The coxsackievirus and adenovirus receptor.

The coxsackievirus and adenovirus receptor (CAR) has been studied extensively since its identification and isolation in 1997. The CAR is an immunoglobulin superfamily protein with two extracellular Ig-like domains, a single membrane-spanning sequence, and a significant cytoplasmic domain. It is structurally and functionally similar to the junctional adhesion molecules. The amino terminal domain, distal from the membrane, has been structurally characterized alone, bound to the adenovirus fiber knob, and, in full-length CAR, docked in the canyon structure of the coxsackievirus capsid. Although the past decade has produced a burst of new knowledge about CAR, significant questions concerning its function in normal physiology and coxsackievirus-related pathology remain unanswered.

Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR.

Binding of virus particles to specific host cell surface receptors is known to be an obligatory step in infection even though the molecular basis for these interactions is not well characterized. The crystal structure of the adenovirus fiber knob domain in complex with domain I of its human cellular receptor, coxsackie and adenovirus receptor (CAR), is presented here. Surface-exposed loops on knob contact one face of CAR, forming a high-affinity complex. Topology mismatches between interacting surfaces create interfacial solvent-filled cavities and channels that may be targets for antiviral drug therapy. The structure identifies key determinants of binding specificity, which may suggest ways to modify the tropism of adenovirus-based gene therapy vectors.

Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection.

Although recombinant adenoviruses are attractive vectors for gene transfer to airway epithelia, they have proven to be relatively inefficient. To investigate the mechanisms of adenovirus-mediated gene transfer to airway epithelia, we examined the role of adenovirus fiber and penton base, the two proteins involved in attachment to and entry of virus into the cell. We used human airway epithelia grown under conditions that allow differentiation and development of a ciliated apical surface that closely resembles the in vivo condition. We found that addition of fiber protein inhibited virus binding and vector-mediated gene transfer to immature airway epithelia, as well as to primary cultures of rat hepatocytes and HeLa cells. However, fiber protein had no effect on vector binding and gene transfer to ciliated airway epithelia. We obtained similar results with addition of penton base protein: the protein inhibited gene transfer to immature epithelia, whereas there was no effect with ciliated epithelia. Moreover, infection was not attenuated with an adenovirus containing a mutation in penton base that prevents the interaction with cell surface integrins. These data suggest that the receptors required for efficient infection by adenovirus are either not present or not available on the apical surface of ciliated human airway epithelia. The results explain the reason for inefficient gene transfer and suggest approaches for improvement.

Adenovirus interaction with its cellular receptor CAR.

Representative adenoviruses from four of the five major virus subgroups have been shown to interact with the 46-kDa coxsackievirus and adenovirus receptor (CAR) that is widely expressed on many human cell types, suggesting that the ability to bind CAR may be a conserved feature of many of the approximately 50 known adenovirus serotypes. Receptor binding is a function of the distal 'knob' domain of the trimeric viral fiber protein. Here we review recent structural characterizations of knob, CAR and knob-CAR complexes, and we discuss how knob architecture may have evolved to accommodate opposing selective pressures to vary antigenic structure while conserving receptor binding specificity. In contrast to the hypervariability of the solvent-exposed surface of knob, the CAR receptor was found to be non-polymorphic.

Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer.

Despite encouraging preclinical studies in many tumor types including head and neck squamous cell carcinoma (HNSCC), initial clinical trials with adenovirus-mediated gene therapy have been disappointing. Although the adenovirus is a "highly efficient vector," it is still limited by the extent of effective in vivo transduction. In our studies with multiple human HNSCC cell lines, we have noted a variation in both in vitro and in vivo responses to the same recombinant adenovirus therapeutic construct. We hypothesize that adenovirus receptor density among tumor cell populations, even of the same histology, greatly influences transduction efficiency and therapeutic results of a variety of adenovirus-based gene therapy strategies. To investigate this hypothesis, the numbers of adenovirus receptors on three well-characterized HNSCC cell lines were determined. Marker and cytokine gene transfer efficiencies as well as therapeutic outcomes after adenovirus-mediated tumor suppressor gene and suicide gene therapies were evaluated and correlated with receptor status. A 5-fold variation in adenovirus receptor density was identified among the HNSCC cell lines (P < 0.002, t test). This variation directly correlated with adenovirus type 5 (Ad5)-mediated green fluorescent protein marker gene and Ad5-interleukin 2 cytokine gene transfer efficiency and resulting protein expression in each individual cell line. The receptor density also directly correlated with therapeutic response after Ad5-thymidine kinase or Ad5-p16 gene transfer in each HNSCC line. The role of the adenovirus receptor in gene transfer efficiency was further supported by recombinant Ad5 fiber knob blocking experiments. The marker gene transfer was increasingly blocked by the same concentration of Ad5 recombinant fiber knob in relation to decreasing levels of adenovirus receptor in the HNSCC lines. An Ad5 recombinant construct that carries the shared coxsackie and adenovirus receptor (CAR) was created and used to up-regulate receptors on each cell line. Ad5-CAR infection significantly increased Ad5-beta-Gal gene transfer efficiency and expression (P = 0.0003, Mann-Whitney test). This increased marker gene expression remained consistent with the established pattern of gene transfer efficiency among the HNSCC cell lines. These data confirm the importance of the adenovirus receptor on individual tumor cell lines with respect to investigating novel adenovirus-mediated gene therapy strategies. This work further supports consideration of assaying adenovirus receptor status, even in tumors of the same histology from patients enrolled in gene therapy clinical trials. Adenovirus receptor status may prove valuable for selecting or stratifying patients as well as assessing outcomes among patients within adenovirus-based cancer gene therapy trials.

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes.

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla--the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen-presenting cells including subsets of dendritic cells.

Purification and cDNA sequence of a murine protein homologous to the human p62 tyrosine phosphoprotein that associates with the Ras GTPase-activating protein p120 GAP.

A 68-kDa protein that binds to the murine dendritic cell-specific monoclonal antibody M342 was purified and its amino acid sequence was partially determined. Corresponding cDNA clones code for a protein that is closely related to a 62-kDa human protein (p62) that associates with the Ras GTPase-activating protein, p120 GAP, suggesting that p62 may have a dendritic cell-specific function or that an M342 cross-reactive epitope may exist on the murine p62 molecule.

Insertion of myoglobin T-cell epitopes into the Escherichia coli alkaline phosphatase.

We are interested in antigen processing mechanisms of antigen-presenting cells, and to what extent the susceptibility of protein antigens to degradation contributes to immunogenicity. Understanding the biochemistry of antigen processing may be essential for reliable prediction of T-cell epitopes and for the design of vaccines that are optimized for T-cell priming. To examine possible effects of protein structural context on antigen presentation, we used genetic engineering techniques to insert helper T-cell epitopes derived from sperm whale myoglobin into surface loops of the highly stable Escherichia coli alkaline phosphatase, with the expectation that presentation of the myoglobin guest epitopes might vary with their position in the carrier protein. Levels of recombinant protein expression in E. coli cells and residual enzyme activity depended on the location of the guest peptides in the alkaline phosphatase carrier. Mutants with insertions between residues 189-190 of the carrier were recovered with yields and activities similar to the wild type protein; however, insertion of the same peptides at a second site, between residues 165-166, led to low recoveries and diminished phosphatase activities. Subcutaneous injection of mice with one of the purified recombinant proteins in complete Freund's adjuvant induced T cells that responded to in vitro challenge with myoglobin. The potential use of this system to dissect processing mechanisms is discussed.

Frameshift mutation in PRKDC, the gene for DNA-PKcs, in the DNA repair-defective, human, glioma-derived cell line M059J.

Anderson, C. W., Dunn, J. J., Freimuth, P. I., Galloway, A. M. and Allalunis-Turner, M. J. Frameshift Mutation in PRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J. Radiat. Res. 156, 2-9 (2001). The glioma-derived cell line M059J is hypersensitive to ionizing radiation, lacks DNA-PK activity, and fails to express protein for the catalytic subunit, DNA-PKcs, while a sister cell line, M059K, derived from the same tumor, has normal DNA-PK activity. Both cell lines are near pentaploid and have multiple copies of chromosome 8, the chromosome on which the DNA-PKcs gene, PRKDC, is located. Sequence analysis of PCR-amplified exons revealed the loss in M059J cells of a single "A" nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC. Loss of the "A" nucleotide would terminate the DNA-PKcs reading frame early in exon 33. DNA from M059K cells had only the wild-type sequence. An analysis of sequences surrounding PRKDC exon 32 from 87 unrelated individuals revealed no polymorphic nucleotides except for a triplet repeat near the 3' end of this exon; no individual had a frameshift mutation in exon 32. No other sequence differences in PRKDC between M059J and M059K cells were observed in approximately 15,000 bp of genomic sequence including the sequences of exons 5 through 38 and surrounding intron sequence, suggesting a possible reduction to homozygosity at this locus prior to acquisition of the mutation leading to the M059J cell line.

A human cell line selected for resistance to adenovirus infection has reduced levels of the virus receptor.

To investigate determinants of host cell susceptibility to infection, cells partially resistant to infection were selected from the rare cells which remained adherent after infection of a culture of A549 cells with Ad2RAE, a mutant of adenovirus type 2 whose vertex capsomers lack an Arg-Gly-Asp (RGD) sequence which mediates binding of wild-type virus to integrins. Integrins promote the internalization of attached virions, whereas adsorption itself results from binding of the viral fibers to an unidentified cellular receptor. Following three rounds of selection, a persistently infected culture was established in which virus replication was detected in approximately 5% of the cells. Uninfected cells were readily cloned from the culture, indicating that at any particular time the majority of cells in the culture were uninfected. The resistance of one clone of uninfected cells to infection was correlated with a 10-fold reduction in the concentration of fiber receptors on these cells compared with the parental A549 cell line, indicating that efficiency of virus adsorption depends on the receptor concentration. Surprisingly, the rate at which host cells internalized RGD-negative virus also was strongly dependent on the fiber receptor concentration. While internalization of wild-type virus is promoted by the binding of integrins to the penton base RGD sequence, these results suggest that virus also can enter cells by an alternate pathway which requires binding of virions to multiple fiber receptors.

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Codon insertion mutants of the adenovirus terminal protein.

A series of codon insertion mutants was isolated following restriction site-directed linker insertion mutagenesis of the open reading frame for the type 5 adenovirus terminal protein precursor. The conditionally lethal mutant H5sub100 bears an insertion mutation upstream of the first AUG in the reading frame, fails to replicate its DNA under nonpermissive conditions, and was assigned to the terminal protein complementation group. These data establish that terminal protein is an essential polypeptide required for DNA replication in vivo and indicate that the NH2-terminal region of the precursor is encoded in an upstream mRNA leader. The extended eclipse period of the viral replication cycle in H5in179-infected cells is probably a consequence of delayed onset of DNA replication. Analysis of DNA replication in coinfections with wild-type virus shows that the in179 mutation has cis and trans effects. The trans-dominant, negative-complementing in179 terminal protein precursor inhibits wild-type DNA replication in a dose-dependent manner. Replication of parental in179 templates is not stimulated by an excess of coinfecting wild-type virus, indicating that the mutant terminal protein covalently bound to the in179 template in some way interferes with the replication of that template. The implications of these results for the structure and function of the terminal protein are discussed.

Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.

The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.

Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.

The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm. We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein. Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base. In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis. In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2. Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.

A single locus on human chromosome 21 directs the expression of a receptor for adenovirus type 2 in mouse A9 cells.

The receptors on human cells which mediate adsorption of adenoviruses have not been identified. We found that murine A9 cells and Chinese hamster ovary (CHO) cells failed to bind significant levels of radiolabeled adenovirus type 2 (Ad2) virions but that derivatives of these cells carrying human chromosome 21 exhibited high levels of virus binding that was specific for the viral fiber protein. G418-resistant A9 cell transformants expressing Ad2 receptors were detected at a frequency of about 10(-4) following cotransfection with high-molecular-weight DNAs from mouse cells containing human chromosome 21 and plasmid DNA containing a neomycin resistance gene. The Ad2 receptors on the transformed A9 cells were similar to those on human cells with respect to their concentration on the cell membrane, their affinity for the viral fiber protein, and their ability to direct virus into cells along a pathway leading to delivery of the viral DNA genome into the cell nucleus. Furthermore, identical human DNA fragments were present in three independent mouse cell transformants expressing Ad2 receptors, supporting the conclusion that these human DNA fragments correspond to a gene or locus on chromosome 21 that directs the expression of Ad2 receptors in these cells.

Shope fibroma virus. II. Role of the virion-associated nucleases.

The effect of Shope fibroma virus (SFV) infection on host DNA synthesis was investigated. The cytocidal strain, SFV-I, inhibited the incorporation of [3H]thymidine into nuclear DNA very shortly (2 h) after infection, whereas the noncytocidal strain, SFV-W, did so later (10 h postinfection) and to a lesser extent. Furthermore, a two- to threefold stimulation of host DNA synthesis was recorded in SFV-W-infected cells 3 to 4 h after infection. Since virion-associated nucleases have been implicated in the shutoff of host synthesis, these and other enzymatic activities were measured in purified virion preparations. The SFV strains and vaccinia virus contained equivalent amounts of DNA-dependent RNA polymerase, ATPase, and protein kinase activities. However, in SFV-W the pH 4.5 exonuclease activity was lower than in SFV-I and vaccinia virus, and the level of pH 7.8 endonuclease was almost undetectable. To test whether the lack of endonucleolytic activity had some effect on the removal of the cross-links in the parental DNA that occurs after viral penetration, the fate of the virion SFV DNA was followed. The majority (80%) of the SFV-I and SFV-W DNA molecules extracted after viral adsorption sedimented in alkaline sucrose gradients as cross-linked. After 3 h of infection, 75% of the SFV-I DNA molecules lacked cross-links, whereas 78% of the SFV-W DNA still remained cross-linked. The same results were obtained when the presence of cross-links was tested in restriction fragments. Taken together, these results indicate that virion-associated nucleases are involved in the early shutoff of host DNA synthesis and in the elimination of cross-links from the parental viral DNA.

Shope fibroma virus. I. Biological and molecular properties of a cytocidal and a noncytocidal strain.

The biological and molecular properties of two strains of Shope fibroma virus (SFV) were compared. SFV-I was highly cytocidal to most of the cell lines tested and produced pocks in the chorioallantoic membrane of chick embryos. By contrast, SFV-W did not produce cytopathic effects in any of the cell lines or in the chorioallantoic membrane, but it induced characteristic foci 3 to 4 days after infection. Both strains produced tumors when inoculated into the skin of susceptible rabbits. Maximal infectivity in BSC-1 cells was reached by both strains between 24 to 48 h after inoculation. Viral DNA synthesis also took place at the same time, although cells infected with SFV-I incorporated three times more [(3)H]thymidine than cells infected with SFV-W. Sedimentation analysis and hydroxylapatite chromatography of the two viral DNAs indicated that their molecular weights were similar and that both were naturally cross-linked. Digestion with three restriction endonucleases, however, revealed that they had different restriction sites. When SFV-I and vaccinia DNA were compared, the restriction patterns were more alike. Analysis of the virion structural proteins by gel electrophoresis indicated that SFV-I, SFV-W, and vaccinia virus had many polypeptides in common, although there were distinctive differences among the three viruses. Finally, the results of plaque neutralization tests with different antisera showed that SFV-I and SFV-W shared common antigens and that vaccinia antiserum inhibited SFV-I but not SFV-W. We conclude that the SFV-I genome contains information for both cytolysis and tumorigenesis. This unusual virus may be a recombinant between an orthopoxvirus and a leporipoxvirus.

Introduction of guest peptides into Escherichia coli alkaline phosphatase. Excision and purification of a dynorphin analogue from an active chimeric protein.

High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution not only to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichia coli alkaline phosphatase polypeptide by linker insertion mutagenesis of the phoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues (Sowadski, J. M., Foster, B. A., and Wycoff, H. W. (1981) J. Mol. Biol. 150, 245-272). Mutant protein enzymatic function was generally tolerant of insertions in exterior loops, but was inactivated by insertion within alpha-helical or beta-strand structural elements. We further demonstrate that these tolerant surface loops can serve as vehicles for high level expression and stabilization of larger foreign peptide sequences, using a 15-residue analogue of dynorphin as an example. Insertion of the dynorphin "guest" peptide probably caused only a local structural perturbation of the alkaline phosphatase carrier since the hybrid protein retained enzymatic activity, was exported efficiently to the periplasmic space, and could be purified by anion-exchange chromatography using a protocol developed for alkaline phosphatase itself. The gust peptide was recovered from one of these fusion proteins intact and in high yield by protease digestion in vitro and was then purified by cation-exchange chromatography to near homogeneity in a single step.

Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA.

Adenovirus DNA is tightly bound to the nuclear matrix throughout the course of infection. Analysis of adenovirus DNA from infected HeLa cell nuclei after extraction with lithium diiodosalicylate and digestion with restriction enzymes demonstrated that the sites of tightest attachment occur in the terminal fragments of the linear viral chromosome. Analysis of viruses mutated in the precursor terminal protein coding sequence demonstrated that the terminal protein, which is covalently attached to the 5' end of each DNA strand, mediates the tight binding. Virions containing chromosomes with mutant terminal proteins were unpackaged and viral DNA accumulated in the nucleus at a normal rate and competed for the limiting component during transcription complex formation, but their early genes were transcribed at reduced efficiency by both RNA polymerases II and III. The transcriptional defects were not complemented by coinfection with a wild-type virus. We propose that the adenovirus chromosome may exist as a single chromatin domain during infection and that binding of DNA to the nuclear matrix may play a critical role in adenovirus transcription.

Coxsackievirus and adenovirus receptor amino-terminal immunoglobulin V-related domain binds adenovirus type 2 and fiber knob from adenovirus type 12.

The extracellular region of the coxsackievirus and adenovirus receptor (CAR) is predicted to consist of two immunoglobulin (Ig)-related structural domains. We expressed the isolated CAR amino-terminal domain (D1) and a CAR fragment containing both extracellular Ig domains (D1/D2) in Escherichia coli. Both D1 and D1/D2 formed complexes in vitro with the recombinant knob domain of adenovirus type 12 (Ad12) fiber, and D1 inhibited adenovirus type 2 (Ad2) infection of HeLa cells. These results indicate that the adenovirus-binding activity of CAR is localized in the amino-terminal IgV-related domain and confirm our earlier observation that Ad2 and Ad12 bind to the same cellular receptor. Preliminary crystallization studies suggest that complexes of Ad12 knob bound to D1 will be suitable for structure determination.