Molecular Characterization of Hemagglutination Domains on the Fibers of Subgenus D Adenoviruses

The adenovirus fiber mediates the agglutination of erythrocytes. Based on differential hemagglutinating properties, subgenus D adenoviruses can be subdivided into clusters DI, DII, and DIII. While subgenus DI adenoviruses agglutinate rat and human erythrocytes, DII adenoviruses simply agglutinate rat erythrocytes and DIII adenoviruses display no or only weak rat erythrocyte agglutination. Amino acid sequence comparisons revealed distinct domains on the fiber knob which could be involved in hemagglutination. In order to localize and characterize the domains responsible for the interaction with rat and human erythrocytes, potential hemagglutination domains of the adenovirus type 9 (Ad9) (subgenus DI) fiber knob were introduced into Ad17 (subgenus DII) and Ad28 (subgenus DIII) fiber knobs by primer-directed mutagenesis. Furthermore, rat erythrocyte hemagglutination domains were also introduced into the Ad3 (subgenus B) fiber knob, which only agglutinated monkey erythrocytes. Altogether, 27 chimeric and mutated fiber proteins were expressed in Escherichia coli and subsequently tested for hemagglutination activity. The hemagglutination tests revealed that at least two domains can mediate the agglutination of rat erythrocytes. While one domain is located on the GH loop, the other domain extends from the C β strand to the CD loop. The domain on the GH loop was partially conserved in all adenoviruses showing an incomplete hemagglutination pattern with rat erythrocytes. The domains involved in the agglutination of human erythrocytes are located on the CD and HI loops of the subgenus DI fiber knob.Besides being associated with a variety of diseases, including respiratory, ophthalmic, and gastrointestinal infections, adenoviruses have recently received special attention as potential viral vectors for gene therapy. Since the fiber protein is responsible for the attachment of the virion to specific receptors on the cell surface (5, 30), thus also being of significant importance for tissue tropism, a detailed understanding of the molecular structure of this protein could be helpful in developing a new, tissue-specific generation of adenovirus vectors.The fiber protein, protruding outward from the 12 vertices of the capsid, comprises a short N-terminal tail, a shaft of variable length, and a globular C-terminal knob (12). The conserved N terminus contains the sequences responsible for association with the penton base as well as the nuclear localization signal (19, 29). The shaft consists of repeating motifs of a 15-amino-acid β structure, with the number of repeats varying among virus serotypes. A conserved amino acid sequence (TLWT) marks the boundary between the shaft and the knob domain, which is responsible for interaction with the host cell receptor. The published crystal structure of the adenovirus type 5 (Ad5) fiber knob domain allows the mapping of functional domains (40, 41). It was shown that the Ad5 knob can block virus infection (14) and that the receptor binding specificity of adenovirus fibers can be altered by exchanging the knob domains (11, 37). While subgenus C and B adenovirus serotypes recognize distinct receptors (6, 24, 38), subgenus C adenoviruses and Ad9 (subgenus D) share the same fiber receptor (33). It was recently demonstrated that a 46-kDa HeLa cell surface protein serves as a common receptor for subgenus C adenoviruses and coxsackie B viruses (3). Furthermore, it was reported that the class I major histocompatibility complex could also serve as an adenovirus receptor (21). The fiber knob also carries the type-specific γ antigen (9, 27), which determines, together with the ɛ antigen of the hexon, the serotype specificity of an adenovirus. The γ determinant is composed of at least 17 amino acids that are not restricted to a distinct region on the fiber knob (10).Since hemagglutination (HA) by human adenoviruses was first demonstrated by Rosén in 1958 (34), it has been shown that members of the six subgenera (A to F) display different HA properties (2, 26). While, e.g., subgenus B adenoviruses only agglutinate monkey erythrocytes, subgenus D adenoviruses can be classified into three clusters: cluster DI adenoviruses agglutinate rat and human erythrocytes, cluster DII adenoviruses agglutinate only rat erythrocytes, and cluster DIII adenoviruses show no or only weak agglutination of rat erythrocytes. The agglutination of erythrocytes is fiber mediated, and specific receptors seem to be present on the erythrocyte membrane. Since intact virions carry several fibers, they can establish a bridge between erythrocytes, leading to HA. In contrast, fibers alone cannot cause HA, as they are monovalent. However, it was shown that fibers obtained from tissue cultures (28) and recombinant fibers (25) can form polymers which are able to agglutinate erythrocytes.Amino acid sequence comparisons revealed distinct domains on the fiber knob which could be expected to mediate the agglutination of rat and human erythrocytes. To localize and characterize these domains, 27 chimeric and mutated Ad9 (subgenus DI), Ad17 (subgenus DII), Ad28 (subgenus DIII), and Ad3 (subgenus B) fiber proteins were expressed in Escherichia coli. The recombinant proteins were tested in HA tests.     

Epitopes and hemagglutination binding domain on subgenus B:2 adenovirus fibers.

The adenovirus fiber serves as a ligand between the virus and the host cell receptor and manifests hemagglutination (HA) activity and antigenic domains. We have screened both the antigenic and immunogenic epitopes on the adenovirus fibers of subgenus B:2 by using recombinant fiber proteins (rfibers) expressed in Escherichia coli, synthesized peptides (P1 to P8), and the corresponding antisera. The results indicated that P4 (amino acids [aa] 201 to 220), P5 (aa 231 to 250), and P7 (aa 275 to 295) presented both antigenic and immunogenic epitopes in adenovirus type 11 prototype (Ad11p), Ad34a, and Ad11a fibers. P6 (aa 251 to 270) presented both epitopes in Ad11a fiber but only an antigenic epitope in other fibers. The C-terminal 20 amino acids of the fiber, corresponding to P8, manifested an epitope of low-level immunogenicity. P5, localized at the N-terminal aa 231 to 250, displayed an epitope that reacted with fibers of all the members of subgenus B analyzed. The rfibers of Ad11p and Ad34a displayed HA activity with monkey erythrocytes, though those of Ad11a did not. Mutagenesis of the rfibers revealed that neither the fragment replacements, 11p20211a, llp26011a,and 11a28011p, nor the Ad11p rfiber with the substitutions of Tyr-260-->H (Tyr260H)and Arg279Q displayed HA activity. The Ad11a fiber knob was sensitive to proteolytic digestion, whereas that of Ad11p was resistant. The results demonstrated that the decisive HA binding domain was presented at aa 260 to 280 and was conformation dependent. Nearby amino acids, aa 283 and 284, may also affect the HA function.     

Adenovirus Serotype 30 Fiber Does Not Mediate Transduction via the Coxsackie-Adenovirus Receptor

Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR.     

Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

The Arg279Gln [corrected] substitution in the adenovirus type 11p (Ad11p) fiber knob abolishes EDTA-resistant binding to A549 and CHO-CD46 cells, converting the phenotype to that of Ad7p

The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Gln [corrected] substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46.     

Mouse adenovirus type 1 attachment is not mediated by the coxsackie-adenovirus receptor

Common human adenovirus (Ad) vectors are derived from serotype 2 or 5, which use the coxsackie-adenovirus receptor (CAR) as their primary cell receptor. We investigated the receptor usage of mouse adenovirus type 1 (MAV-1), which in vivo is characterized by a pronounced endothelial cell tropism. Alignment of the fiber knob sequences of MAV-1 and those of CAR-using adenoviruses, revealed that amino acid residues, critical for interaction with CAR, are not conserved in the MAV-1 fiber knob. Attachment of MAV-1 to Chinese hamster ovary (CHO) cells was not increased by stable transfection with mouse CAR, whereas the binding efficiency of Ad2 was 20-fold higher in the mouse CAR-transfectant compared to the wild type cells. Also, purified fiber knob of Ad5, which is interchangeable with the Ad2 fiber knob, did not compete with MAV-1 for receptor binding, indicating that MAV-1 binds to a receptor different from CAR. These results support further exploration of an MAV-1-derived vector as a potential vehicle for gene delivery to cell types which are not efficiently transduced by human adenovirus vectors.     

Adenovirus Type 9 Fiber Knob Binds to the Coxsackie B Virus-Adenovirus Receptor (CAR) with Lower Affinity than Fiber Knobs of Other CAR-Binding Adenovirus Serotypes

The coxsackie B virus and adenovirus (Ad) receptor (CAR) functions as an attachment receptor for multiple Ad serotypes. Here we show that the Ad serotype 9 (Ad9) fiber knob binds to CAR with much reduced affinity compared to the binding by Ad5 and Ad12 fiber knobs as well as the knob of the long fiber of Ad41 (Ad41L). Substitution of Asp222 in Ad9 fiber knob with a lysine that is conserved in Ad5, Ad12, and Ad41L substantially improved Ad9 fiber knob binding to CAR, while the corresponding substitution in Ad5 (Lys442Asp) significantly reduced Ad5 binding. The presence of an aspartic acid residue in Ad9 therefore accounts, at least in part, for the reduced CAR binding affinity of the Ad9 fiber knob. Site-directed mutagenesis of CAR revealed that CAR residues Leu73 and Lys121 and/or Lys123 are critical contact residues, with Tyr80 and Tyr83 being peripherally involved in the binding interaction with the Ad5, Ad9, Ad12, and Ad41L fiber knobs. The overall affinities and the association and dissociation rate constants for wild-type CAR as well as Tyr80 and Tyr83 CAR mutants differed between the serotypes, indicating that their binding modes, although similar, are not identical.     

Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor

The majority of adenovirus serotypes can bind to the coxsackievirus and adenovirus receptor (CAR) on human cells despite only limited conservation of the amino acid residues that comprise the receptor-binding sites of these viruses. Using a fluorescence anisotropy-based assay, we determined that the recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2 binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold greater affinity than does the recombinant knob domain from Ad12. Homology modeling predicted that the increased affinity of Ad2 knob for CAR D1 could result from additional contacts within the binding interface contributed by two residues, Ser408 and Tyr477, which are not conserved in the Ad12 knob. Consistent with this structural model, substitution of serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas the double mutation increased binding affinity 10-fold. X-ray structure analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted residues potentially could form additional hydrogen bonds across the knob-CAR interface. Structural changes resulting from these mutations were highly localized, implying that the high tolerance for surface variation conferred by the stable knob scaffold can minimize the impact of antigenic drift on binding specificity and affinity during evolution of virus serotypes. Our results suggest that the interaction of knob domains from different adenovirus serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1 crystal structure as a template.     

Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v) integrins

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.     

Dependence of adenovirus infectivity on length of the fiber shaft domain

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.     

Novel fiber-dependent entry mechanism for adenovirus serotype 5 in lacrimal acini

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to alpha(v) integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.     

Structural and Functional Studies on the Interaction of Adenovirus Fiber Knobs and Desmoglein 2

Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. Our findings have practical relevance for cancer therapy. We have recently reported that an Ad3 fiber knob-containing recombinant protein (JO-1) is able to trigger opening of junctions between epithelial cancer cells which, in turn, greatly improved the intratumoral penetration and efficacy of therapeutic agents (I. Beyer, et al., Clin. Cancer Res. 18:3340–3351, 2012; I. Beyer, et al., Cancer Res. 71:7080–7090, 2011). Here, we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models.     

一种高效感染血液细胞的新型靶向性腺病毒载体的构建

人C组5型腺病毒(Ad5)载体能够有效感染上皮来源的细胞,但对造血细胞的感染效率很低,限制了其在造血调控基础研究以及血液病基因治疗中的应用。为了建立高效感染血液细胞的新型靶向性腺病毒载体系统,对5型腺病毒载体的纤维顶球进行了改造,以AdEasy系统为基础,应用递归PCR的方法人工合成人B组11p型腺病毒的部分纤维(fiber)基因,采用一系列分子生物学方法将其替换AdEasy骨架质粒中的人5型腺病毒的fiber基因,得到新的腺病毒骨架质粒命名为pAdEasy-1/F11p,应用带有GFP报告基因的穿梭质粒pShuttle-GFP与AdEasy-1/F11p腺病毒DNA在BJ5183细菌内重组得到重组腺病毒质粒,将其转染293细胞获得重组腺病毒,命名为Ad5F11p-GFP。以Ad5-GFP作对照,同时感染K562、U937等白血病细胞系,流式细胞仪检测GFP的表达。初步检测结果显示:在10MOI时,Ad5F11p-GFP能够有效感染K562、U937等白血病细胞系,感染细胞效率>90%,对照Ad5-GFP感染细胞效率<30%,这表明改建后的腺病毒AdEasy-1/F11p可以高效介导基因转移到血液细胞,是一种很好的血液细胞靶向性腺病毒载体。     

A single amino acid in the adenovirus type 37 fiber confers binding to human conjunctival cells

A 46-kDa receptor, coxsackievirus-adenovirus (Ad) receptor (CAR), mediates cell attachment of a number of different Ad serotypes; however, not all Ad serotypes utilize this receptor for infection. Moreover, the precise amino acid sequences in the Ad fiber protein that mediate cell attachment have yet to be identified. We investigated the interaction of subgroup D Ads with human ocular cells. Ad serotype 37 (Ad37), a virus associated with epidemic keratoconjunctivitis, but not a closely related virus serotype, Ad19p, exhibited preferential binding to and infection of human conjunctival cells. A single amino acid substitution in the Ad19p fiber distal domain (knob), Glu240 to Lys, conferred binding to conjunctival cells, while the reverse substitution in the Ad37 fiber abrogated cell binding. These findings provide new information on the fiber sequences that regulate Ad host cell tropism.     

Flexibility of the adenovirus fiber is required for efficient receptor interaction

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.     

A novel vector for a rapid generation of fiber-mutant adenovirus based on one step ligation and quick screening of positive clones

The generation of fiber-modified adenoviral vector has proven difficult. In the paper, we developed a new system for rapid construction of fiber-modified adenoviral vector containing foreign peptides in the HI loop or C-terminal of the fiber knob. The new system was established through the following processes. First, a unique BamHI mutation was made in the genome of Ad5 without causing amino acid change. Second, two unique restriction enzymes BamHI and SfuI, both with sticky end, were introduced in the HI loop or C-terminal of Ad5 fiber knob. Third, a lacza expression cassette was placed between BamHI and SfuI sites for a quick identification of positive cloning based on white-blue color screening. This system allows generation of recombinant adenoviral vector by a single step, in vitro ligation followed by quick white-color positive clone screening. To prove the principle of the method, Ad5HI-RGD by modifying HI-loop of the fiber knob with RGD motif and Ad5Cter-PK7 by modifying C-terminal of the knob with poly-lysine (pK7) were successfully generated in vitro. Ad5 with a knob modified in the HI loop of the fiber with Tat-PTD, NGR or SIKVAV peptide were also successfully developed. The transduction of the modified viruses for Hela, U87 MG and MDA-MB-231 cells was investigated in vitro compared with unmodified Ad5. In conclusion, the new vector system allows for a rapid generation of fiber-mutant adenovirus and provides useful tool for gene function analysis and cancer gene therapy.     

Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.     

Comparative analysis of adenovirus fiber-cell interaction: adenovirus type 2 (Ad2) and Ad9 utilize the same cellular fiber receptor but use different binding strategies for attachment.

We have analyzed the binding of adenovirus (Ad) serotypes from subgroups B, C, and D through fiber-virus and fiber-fiber cross-competition experiments. Since viruses in these distinct subgroups display markedly different tropisms, it was unexpected that the subgroup C viruses Ad2 and 5 and the subgroup D virus Ad9 cross-competed for the same cellular fiber receptor. The subgroup B serotype Ad3 recognized a receptor distinct from the Ad2, 5, and 9 fiber receptor. However, despite sharing the same fiber receptor, Ad2 and Ad9 displayed markedly different binding characteristics that appeared to result from direct Ad9 binding to cells via alpha(v)-integrins. Unlike Ad2, Ad9 binding to many cell lines was not abrogated by competition with the fiber 9 knob (F9K). Ad9 binding to fiber receptor-deficient cells was blocked by a monoclonal antibody to alpha(v)-integrins. In contrast, Ad9 binding to alpha(v)-deficient cells that express fiber receptor was blocked by F9K. Transfection of an alpha(v)-integrin-deficient cell line with a plasmid that expresses alpha(v)beta5 resulted in Ad9 binding that was not significantly blocked by F9K but was blocked with a combination of F9K and penton base. These results imply that the shorter length of fiber 9 (11 nm) relative to fiber 2 (37 nm) permits fiber-independent binding of Ad9 penton base to alpha(v)-integrins. The difference in fiber length may explain the different binding characteristics and tissue tropisms of each virus despite both utilizing the same fiber and penton base receptors.     

Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.     

A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid.