Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10

The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMV beta gal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMV beta gal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or alpha(v) integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.     

Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types

Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and alphav integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other types of glial cells. Combined mutations of fiber and penton base that ablate both CAR and integrin binding almost abolished brain transduction. Thus, doubly-ablated capsids engineered to express new ligands should allow complete vector retargeting in the central nervous system. Although transduction by the doubly-ablated vectors was reduced by greater than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors.     

Reduction of natural adenovirus tropism to the liver by both ablation of fiber-coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber

The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and alphav integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.     

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.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Reduction of natural adenovirus tropism to mouse liver by fiber-shaft exchange in combination with both CAR- and alphav integrin-binding ablation

The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.     

Fc Receptor-Mediated Phagocytosis,Superoxide Production and Calcium Signaling of β2 Integrin-Deficient Bovine Neutrophils

Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca²⁺]ⁱ) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca²⁺]ⁱ signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca²⁺]ⁱ concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces.     

Lipopolysaccharide-coated erythrocytes activate human neutrophils via CD14 while subsequent binding is through CD11b/CD18

Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.     

Effective targeting of pathogens to neutrophils via chimeric surfactant protein D/anti-CD89 protein

Targeting of specific pathogens to FcRs on immune effector cells by using bispecific Abs was reported to result in effective killing of the pathogens, both in vitro and in vivo. Instead of targeting a specific pathogen to an FcR, we assessed whether a broad spectrum of pathogens can be targeted to an FcR using surfactant protein D (SP-D). SP-D is a collectin that binds a great variety of pathogens via its carbohydrate recognition domain. A recombinant trimeric fragment of SP-D (rfSP-D), consisting of the carbohydrate recognition domain and neck domain of human SP-D, was chemically cross-linked to the Fab' of an Ab directed against the human Fc alpha RI (CD89). In vitro, the chimeric rfSP-D/anti-CD89 protein enhanced uptake of Escherichia coli, Candida albicans, and influenza A virus by human neutrophils. Blocking of the interaction between rfSP-D/anti-CD89 and either the pathogen or CD89 abolished its stimulatory effect on pathogen uptake by neutrophils. In addition, rfSP-D/anti-CD89 stimulated killing of E. coli and C. albicans by neutrophils and enhanced neutrophil activation by influenza A virus. In conclusion, rfSP-D/anti-CD89 effectively targeted three structurally unrelated pathogens to neutrophils. (Col)lectin-based chimeric proteins may thus offer promise for therapy of infectious disease.     

Subgroup B and F fiber chimeras eliminate normal adenovirus type 5 vector transduction in vitro and in vivo

Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types. In this study we have explored a novel approach to altering Ad type 5 (Ad5) vector targeting based on serotypic differences in fiber function. The subgroup B viruses bind to an unidentified receptor that is distinct from CAR. The subgroup F viruses are the only adenoviruses that express two distinct terminal exons encoding fiber open reading frames. We have constructed chimeric fiber adenoviruses that utilize the tandem fiber arrangement of the subgroup F genome configuration. By taking advantage of serotypic differences in fiber expression, fiber shaft length, and fiber binding efficiency, we have developed a tandem fiber vector that has low binding efficiency for the known fiber binding sites, does not rely on an Ad5-based fiber, and can be grown to high titer using conventional cell lines. Importantly, when characterizing these vectors in vivo, we find the subgroup B system and our optimal tandem fiber system demonstrate reduced liver transduction by over 2 logs compared to an Ad5 fiber vector. These attributes make the tandem fiber vector a useful alternative to conventional strategies for fiber manipulation of adenovirus vectors.     

CD46 is a cellular receptor for group B adenoviruses

Group B adenoviruses, a subgenus of human Adenoviridae, are associated with a variety of often-fatal illnesses in immunocompromised individuals, including bone marrow transplant recipients and cancer and AIDS patients. Recently, group B adenovirus derivatives have gained interest as attractive gene therapy vectors because they can transduce target tissues, such as hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors. Whereas many adenoviruses infect cells through the coxsackievirus and adenovirus receptor (CAR), group B adenoviruses use an alternate, as-yet-unidentified cellular attachment receptor. Using mass spectrometric analysis of proteins interacting with a group B fiber, we identified human CD46 as a cellular attachment receptor for most group B adenoviruses. We show that ectopic expression of human CD46 rendered nonhuman cells susceptible to infection with group B viruses in vitro and in vivo. In addition, both siRNA-mediated knockdown of CD46 and a soluble form of CD46 blocked infection of human cell lines and primary human cells. The discovery that group B adenoviruses use CD46, a ubiquitously expressed complement regulatory protein, as a cellular attachment receptor elucidates the diverse clinical manifestation of group B virus infections, and bears directly on the application of these vectors for gene therapy.     

Adhesiveness for Extracellular Matrices and Lysosomal Enzyme Release from Normal and β2 Integrin-Deficient Bovine Neutrophils

The adhesiveness of control and CD18-deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release of N-acetyl-β-D -glucosaminidase (NAG_ase) from control and CD18-deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18-deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P < 0.05); however, similar adhesion to laminin was observed in CD18-deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P < 0.05) with the presence of PMA. The mean activities for NAG_ase release from CD18-deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg-IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg-IgG-induced NAG_ase release from control and CD18-deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors

After intravenous administration, adenovirus (Ad) vectors are predominantly sequestered by the liver. Delineating the mechanisms for Ad accumulation in the liver is crucial for a better understanding of Ad clearance and Ad-associated innate toxicity. To help address these issues, in this study, we used Ad vectors with different fiber shaft lengths and either coxsackievirus-Ad receptor (CAR)-interacting Ad serotype 9 (Ad9) or non-CAR-interacting Ad35 fiber knob domains. We analyzed the kinetics of Ad vector accumulation in the liver, uptake into hepatocytes and Kupffer cells, and induction of cytokine expression and release in response to systemic vector application. Immediately after intravenous injection, all Ad vectors accumulated equally efficiently in the liver; however, only genomes of long-shafted Ads were maintained in the liver tissue over time. We found that Kupffer cell uptake of long-shafted Ads was mediated by the fiber knob domain and was CAR independent. The short-shafted Ads were unable to efficiently interact with hepatocellular receptors and were not taken up by Kupffer cells. Moreover, our studies indicated that Kupffer cells were not the major reservoir for the observed accumulation of Ads (used in this study) in the liver within the first 30 min after virus infusion. The lower level of liver cell transduction by short-shafted Ads correlated with a significantly reduced inflammatory anti-Ad response as well as liver damage induced by the systemic administration of these vectors. This study contributes to a better understanding of the biology of systemically applied Ad and will help in designing safer vectors that can efficiently transduce target tissues.     

Mechanism of ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells

Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs) was independent of the coxsackievirus and adenovirus receptor (CAR), its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines.     

腺病毒载体衣壳蛋白质的遗传修饰

腺病毒载体是最早用于基因治疗研究的病毒载体之一,也是目前肿瘤基因治疗中最为常见的病毒载体之一,其主要通过靶细胞表面的天然柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)感染宿主细胞。由于大多数肿瘤细胞表面该受体表达水平较低,降低了腺病毒载体对靶细胞的感染效率,从而制约了腺病毒载体在肿瘤基因治疗中的应用。因此,如何提高腺病毒载体对靶细胞的感染效率是腺病毒载体应用于肿瘤基因治疗的关键。目前对腺病毒载体衣壳蛋白质(capsid protein)的遗传修饰是提高其对宿主细胞感染效率的主要途径。本文将对这一领域的主要研究进展作一综述,为该方面的研究提供有用的信息。     

Coxsackievirus and adenovirus receptor expression in non-malignant lung tissues and clinical lung cancers

Adenoviral vector mediated gene delivery has been applied in clinical trials and mechanistic studies to explore new treatment approaches for lung cancers. The expression of coxsackievirus adenovirus receptor (CAR), the primary receptor for the most commonly used adenovirus serotype 5 (Ad5)-based vectors, predominantly determines the permissiveness of lung cancer cells. CAR expression is also suggested to modulate tumor cell proliferation capacity. Here, we studied CAR expression in archival lung cancer specimens by using well-characterized CAR 72 antibodies. High levels of CAR expression were observed in most of the 32 cases of squamous cell carcinoma lung cancers and in all the five cases of small cell lung cancers investigated. In contrast, high levels of CAR expression were detected only in 6 of 22 adenocarcinoma lung cancers. The relative levels of CAR expression did not correlate with the pathologic grade in lung cancers, and was thus inconsistent with a role of modulating cancer cell proliferation. Of note, CAR expression was not detected in non-malignant alveolar cells. Our data suggest a preferred utility of Ad5 vector mediated gene delivery to squamous cell carcinoma lung cancers, small cell lung cancers, but not to the majority of adenocarcinoma lung cancers.     

Localization of regions in CD46 that interact with adenovirus

A variety of pathogens use CD46, a ubiquitously expressed membrane protein that regulates complement activation, as a cellular attachment receptor. While the CD46 binding sites of several pathogens, including measles virus, Neisseria gonorrhea, and human herpesvirus 6, have been described, the region of CD46 responsible for adenovirus binding has not been determined. In this study, we used competition experiments with known CD46 ligands, CD46-specific antibodies, and a set of CD46 mutants to localize the binding domain for the group B adenovirus serotype 35 (Ad35). Our results show that Ad35 competes with measles virus for binding to CD46 but not with complement protein C3b. We further show that this interaction is a protein-protein interaction and that N glycosylations do not critically contribute to infection with Ad35 fiber-containing Ad vectors. Our data demonstrate that the native conformation of the CCP2 domain is crucial for Ad35 binding and that the substitution of amino acids at positions 130 to 135 or 152 to 156 completely abolishes the receptor function of CD46. These regions localize to the same planar face of CD46 and likely form an extended adenovirus binding surface, since no single amino acid substitution within these areas eliminates virus binding. Finally, we demonstrate that the infection with a virus possessing human group B serotype Ad11 fibers is also mediated by the CCP2 domain. This information is important to better characterize the mechanisms of the receptor recognition by adenovirus relative to other pathogens that interact with CD46, and it may help in the design of antiviral therapeutics against adenovirus serotypes that use CD46 as a primary cellular attachment receptor.