Knob Protein Research Articles

Trimeric knob protein specifically distinguishes neutralizing antibodies to different human adenovirus species: potential application for adenovirus seroepidemiology.

Adenoviruses (Ads) are non-enveloped DNA viruses that have been extensively studied and used as vectors for gene therapy and several potential vaccines. There are 57 Ad serotypes in seven species (A-G), and Ad neutralizing antibody (NAb) titres can vary by serotype and geographical location. Until now serotype- and species-specific antibodies have been detected by neutralization or haemagglutination inhibition assays. These expensive and cumbersome methods of adenovirus typing have mainly been used in epidemiological studies. Our prior work demonstrated that NAbs against the fiber protein are commonly generated during natural Ad infection in humans and the trimeric knob is preferentially recognized by fiber-induced NAbs. In this study, we expressed nine trimeric knob proteins from representative Ad serotypes of human Ad (HAdV)-A-F in Escherichia coli and found no cross-reactivity of these recombinant proteins with rabbit hyperimmune sera (among HAdV-A-F or within HAdV-C). Results of the ELISA based on Ad2 and Ad5 (both HAdV-C) knob proteins were consistent with those of neutralization assays, indicating that the trimeric knob protein would be a good candidate antigen for detecting Ad serotype-specific NAbs in sera from naturally infected subjects. We also demonstrated the primary seroepidemiology of nine Ad serotypes in 274 children using the knob-based ELISA. These results have potential implications for epidemiology of Ad serotypes and future development of Ad-based vaccines and gene therapy.

Retargeting Adenovirus Serotype 48 Fiber Knob Domain by Peptide Incorporation

Adenovirus type 5 (Ad5) is a commonly used vector for gene therapy, but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modeling to assess the CD, DG, HI, and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ανβ6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20, and Knob48-IJ-A20 were engineered and purified after expression in Escherichia coli. We confirmed that Knob48, Knob48-CD-A20, and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ανβ6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-factor-X-binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favorable characteristics for targeted delivery in vivo.

Proteome of the nematode-trapping cells of the fungus Monacrosporium haptotylum.

Many nematophagous fungi use morphological structures called traps to capture nematodes by adhesion or mechanically. To better understand the cellular functions of adhesive traps, the trap cell proteome of the fungus Monacrosporium haptotylum was characterized. The trap of M. haptotylum consists of a unicellular structure called a knob that develops at the apex of a hypha. Proteins extracted from knobs and mycelia were analyzed using SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The peptide sequences were matched against predicted gene models from the recently sequenced M. haptotylum genome. In total, 336 proteins were identified, with 54 expressed at significantly higher levels in the knobs than in the mycelia. The upregulated knob proteins included peptidases, small secreted proteins with unknown functions, and putative cell surface adhesins containing carbohydrate-binding domains, including the WSC domain. Phylogenetic analysis showed that all upregulated WSC domain proteins belonged to a large, expanded cluster of paralogs in M. haptotylum. Several peptidases and homologs of experimentally verified proteins in other pathogenic fungi were also upregulated in the knob proteome. Complementary profiling of gene expression at the transcriptome level showed poor correlation between the upregulation of knob proteins and their corresponding transcripts. We propose that the traps of M. haptotylum contain many of the proteins needed in the early stages of infection and that the trap cells can tightly control the translation and degradation of these proteins to minimize the cost of protein synthesis.

Characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera

Although it is known that Ad5-specific neutralizing antibodies (NAbs) against three major viral capsid components (hexon, penton and fiber) are generated, differences in the frequency and nature of these pre-existing NAbs remain unclear. The results emphasized the contribution of anti-fiber antibodies to Ad5 neutralization responses generated during natural viral infection. Additionally, Ad5-specific NAbs against the fiber knob protein were present in over 90% of the positive serum samples while 42% of the sera had NAbs against hexon in this study based on neutralization assay of anti-HVR and anti-knob subtracted sera and Western blotting analysis. We also found that the trimeric knob was preferentially recognized by fiber-induced NAbs and it was serotype-specific in human adenovirus species C. Results indicated that the trimeric knob protein would be a good candidate antigen for detecting adenovirus serotype-specific NAbs in naturally infected sera.

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.

Modifications in erythrocyte membrane zeta potential by Plasmodium falciparum infection

The zeta potential (ZP) is an electrochemical property of cell surfaces that is determined by the net electrical charge of molecules exposed at the surface of cell membranes. Membrane proteins contribute to the total net electrical charge of cell surfaces and can alter ZP through variation in their copy number and changes in their intermolecular interactions. Plasmodium falciparum extensively remodels its host red blood cell (RBC) membrane by placing ‘knob’-like structures at the cell surface. Using an electrophoretic mobility assay, we found that the mean ZP of human RBCs was −15.7mV. In RBCs infected with P. falciparum trophozoites (‘iRBCs’), the mean ZP was significantly lower (−14.6mV, p<0.001). Removal of sialic acid from the cell surface by neuraminidase treatment significantly decreased the ZP of both RBCs (−6.06mV) and iRBCs (−4.64mV). Parasite-induced changes in ZP varied by P. falciparum clone and the presence of knobs on the iRBC surface. Variations in ZP values were accompanied by altered binding of iRBCs to human microvascular endothelial cells (MVECs). These data suggest that parasite-derived knob proteins contribute to the ZP of iRBCs, and that electrostatic and hydrophobic interactions between iRBC and MVEC membranes are involved in cytoadherence.

Attenuation of CD4+ T-cell function by human adenovirus type 35 is mediated by the knob protein

The complement-regulatory protein CD46 is the primary receptor for human adenovirus type 35 (HAdV-35) and can regulate human immune-cell activation. CD4(+) T-cells are critical for initiating and maintaining adaptive immunity elicited by infection or vaccination. It was reported previously that HAdV-35 can bind these cells and suppress their activation. The data reported here demonstrate that recombinant trimeric HAdV-35 knob proteins alone can induce CD46 receptor downregulation and inhibit interleukin-2 production and proliferation of human CD4(+) T-cells in vitro similarly to mAbs specific to the CD46 region bound by HAdV-35 knobs. A mutant knob protein with increased affinity for CD46 compared with the wild-type knob caused equivalent effects. In contrast, a CD46-binding-deficient mutant knob protein did not inhibit T-cell activation. Thus, the capacity of HAdV-35 to attenuate human CD4(+) T-cell activation depends predominantly on knob interactions with CD46 and can occur independently of infection.

Sorafenib Affects Membrane Complement Inhibitors and Improves Antitumor Activity of Rituximab,

Abstract 3723Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels.The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis. [Display omitted] Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs. Disclosures:No relevant conflicts of interest to declare.

Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions

Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96-104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered the transient opening of intercellular junctions in epithelial cells. The pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g., CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased the transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, the pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased the killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and the infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues.

Plasmodium falciparum malaria and carbohydrate blood group evolution

Plasmodium falciparum malaria, as ancient as hominid evolution itself, has provoked more change within the human genome than any other pathogen. JBS Haldane observed the overlapping distributions for thalassaemia and malaria endemicity and proposed ‘balanced polymorphisms’ as advantageous heterozygous mutant states. We now appreciate the wider range of haemoglobinopathies, membranopathies, and enzymopathies as distinct evolutionary adjustments to the erythrocyte, the very compartment which P. falciparum hijacks to sicken the host. Unlike other Plasmodium species, P. falciparum’s power over the erythrocyte consists of its limitless red cell infectivity and its capacity to render the infected red blood cell (iRBC) adhesive enough to arrest in the circulation. This latter cytoadhesivity is achieved by sticky knob proteins known as ‘Plasmodium falciparum erythrocyte membrane protein‐1’ (PfEMP‐1), trafficked to the red cell exterior from the parasite within. PfEMP‐1 is designed to latch onto endothelial cells of the post‐capillary venules (‘sequestration’), as well as onto other uninfected red blood cells and platelets (‘rosetting’). In so stalling their flow towards the spleen, the iRBC doubly harms the host by resisting the first defence of reticuloendothelial clearance and congesting the host’s microvasculature. The youngest, most malaria‐naïve suffer malaria’s highest case fatality rates, revealing just how critical this innate (pre‐adaptive) immune control of parasitaemia is. The biochemical means by which PfEMP‐1 achieves its cytoadhesive promiscuity is in part through one particular lectin‐like domain, DBL1α. This domain binds not only to heparan sulphate‐like glycosaminoglycans, but to two blood group antigens expressed densely on erythrocytes: the group A carbohydrate in the ABO system and antigens (including those of the Knops system) on CR1 (CD35). If indeed these ligands are critical in the molecular pathogenesis of malaria fatalities, then we might expect to observe non‐adhesive variants ascending to higher prevalence in the most malaria‐endemic parts of the world. The cytoadhesivity of wildtype group A hosts is theoretically, and in vitro, demonstrably mitigated by what we now know are the mutant phenotypes which define the polymorphisms of the ABO system. These include the group O or B alleles, the weaker A types and the genetics influencing the quantity of secreted (competitive) free A antigen in group A hosts. Each of these phenotypes is observed at higher frequencies in malaria‐endemic areas. Certain CR1 polymorphisms are also more frequently found in these parts of the world. The assembly of in vitro, geographic and clinical evidence weighs heavily towards ABO evolution being a highly specific response to P. falciparum. Rather than bypassing invasion or enhancing clearance, these mutations are special because they highlight the importance of escape from cytoadhesion. Forthcoming are the results of the first prospective study powered to confirm the impact of ABO on malaria mortality (NCT 00707200, http://www.clinicaltrials.gov). Should the results of emerging studies confirm a survival advantage among group O individuals, the basic strategy for transfusion support in malaria may shift to greater use of group O red cells. The clinical value of this approach, more immediately available than any new drug or vaccine development, will need to be tested in clinical trials.

A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy

AbstractMany tumors, including lymphomas, up-regulate expression of CD46 to escape destruction by complement. Tumor cells are therefore relatively resistant to therapy by monoclonal antibodies, which act through complement-dependent cytotoxicity (CDC). From an Escherichia coli expression library of adenovirus type 35 fiber knob mutants, we selected a variant (Ad35K++) that had a higher affinity to CD46 than did the natural Ad35 fiber knob. We demonstrated that incubation of lymphoma cells with recombinant Ad35K++ protein resulted in transient removal of CD46 from the cell surface. Preincubation of lymphoma cells with Ad35K++ sensitized cells to CDC, triggered by the CD20-specific monoclonal antibody rituximab. In xenograft models with human lymphoma cells, preinjection of Ad35K++ dramatically increased the therapeutic effect of rituximab. Blood cell counts and organ histology were normal after intravenous injection of Ad35K++ into mice that express human CD46. The presence of polyclonal anti-Ad35K++ antibodies did not affect the ability of Ad35K++ to enhance rituximab-mediated CDC in in vitro assays. The Ad35K++-based approach has potential implications in monoclonal antibody therapy of malignancies beyond the combination with rituximab.

Engineering fibrin matrices: The engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins

Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob–protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants ( K D) obtained using surface plasmon resonance indicate that these fusions have μ m binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob:pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.

Computational Study of a Nanobiosensor: A Single-Walled Carbon Nanotube Functionalized with the Coxsackie-Adenovirus Receptor

Combining single-walled carbon nanotubes (CNT) with biological molecules provides a route to novel nanoscale materials with many promising applications in nanotechnology and nanomedicine. Recent experiments show that CNTs covalently functionalized with the coxsackie-adenovirus receptor (CAR) serve as biosensors capable of specifically recognizing Knob proteins from the adenovirus capsid. These experiments suggest that CAR retains its biologically active form when bound to CNT, but a detailed understanding of the structural changes that occur within CAR after CNT attachment is lacking. To address this, we have performed all-atom classical molecular dynamics (MD) simulations of CAR and the CAR-Knob complex in aqueous solution alone and also when covalently linked to CNT. The MD results show that the CNT damps structural fluctuations in CAR and reduces the internal mobility of the protein. However, CNT induces very little structural deformation and does not affect CAR's ability to specifically bind Knob. This MD study verifies that CAR retains its biological functionality when attached to CNT and provides a computational approach to rationalize nanobiosensing devices.

Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.

Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin-independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin-dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid-phase uptake. It was sensitive to macropinocytosis inhibitors targeting F-actin, protein kinase C, the sodium-proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21-activated kinase 1 (PAK1) and the C-terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and alpha v integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation-defective S147A-CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.

Gold Nanoparticle Size Controlled by Polymeric Au(I) Thiolate Precursor Size

We developed a method in preparing size-controllable gold nanoparticles (Au NPs, 2-6 nm) capped with glutathione by varying the pH (between 5.5 and 8.0) of the solution before reduction. This method is based on the formation of polymeric nanoparticle precursors, Au(I)-glutathione polymers, which change size and density depending on the pH. Dynamic light scattering, size exclusion chromatography, and UV-vis spectroscopy results suggest that lower pH values favor larger and denser polymeric precursors and higher pH values favor smaller and less dense precursors. Consequently, the larger precursors led to the formation of larger Au NPs, whereas smaller precursors led to the formation of smaller Au NPs. Using this strategy, Au NPs functionalized with nickel(II) nitriloacetate (Ni-NTA) group were prepared by a mixed-ligand approach. These Ni-NTA functionalized Au NPs exhibited specific binding to 6x-histidine-tagged Adenovirus serotype 12 knob proteins, demonstrating their utility in biomolecular labeling applications.

Functionalized Carbon Nanotubes for Detecting Viral Proteins

We investigated the biocompatibility, specificity, and activity of a ligand-receptor-protein system covalently bound to oxidized single-walled carbon nanotubes (SWNTs) as a model proof-of-concept for employing such SWNTs as biosensors. SWNTs were functionalized under ambient conditions with either the Knob protein domain from adenovirus serotype 12 (Ad 12 Knob) or its human cellular receptor, the CAR protein, via diimide-activated amidation. We confirmed the biological activity of Knob protein immobilized on the nanotube surfaces by using its labeled conjugate antibody and evaluated the activity and specificity of bound CAR on SWNTs, first, in the presence of fluorescently labeled Knob, which interacts specifically with CAR, and second, with a negative control protein, YieF, which is not recognized by biologically active CAR proteins. In addition, current-gate voltage (I-V(g)) measurements on a dozen nanotube devices explored the effect of protein binding on the intrinsic electronic properties of the SWNTs, and also demonstrated the devices' high sensitivity in detecting protein activity. All data showed that both Knob and CAR immobilized on SWNT surfaces fully retained their biological activities, suggesting that SWNT-CAR complexes can serve as biosensors for detecting environmental adenoviruses.

Downregulation of human CD46 by adenovirus serotype 35 vectors

Human CD46 (membrane cofactor protein), which serves as a receptor for a variety of pathogens, including strains of measles virus, human herpesvirus type 6 and Neisseria, is rapidly downregulated from the cell surface following infection by these pathogens. Here, we report that replication-incompetent adenovirus (Ad) serotype 35 (Ad35) vectors, which belong to subgroup B and recognize human CD46 as a receptor, downregulate CD46 following infection. A decline in the surface expression of CD46 in human peripheral blood mononuclear cells was detectable 6 h after infection, and reached maximum (72%) 12 h after infection. Ad35 vector-induced downregulation of surface CD46 levels gradually recovered after the removal of Ad35 vectors, however, complete recovery of CD46 expression was not observed even at 96 h after removal. The surface expression of CD46 was also reduced after incubation with fiber-substituted Ad serotype 5 (Ad5) vectors bearing Ad35 fiber proteins, ultraviolet-irradiated Ad35, vectors and recombinant Ad35 fiber knob proteins; in contrast, conventional Ad5 vectors did not induce surface CD46 downregulation, suggesting that the fiber knob protein of Ad35 plays a crucial role in the downregulation of surface CD46 density. These results have important implications for gene therapy using CD46-utilizing Ad vectors and for the pathogenesis of Ads that interact with CD46.

Detection of Viral Proteins using Human Receptor Functionalized Carbon Nanotubes

ABSTRACTWe present proof-of-concept experiments for developing a highly-sensitive and fast-response miniaturized single-walled carbon nanotube field-effect transistor (SWNT-FET) biosensor for electrically detecting adenovirus using ligand-receptor-protein specificity. SWNTs are mildly oxidized to form carboxylic groups on the surfaces without compromising the electronic integrity of the nanotubes. Then the human coxsackievirus and adenovirus receptor (CAR) is covalently functionalized onto the nanotube surface via diimide-activated amidation process. Upon exposure of the device to adenovirus protein, Ad12 Knob (Knob), specific binding of Knob to CAR decreases the current that flows through the SWNT-FET device. For control experiment, the CAR-SWNT device is exposed to YieF, which is a virus protein that does not bind specifically to CAR, and no current change is observed. The biological activity of the CAR and Knob proteins that are immobilized on SWNTs has been confirmed by previous fluorescence studies [1]. AFM analysis is done to show height increase of a few nanometers at specific spots where the CAR-Knob complex are covalently linked to the nanotube surface. Therefore, our results show that the human receptor protein CAR does immobilize on SWNT surface while fully retains its biological activity. Moreover, the specific binding of CAR to its complementary adenovirus Knob can be electrically detected using individual SWNT-FET devices. These findings suggest that CAR-functionalized SWNT-FETs can ably serve as biosensors for detection of environmental adenoviruses.

Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector

In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.