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.     

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.     

Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor.     

Functional interaction of the adenovirus IVa2 protein with adenovirus type 5 packaging sequences

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.     

Biochemical analysis of adenovirus type 5 DNA-binding protein mutants

We previously reported the isolation and functional characterization of seven adenovirus type 5 (Ad5) DNA-binding protein (DBP) point mutants (Quinn, C. O., and Kitchingman, G. R. (1986) J. Virol. 60, 653-661). Six of the seven mutants were defective in their ability to help adeno-associated virus replicate its DNA. To determine the level at which the mutations affect this function of the DBP, we analyzed several properties of the mutant proteins. All are transported to the nucleus and are post-translationally phosphorylated to the same extent. The half-lives of the proteins, measured by pulse-chase, were nearly identical to that of the wild-type DBP. The mutant DBPs were examined for their ability to bind to single-stranded DNA (ssDNA). Mutations in amino acids 322, 323, and 470 lowered the affinity of the DBP for ssDNA, while a mutation in amino acid 181 had no affect. Combinations of mutations in amino acid 470 with either 322 or 323 did not further lower the affinity of the protein for ssDNA. These data indicate that the functional defect for adeno-associated virus helper activity of the six mutants is due mainly, if not totally, to their reduced affinity for single-stranded DNA. These experiments have thus identified a functional domain of the adenovirus type 5 DBP potentially involved in DNA-protein interactions. Comparisons with temperature-sensitive DBP mutants indicate that the conserved region mutants are functionally distinct and represent a new class of DBP mutants.     

Identification of active site residues of the adeno-associated virus type 2 Rep endonuclease

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.     

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.     

Radioimmunoassay of human serum antibody specific for adenovirus type 5-purified fiber.

A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with 125I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at minus 20 degrees C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per millimeter was sufficient to inhibit the 125I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.     

Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein.

The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.     

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.     

Importance of the Ser-132 phosphorylation site in cell transformation and apoptosis induced by the adenovirus type 5 E1A protein.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.     

Isolation and characterization of an extremely basic protein from adenovirus type 5.

By starch-gel electrophoresis and a staining method that is highly sensitive for argininyl residues, adenovirus type 5 was found to contain two minor basic polypeptides of extreme cathodic mobility in addition to the two known core proteins. The fastest-migrating polypeptide, named mu protein, and the second fastest polypeptide are found in adenovirions and virus-infected KB cells but not in top components or in uninfected cells. The top components and infected cells contain an additional basic polypeptide, presumably P-VII, that migrates slightly slower than polypeptide VII. None of the basic polypeptides of adenovirions was electrophoretically identical to the host histone. The basic proteins of adenovirions were purified by urea phosphocellulose column chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two minor basic core proteins, mu and another component, have similar mobilities in sodium dodecyl sulfate-polyacrylamide gels as a complex of polypeptides X-XII. After further purification on a Sephadex G-75 column, the mu protein was found to have a molecular weight of about 4,000. Amino acid analysis showed that the mu protein lacks tryptophan and 69% of the total amino acid residues are basic, that is, 54% arginine, 13% histidine, and 2% lysine. Only eight amino acids seem to contribute to make the mu polypeptide. There are 125 copies of the mu polypeptide per 1,000 copies of polypeptide VII in a virion.     

Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3

Usefulness of adenoviral vectors derived from human adenovirus (HAd) type 5 (HAd5) is mainly limited by wide prevalence of preexisting anti-HAd5 immunity as well as non-specific tissue tropism of these vectors. As an alternative, non-human adenoviral vectors including bovine adenovirus type 3 (BAd3) are currently being investigated. Non-prevalence of BAd3 in humans and its ability to evade preexisting HAd immunity are some of the features that make BAd3 a promising vector for human gene delivery. BAd3 appears to have a tissue tropism distinct from that of HAd5 and also the repertoire of cells efficiently transduced by BAd3 is different. We performed antibody-mediated receptor blocking experiments to show that BAd3 internalization was independent of coxsackievirus-adenovirus receptor, the primary determinant of HAd5 tropism, or integrin alpha(v)beta3, a secondary molecule involved in HAd5 entry. Using homologous and heterologous knob-mediated competition assays with recombinant knobs of HAd5, porcine adenovirus type 3 (PAd3), or BAd3, we observed that BAd3 internalization was independent of the primary receptors of HAd5 and PAd3. These results provide support for further exploration of BAd3 vectors for designing targeted vectors for human gene therapy.     

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5

We have determined the nucleotide sequence of a region of adenovirus type 5 (Ad5) DNA located between map positions 61.7 and 71.4, which covers the gene form the 72 kD DNA binding protein (DBP) and the sequence encoding the amino-terminal part of the 100 kD protein. Sequence analysis of cDNA copies of DBP mRNA revealed the existence of two abundant species of spliced mRNA molecules. One species consists of two short leader sequences from positions 75.2 (67 and 68 nucleotides long) and 68.8 (77 nucleotides long), respectively, and the main body of the RNA molecules. The other species contains only the leader sequence from position 75.2 and the main body. The amino acid sequence of DBP is encoded entirely by a long open reading frame of 1587 nucleotides in the main body of DBP mRNA. From the nucleotide sequence of the DBP gene it can be derived that DBP contains 529 amino acid residues and has an actual molecular weight of 59,049 daltons. The sites of mutation in the mutants H5hr404 and H5ts125 were determined at the nucleotide level. Single nucleotide alterations were detected in H5hr404 and H5ts125 in the sequences corresponding to the amino-terminal part and the carboxy-terminal part of DBP, respectively. The implications of these mutations are discussed.