Hexon Capsomers Research Articles

Human Full-Length Coagulation Factor X and a GLA Domain-Derived 40-mer Polypeptide Bind to Different Regions of the Adenovirus Serotype 5 Hexon Capsomer

The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, resulting in high levels of hepatotropism and potential hepatotoxicity. In this study, we tested the possibility of preventing these undesirable effects by using a GLA-mimicking peptide as a competitor. An FX GLA domain-derived, 40-mer polypeptide carrying 12 carboxyglutamate residues was synthesized (GLA(mim)). Surface plasmon resistance (SPR) analysis showed that GLA(mim) reacted with free and capsid-embedded hexon with a nanomolar affinity. Unexpectedly, GLA(mim) failed to compete with FX for hexon binding, and instead significantly increased the formation of FX-hexon or FX-adenovirion complexes. This observation was confirmed by in vitro cell transduction experiments using HAdV-C5-Luciferase vector (HAdV5-Luc), as preincubation of HAdV5-Luc with GLA(mim) before FX addition resulted in a higher transgene expression compared with FX alone. HAdV-C5 virions complexed with GLA(mim) were analyzed by cryoelectron microscopy. Image reconstruction demonstrated the bona fide hexon-GLA(mim) interaction, as for the full-length FX, although with considerable differences in stoichiometry and relative location on the hexon capsomer. Three extra densities were found at the periphery of each hexon, whereas one single FX molecule occupied the central cavity of the hexon trimeric capsomer. A refined analysis indicated that each extra density is found at the expected location of one highly variable loop 1 of the hexon, involved in scavenger receptor recognition. HAdV5-Luc complexed with a bifunctional GLA(mim)RGD peptide showed a lesser hepatotropism, compared with control HAdV5-Luc alone, and efficiently targeted αβ-integrin-overexpressing tumor cells in an in vivo mouse tumor model. Collectively, our findings open new perspectives in the design of adenoviral vectors for biotherapy.

Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon∶FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

Modifications of Adenovirus Hexon Allow for Either Hepatocyte Detargeting or Targeting With Potential Evasion From Kupffer Cells

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

Enhanced transduction of CAR-negative cells by protein IX-gene deleted adenovirus 5 vectors

In human adenoviruses (HAdV), 240 copies of the 14.3-kDa minor capsid protein IX stabilize the capsid. Three N-terminal domains of protein IX form triskelions between hexon capsomers. The C-terminal domains of four protein IX monomers associate near the facet periphery. The precise biological role of protein IX remains enigmatic. Here we show that deletion of the protein IX gene from a HAdV-5 vector enhanced the reporter gene delivery 5 to 25-fold, specifically to Coxsackie and Adenovirus Receptor (CAR)-negative cell lines. Deletion of the protein IX gene also resulted in enhanced activation of peripheral blood mononuclear cells. The mechanism for the enhanced transduction is obscure. No differences in fiber loading, integrin-dependency of transduction, or factor-X binding could be established between protein IX-containing and protein IX-deficient particles. Our data suggest that protein IX can affect the cell tropism of HAdV-5, and may function to dampen the innate immune responses against HAdV particles.

321. Twenty-Fold Increased In Vivo Liver Gene Transfer with a Geneti-Chemically Modified Ad Vector Carrying a High-Affinity Protein Ligand on the Hexon Capsomere

321. Twenty-Fold Increased In Vivo Liver Gene Transfer with a Geneti-Chemically Modified Ad Vector Carrying a High-Affinity Protein Ligand on the Hexon Capsomere

Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.

The 100K-Chaperone Protein from Adenovirus Serotype 2 (Subgroup C) Assists in Trimerization and Nuclear Localization of Hexons from Subgroups C and B Adenoviruses

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.

29. Redirection of Adenoviral Tropism through Capsomers Other Than Fiber Is Generally Inefficient and Dependent on Ligand-Receptor Biology

We have generated a panel of metabolically biotinylated adenoviral vectors based on the genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, and hexon capsomers of the Ad5 virion. Here we demonstrate that avidin-based redirection of Ad transduction through biotinylated ligand:receptor interactions is greatly dependent on the nature of the BAP-modified capsomer. While transduction of Ad-Fiber-BAP can generally be targeted through a wide range of ligand:receptor pairs, Ad-IX-BAP and Ad-Hexon-BAP are surprisingly inefficient as targeting platforms, despite having a higher display of biotins than Ad-Fiber-BAP. We reason this difference is due to the nature of the interactions between viral capsomers and cell surface receptors. Ad5 fiber, the natural cell-binding capsomer, has evolved to bind very tightly to cell surface CAR. Upon endosomal uptake fibers then dissociate from the capsid, releasing the virion from the receptor and enabling trafficking to continue. We believe that targeting Ad through IX or hexon capsomers results in the entrapment of virions on cellular receptors thereby leading to very inefficient transduction. Indeed, preliminary evidence suggests that protein IX stays tightly associated with the virion and does not dissociate at early stages of vector trafficking like fiber. We reason that targeted transduction through IX or hexon may be feasible with ligands that naturally release from their receptors upon endocytosis, resulting in release of the incoming virions. These initial results highlight the importance of knowledge of capsomer, ligand, and receptor biology when engineering new tropism into viral capsids and may be of great importance for vectors like AAV, which do not shed capsomers as they traffic. Current progress on the analysis of the differences in trafficking between the different BAP-modified vectors will be presented.

The Coiled-Coil Domain of the Adenovirus Type 5 Protein IX Is Dispensable for Capsid Incorporation and Thermostability

The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXDelta100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXDelta60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.

134. Structural Requirements of Adenovirus Targeting Via by Modification of Minor Capsid Protein IX

In mastadenoviruses, the hexon-associated protein IX is a 14.3kDa protein that is strongly conserved between the different members of the genus. It associates with nine hexon capsomers to form group-of-nine hexons (GON). These make up the central region of each of the facets of the icosahedron. Protein IX is believed to function as a capsid 'cement'. So far, its structure in not known. Although viruses lacking pIX do not form GONs and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains a putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. To get more insight into the structure/function relationship we studied the incorporation of heterologous pIX molecules into the HAdV-5 capsid. Our data show that the pIX proteins of HAdV-12 (species A) and HAdV-7 (species B) can be efficiently incorporated into the capsid of pIX-deleted HAdV-5 (species C). In addition, further deletion mutagenesis confirmed that the N-terminus of the HAdV-5 pIX is required and sufficient for incorporation into the capsid. These data further our knowledge on pIX and facilitated the development of pIX as an anchor for the incorporation of heterologous peptides into the capsid. In this way receptor-binding ligands were incorporated in the capsid. Inclusion of alphahelical spacers of up to 7.5 nm between the pIX anchor and the ligand resulted in more efficient binding of the virions to antibodies and cells. These data conform that modification of pIX can be used to incorporate heterologous peptides into the adenoviral capsid, and support the feasibility of this approach to amend the tissue tropism of the adenovirus vectors.

Delineation of Antigenic Determinants of Adenovirus Hexons by Means of Monoclonal Antibodies

60 hexon-specific mouse hybridoma clones were selected by using crystallized hexon capsomers of human adenovirus type 1 as immunizing and selecting antigen. The reactivities of the monoclonal antibodies were tested with purified hexon preparations from 10 human adenovirus types by enzyme-linked immunosorbent assay and passive hemagglutination techniques. The reactivity patterns delineated a total of 41 adenovirus-type-1-related determinants present on a number of heterologous hexons in different interspecies combinations. The pattern of interspecies specificities did not coincide with the current grouping of adenoviruses into subgenera.

Mutual orientation of peripentonal hexons and polypeptide subunits in the adenovirus capsid.

The mutual rotational orientation of the three hexon polypeptide subunits of the peripentonal hexons has been determined in relation to each other, to the penton and to the neighbouring hexons. The possible mutual orientation of all the polypeptide subunits of the adenovirus-building hexon capsomers are presented on a tentative virus model.

Analysis of Ad5 Hexon and 100K is mutants using conformation-specific monoclonal antibodies

Adenovirus type 5 is mutants deficient in hexon metabolism were investigated using conformation-specific monoclonal antibodies directed against hexon capsomeres and the viral 100K protein. The is mutants map either in the hexon structural gene or in the gene encoding the 100K protein, a major, late nonstructural protein. All of the mutants examined (tsl, ts2, ts3, ts4, ts17, and ts20 of J. F. Williams, M. Gharpure, S. Ustacelebi, and S. McDonald (1971). J. Gen. Virol. 11, 95–101 ) were unable to produce the capsomeric form of hexon (a trimer of three hexon monomers) at the nonpermissive temperature. However, all of the mutants retained the ability to produce a complex of 100K and hexon which has been demonstrated to play a major role in the assembly of hexon trimers. The mutants accumulated nontrimerized hexon in this is complex in the perinuclear region of the cell. Several of the mutants (tsl, ts2, ts3) were found to successfully assemble hexon synthesized at the nonpermissive temperature upon shift down to the permissive temperature, even in the presence of a protein synthesis inhibitor. The mutant, ts2, which maps in the hexon structural gene, was found to be dependent on protein synthesis for transport of hexon trimers into the nucleus during temperature shift down, while the 100K is mutants, tsl and ts3, were independent of protein synthesis for both hexon assembly and transport.

Assembly of adenovirus major capsid protein is mediated by a nonvirion protein.

The assembly of hexon, the major capsid protein of adenovirus, was investigated with the use of conformation-specific monoclonal antibodies. The hexon capsomere is a trimer of three identical monomers folded into a highly conserved and stable structure. The unique nature of this structure is indicated by the lack of common antigenic determinants between the capsomere and either monomeric or denatured hexon. The assembly of the trimer requires the action of a nonvirion protein, the 100K protein. The virus-encoded 100K protein forms a tight complex with hexon polypeptides. This 100K-hexon complex can form on the polyribosomes while hexon is a nascent chain. Exclusion chromatography revealed that the complex has a molecular weight of 800,000. The complex contains only pretrimer hexon; no mature hexon capsomeres can be found bound to 100K. Kinetic analysis of hexon trimerization and hexon-100K binding indicated that trimerization and the release of hexon from the complex occur concomitantly.

Inter- and intrahexonal connections between adenovirus hexon polypeptides in the two-dimensional crystalline array

High-resolution micrographs revealed the presence of linkers between adenovirus hexon capsomers organized into two-dimensional, closely packed arrays. Examination of electron micrographs directly and by Markham's rotational integration technique permitted the establishment of the arrangement of the interhexonal connective elements within small, distinct parts of the crystalline array. Every hexon is joined to its six nearest neighbors by six times two parallel connections. Intrahexonal connections were found also among the three polypeptide subunits constituting one complete hexon.

Electron microscopic studies on the crystallization of adenovirus hexon capsomers.

Highly purified soluble adenovirus type 1 hexons were crystallized by dialyzing against 0.5 M acetate buffer, pH 4.5, for 3 days. Crystals of tetrahedral shape were formed after storage at 4 degrees within 1--10 days and ranged in size from 0.01 to 0.1 mm. In early phase of crystallization, diffusely dispersed hexons were observed by electron microscopy. Later, continuous monolayers of two-dimensional hexon paracrystalline arrays developed. Direct analysis of the electron micrographs and their optical diffraction patterns showed that the structure of the dense two-dimensional crystalline arrays of hexon capsomers was characterized by slightly skew and irregular hexagonal packing. Multilayered microcrystals with regular linear internal structure were also found. In addition, unique forms of aggregated hexons, such as holey hexon lattice and filamentous forms, were also found.

Direct evidence for 6-fold symmetry of the herpesvirus hexon capsomere.

Rotational power spectrum analysis of scanning transmission electron micrographs of negatively stained herpesvirus capsids has given direct evidence for the 6-fold symmetry of the herpesvirus hexon capsomere. Individual hexons have been analyzed in situ without interference from other parts of the capsid because only one level of the capsid was in focus in the micrograph. This study found hexons to have 6-fold symmetry. The majority of the mass of each hexon subunit was seen to lie on a line connecting the centers of adjacent capsomeres. This agrees with analysis of conventional micrographs of capsid fragments, which are also presented, as well as with biochemical data and theoretical expectations.

Comparative optical properties of free and assembled hexon capsomeres of human adenovirus type 2

Comparative optical properties of free and assembled hexon capsomeres of human adenovirus type 2

Genetic Analysis of Adenovirus Type 2. Pleiotropic Effects in an Assembly Mutant

SUMMARY A new temperature sensitive mutant of adenovirus type 2 was isolated and characterized. H2ts48 is DNA positive but fails to synthesize stable 95K, 80K and Va (Pre-VI) polypeptides. In addition, many polypeptides exhibit reduced stability during pulse-chase experiments. No virion particles of any type are assembled although a new type of intranuclear core-like structure was observed in thin sections by electron microscopy. Hexon polypeptides (120K) are synthesized as demonstrated by SDS-polyacrylamide gel electrophoresis, but immunofluorescence, immunodiffusion and sucrose velocity gradient analyses show that no hexon capsomere antigens (360K) are assembled. Similarly, fibre polypeptides are synthesized normally, but the immunological and sedimentation properties of fibre are abnormal. Because this mutant failed to complement several complementing adenovirus mutants, and depressed the growth of wild type virus, it was concluded that ts48 may be a novel, trans-dominant mutation.

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.