the DNA-binding protein gene of a human subgroup B adenovirus (type 7). Comparisons with subgroup C (type 5) and subgroup A (type 12)

The adenovirus type 7 (Ad7) single-stranded DNA-binding protein (DBP) structural gene has been sequenced and located between 66.7 and 62.3 map units. This region codes for a protein that contains 517 amino acid residues with a calculated molecular mass of 58,240 daltons. We compared the Ad7 amino acid sequence with those reported for the Ad5 (Kruijer, W., van Schaik, F.M.A., and Sussenbach, J.S. (1981) Nucleic Acids Res. 9, 4439-4457) and Ad12 (Kruijer, W., van Schaik, F.M.A., Speijer, J.G., and Sussenbach, J.S. (1983) Virology 128, 140-153) DNA-binding proteins. A greater amount of amino acid sequence homology was found in the carboxyl-terminal DNA-binding domain of the molecule. This homology is 61% between Ad7 and Ad5 and 49% when Ad12 was included in the comparison. The NH2-terminal domain of DBP retained a 49% homology between Ad7 and Ad5 and a 23% homology for all three serotypes. The greatest difference between the Ad7 and Ad5 DBPs is the absence, in the Ad7 protein, of 12 amino acids located between the two functional domains in the Ad5 protein (amino acids 151-162). In addition, three regions of high amino acid conservation between Ad5, Ad7, and Ad12 consisting of 9 (178-186), 9 (322-330), and 12 (464-475) consecutive amino acids (numbers refer to Ad5) in the DNA-binding portion of the molecule were revealed. These three regions contain a centrally located basic amino acid (183, 326, and 470) as well as an aromatic amino acid residue (181, 324, and 469). Since basic and aromatic amino acids have been implicated in other single-stranded DNA-binding protein/DNA interactions (Anderson, R.A., Nakashima, V., and Coleman, J.E. (1975) Biochemistry 14,907-917; Kowalczykowski, S.C., Lonberg, N., Newport, J.W., and von Hippel, P.H. (1981). J. Mol. Biol. 145, 75-104), these three conserved regions may represent DBP/DNA contact points.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

Identification of the glycosylation site of the adenovirus type 5 fiber protein

The fiber protein purified from the pool of nonincorporated viral protein after infection of cells with adenovirus type 5 exists as two forms separable by reverse-phase HPLC. As determined by mass spectrometry, this heterogeneity results from a difference in one O-linked N-acetylglucosamine (GlcNac). A western blot analysis using a monoclonal antibody directed against the GlcNac motif showed that only one of the two forms reacted with the antibody, suggesting that one form carries a single GlcNac and the other form has none. The ratio of glycosylated to nonglycosylated forms of fiber, which is about 1, is conserved in assembled viruses. After digestion of glycosylated fiber with endoproteinase GluC, isolation of the glycosylated peptide by reverse-phase HPLC, and chemical derivatization using dimethylamine, the site of glycosylation was located in the fiber shaft at serine 109 by mass spectrometry. Elimination of glycosylation by site-directed mutagenesis of fiber should help to understand the function of this postranslational modification.     

Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C. The synthesized protein constituted about 1% of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.