Interaction between HeLa cells and adenovirus type 2 virions neutralized by different antisera.

Three adenovirus type 2-specified immunogens elicited neutralizing antibodies when injected into rabbits; these were the fiber, the hexon, and the penton base. Adenovirus type 2 virions, neutralized by antihexon- or anti-penton base antisera, attached to HeLa cells to the same extent as untreated control virus, and after attachment, neutralized viruses also became sensitive to DNase treatment. A fraction of 75 to 80% of the attached antibody-treated virions penetrated the plasma membrane, which should be compared with an 84 to 88% penetration level in the control series. A majority of the antihexon-neutralized virions was found in intracellular vesicles, as revealed with an electron microscope, but in the case of anti-penton base neutralization, a maximum of 50% of the virions was retained within vesicles, and ca. 30% was free in the cytoplasmic compartment. A value greater than 45% was never obtained for neutralization with a monospecific anti-penton base antiserum, which could imply the existence of alternative pathways for virus penetration into HeLa cells--one of these being sensitive to treatment with anti-penton base antiserum. Antisera containing antifiber specificities efficiently aggregated virions, and the aggregation data mirrored the degree of neutralization. Antifiber-neutralized virions attached to cells to a three- to five times greater extent than untreated control virus, but the former virions had a reduced ability to become sensitive to DNase treatment. Around 15% of the attached antifiber-treated virions was found as large aggregates inside multivesicular bodies or lysosomes.     

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.     

Antibody-mediated uncoating of adenovirus in vitro

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.     

Molecular composition of the adenovirus type 2 virion

The representation of the different structural polypeptides within the adenovirus virion has been accurately determined, and the particle molecular weight has been derived. A stoichiometric analysis was performed with [35S]methionine as a radiolabel, and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the polypeptides. The recently available sequence of the adenovirus type 2 genome was used to determine the number of methionines in each polypeptide. The resulting relative representation was placed on an absolute scale by using the known number of hexon polypeptides per virion. The analysis provides new information on the composition of the vertex region, which has been the subject of some controversy. Penton base was found to be present in 60 copies, distributed as pentamers at each of the 12 vertices. Three fiber monomers were associated with one penton base to form the penton complex. Polypeptide IX was present in 240 copies per virion and 12 copies per group-of-nine hexons, supporting a model proposed earlier for the distribution of this protein. The location of polypeptide IX explains the dissociation of the virus outer capsid into groups-of-nine hexons. The penton base was microheterogeneous, and the relative amounts suggest that the symmetry mismatch, which occurs within the penton complex between base and fiber, is resolved by the synthesis of penton base polypeptides from two closely spaced start codons.     

Role of vesicles during adenovirus 2 internalization into HeLa cells.

In this investigation, the early period of adenovirus type 2 (Ad2)-HeLa cell interaction was analyzed by electron microscopy and biochemical techniques. Events observed in this period ranged from the disappearance of virions from the cell surface to their subsequent association with the cell nucleus. Destabilization of the virions attached to the intact cell was necessary for virions to escape from intracellular vesicles. Strong temperature dependence and rapid escape from a vesicular compartment were shown in temporal kinetic experiments. These vesicles appeared to be acidic, since lysosomotropic agents partly inhibited the release of virions from vesicles. Studies of Ad2 binding to cells in buffers of different pH values suggested that adenovirus binds to cells by two different mechanisms. At low pH the binding was most probably mediated by the penton base and at neutral pH by the fiber protein. The number of receptor sites per cell was 25,000 and 6,000 at low and neutral pH, respectively. This study suggests that the low-pH affinity between the penton base and a vesicular membrane is important inside acid vesicles when Ad2 quickly enters the cytoplasm. However, a significant fraction of the virions was possibly internalized by a pathway not requiring a passage through such vesicles.     

Molecular weight of adenovirus serotype 2 capsomers a new characterization

We have determined the molecular weight of some of the adenovirus serotype 2 structural proteins: penton, penton base and fibre. Physical techniques, namely neutron scattering and hydrodynamical measurements, indicate that the penton base is a trimer. This is confirmed by analysis of the virion composition based on quantitative gel scanning. This finding implies either that other proteins (e.g. protein IIIa) are essential in the architecture of the fivefold vertex of the virion, or that the usual assumption that icosahedral symmetry involves identical interactions related to the symmetry of the virion does not hold.     

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.     

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.     

Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction.     

Antigenic determinants of adenovirus capsids. II. Homogeneity of hexons, and accessibility of their determinants, in the virion.

We have tested the two principal theories which explain the previous finding that small amounts of type-specific antibody to the adenovirus hexon can neutralize infectivity, whereas even large amounts of cross-reactive antibody do not. a) It has been suggested that the type-specific determinants are especially prominent in the virion. We have therefore measured the capacity of whole virus to bind appropriate antibodies, using a sensitive radioimmunoprecipitation (RIP) system. In fact, virions bound type-specific and cross-reactive antibodies impartially. Moreover, they bound both much less effectively than did free hexon or disrupted virus, suggesting that many of each kind of determinant are inaccessible in virions. b) It has been suggested that the type-specific determinants are confined to those hexons located next to the pentons, and that they are the targets for neutralizing antibody. We have therefore studied the antigenicity of peripentonal and nonamer hexons isolated from virions, and found that each possessed both kinds of determinants. Furthermore, these were present in the same proportion as in hexons purified from the soluble antigens in infected cells ("free hexons"). We concluded that the mechanism of neutralization by antibody is complicated, and that the type-specific determinants exposed on the virion must play a crucial role.     

Restricted occupancy models for neutralization of HIV virions and populations

HIV virions infect cells by attaching to target cell receptors, fusing membranes with the cell and by finally releasing their genetic material into the target cells. Antibodies can hinder the infection by attaching to the HIV envelope glycoprotein trimers before or during attachment. The exact mechanisms and the quantitative requirements of antibody neutralization are still debated. Recently, the number of antibodies rendering one trimer non-functional, called stoichiometry of (trimer) neutralization, was studied with mathematical models. Here we extend this theoretical framework to calculate the stoichiometries of neutralizing a single virion and a whole virion population. We derive mathematical equations for antibody neutralization based on restricted occupancy theory. Additionally we simulate these processes when a direct calculation is not possible. We find that the number of trimers needed for cell entry and the number of antibodies neutralizing one trimer strongly influence the mean number of antibodies needed for virion and population neutralization. Further we show that the mean number of antibodies needed to neutralize a virion population exceeds the product of the number of virions in the population and the mean number of antibodies needed to neutralize one virion.     

CryoEM structure at 9A resolution of an adenovirus vector targeted to hematopoietic cells

We report a sub-nanometer resolution cryo-electron microscopy (cryoEM) structural analysis of an adenoviral vector, Ad35F, comprised of an adenovirus type 5 (Ad5) capsid pseudo-typed with an Ad35 fiber. This vector transduces human hematopoietic cells via association of its fiber protein with CD46, a member of the complement regulatory protein family. Major advances in data acquisition and image processing allowed a significant improvement in resolution compared to earlier structures. Analysis of the cryoEM density was enhanced by docking the crystal structures of both the hexon and penton base capsid proteins. CryoEM density was observed for hexon residues missing from the crystal structure that include hypervariable regions and the epitope of a neutralizing monoclonal antibody. Within the penton base, density was observed for the integrin-binding RGD loop missing from the crystal structure and for the flexible beta ribbon of the variable loop on the side of the penton base. The Ad35 fiber is flexible, consistent with the sequence insert in the third beta-spiral repeat. On the inner capsid surface density is revealed at the base of the hexons and below the penton base. A revised model is presented for protein IX within the virion. Well-defined density was assigned to a conserved domain in the N terminus of protein IX required for incorporation into the virion. For the C-terminal domain of protein IX two alternate conformations are proposed, either binding on the capsid surface or extending away from the capsid. This model is consistent with the tolerance of the C terminus for inserted ligands and its potential use in vector retargeting. This structural study increases our knowledge of Ad capsid assembly, antibody neutralization mechanisms, and may aid further improvements in gene delivery to important human cell types.     

Immunoenzyme analysis of adenovirus hexon. Determination of antibodies from hyperimmunized chickens

We have elaborated three systems of enzyme-linked immunosorbent assay (ELISA) for detection of chicken IgG antibodies specific for hexon antigens of three immunologically distinct adenovirus groups: those of mammalian adenoviruses (Mastadenovira), typical avian adenoviruses (Aviadenovira) and of egg-drop syndrome-76 (EDS-76) virus. In each system the antibodies against respective hexons were specifically detected. In mammalian adenovirus hexons the ELISA detects primarily the type-specific (epsilon) and genus-specific (alpha) antigenic determinants. The time course of anti-hexon antibodies content was followed during immunization. The level of anti-hexon antibodies in egg yolk reflects adequately their content in blood serum. The technique is suitable for serological diagnosis of chicken adenoviral infections as well as for characterization of egg-yolk antibodies obtained by preparative hyperimmunization of hens.     

Unfolding studies of human adenovirus type 2 fibre trimers. Evidence for a stable domain.

Adenovirus fibres are trimeric proteins that protrude from the 12 fivefold vertices of the virion and are the cell attachment organelle of the virus. They consist of three segments: an N-terminal tail, which is noncovalently attached to the penton base, a thin shaft carrying 15 amino acid pseudo repeats, and a C-terminal globular head (or knob) which recognizes the primary cell receptor. Due to their exceptional stability, which allows easy distinction of native trimers from unfolded forms and folding intermediates, adenovirus fibres are a very good model system for studying folding in vivo and in vitro. To understand the folding and stability of the trimeric fibres, the unfolding pathway of adenovirus 2 fibres induced by SDS and temperature has been investigated. Unfolding starts from the N-terminus and a stable intermediate accumulates that has the C-terminal head and part of the shaft structure (shown by electron microscopy). The unfolded part can be digested away using limited proteolysis, and the precise digestion sites have been determined. The remaining structured fragment is recognized by monoclonal antibodies that are specific for the trimeric globular head and therefore retains a native trimeric structure. Taken together, our results indicate that adenovirus fibres carry a stable C-terminal domain, consisting of the knob with five shaft-repeats.     

Model of the trimeric fiber and its interactions with the pentameric penton base of human adenovirus by cryo-electron microscopy

Adenovirus invades host cells by first binding to host receptors through a trimeric fiber, which contains three domains: a receptor-binding knob domain, a long flexible shaft domain, and a penton base-attachment tail domain. Although the structure of the knob domain associated with a portion of the shaft has been solved by X-ray crystallography, the in situ structure of the fiber in the virion is not known; thus, it remains a mystery how the trimeric fiber attaches to its underlying pentameric penton base. By high-resolution cryo-electron microscopy, we have determined the structure of the human adenovirus type 5 (Ad5) to 3.6-Å resolution and have reported the full atomic models for its capsid proteins, but not for the fiber whose density cannot be directly interpreted due to symmetry mismatch with the penton base. Here, we report the determination of the Ad5 fiber structure and its mode of attachment to the pentameric penton base by using an integrative approach of multi-resolution filtering, homology modeling, computational simulation of mismatched symmetries, and fitting of atomic models into cryo-electron microscopy density maps. Our structure reveals that the interactions between the trimeric fiber and the pentameric penton base are mediated by a hydrophobic ring on the top surface of the penton base and three flexible tails inserted into three of the five available grooves formed by neighboring subunits of penton base. These interaction sites provide the molecular basis for the symmetry mismatch and can be targeted for optimizing adenovirus for gene therapy applications.     

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.     

Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion

HIV-1 can infect T cells by cell-free virus or by direct virion transfer between cells through cell contact-induced structures called virological synapses (VS). During VS-mediated infection, virions accumulate within target cell endosomes. We show that after crossing the VS, the transferred virus undergoes both maturation and viral membrane fusion. Following VS transfer, viral membrane fusion occurs with delayed kinetics and transferred virions display reduced sensitivity to patient antisera compared to mature, cell-free virus. Furthermore, particle fusion requires that the transferred virions undergo proteolytic maturation within acceptor cell endosomes, which occurs over several hours. Rapid, live cell confocal microscopy demonstrated that viral fusion can occur in compartments that have moved away from the VS. Thus, HIV particle maturation activates viral fusion in target CD4+ T cell endosomes following transfer across the VS and may represent a pathway by which HIV evades antibody neutralization.     

Adenovirus-dependent release of choline from plasma membrane vesicles at an acidic pH is mediated by the penton base protein.

It has been suggested that during receptor-mediated endocytosis of human adenovirus (Ad) type 2 into cells, Ad disrupts the membrane of endocytic vesicles to enter the cytosol. To understand the mechanism of Ad-mediated disruption of the endocytic vesicles, I exposed Ad to plasma membrane vesicles derived from KB cells. Ad caused release of choline from the plasma membrane vesicles preloaded with [3H]choline. The efflux of choline was dependent on (i) the concentration of Ad, with a half-maximal effect at 0.5 microgram/ml; (ii) the pH of the buffer, with the optimum pH of the reaction ranging from 5.5 to 6.0; (iii) the length of the incubation, with a half-maximal release at 2 min; and (iv) the temperature of the incubation, with the optimum temperature being 37 degrees C. The Ad-dependent release of choline was inhibited by anti-penton base, while antihexon did not block the effect. These results suggest roles for a low-pH environment and the penton base protein in the Ad-dependent efflux of choline from plasma membrane vesicles.     

Impact of human adenovirus type 3 dodecahedron on host cells and its potential role in viral infection

During human adenovirus type 3 (Ad3) infection, an excess of penton base and fiber proteins are produced which form dodecahedral particles composed of 12 pentamers of penton base and 12 trimers of fiber protein. No biological functions have yet been ascribed to Ad3 dodecahedra. Here, we show that dodecahedra compete with Ad3 virions for binding to the cell surface and trigger cell remodeling, giving new insights into possible biological functions of dodecahedra in the Ad3 infectious cycle.     

Identification of adenovirus (ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative ad (addl1520) for treatment of liver tumors

Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.