Crystallization and preliminary X-ray diffraction analysis of human adenovirus.

Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in approximately 25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100mM HEPES pH 7.0, 200mM Ca(OAc)(2), 14% PEG 550 MME, 15% glycerol in 100mM Tris-HCl 8.5. Freshly grown crystals diffracted well to 4.5A resolution and weakly to 3.5A at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a=854.03A, b=855.17A, c=865.24A, alpha=119.57 degrees , beta=91.71 degrees , gamma=118.08 degrees with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

Insights into adenovirus host cell interactions from structural studies.

Human adenoviruses cause a significant number of acute respiratory, enteric and ocular infections, however they have also served as useful model systems for uncovering fundamental aspects of cell and molecular biology. In addition, replication-defective forms of adenovirus are being used in gene transfer and vaccine clinical trials. Over the past decade, steady advances in structural biology techniques have helped reveal important insights into the earliest events in the adenovirus life cycle as well as virus interactions with components of the host immune system. This review highlights the continuing use of structure-based approaches to uncover the molecular features of adenovirus-host interactions.

Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus.

Adenovirus type 5 (Ad) based vectors transduce vascular endothelial cells (EC) and have been widely used for vascular gene transfer. However, many cell types express the Ad receptor (cox-sackievirus adenovirus receptor; CAR), preventing selective EC infection and precluding clinical use. We previously isolated the human EC-binding peptides SIGYPLP and LSNFHSS by phage display and demonstrated by means of a bispecific antibody that SIGYPLP directs efficient, high-level, EC-selective Ad-mediated gene transfer. We now generate genetically modified Ad fiber proteins with selective EC tropism by engineering these peptides into the HI loop of the Ad fiber. SIGYPLP, but not LSNFHSS, enhanced EC selectivity, demonstrating maintenance of peptide-cell binding fidelity upon incorporation into virions. Combining fiber mutations that block CAR binding (detargeting) with SIGYPLP insertion (retargeting) generated a novel Ad vector, AdKO1SIG, in a single component system. AdKO1SIG demonstrated efficient and selective tropism for EC compared with control Ad vectors. This is the first demonstration of genetic incorporation of a novel, mammalian, cell-selective ligand that retains its targeting fidelity in the Ad fiber HI loop, in combination with point mutations that abolish fiber-CAR interaction. This study demonstrates the potential for improving the cell-selectivity and safety of adenoviral vectors.

Structural analysis of a fiber-pseudotyped adenovirus with ocular tropism suggests differential modes of cell receptor interactions.

Adenovirus (Ad) entry into cells is initiated by the binding of the fiber knob to a cell surface receptor. The coxsackie- and adenovirus receptor (CAR) functions as the attachment receptor for many, but not all, Ad serotypes. Ad type 37 (Ad37), a subgroup D virus that causes keratoconjunctivitis in humans, does not infect cells via CAR despite demonstrated binding of the Ad37 knob to CAR. We have pseudotyped a fiber deletion Ad5 vector with the Ad37 fiber (Ad37f), and this vector retains the ocular tropism of Ad37. Here we present a cryo-electron microscopy reconstruction of Ad37f that shows the entire Ad37 fiber, including the shaft and knob domains. We have previously proposed that Ad37 may not utilize CAR for cell entry because of the geometric constraints imposed by a rigid fiber (E. Wu, J. Fernandez, S. K. Fleck, D. Von Seggern, S. Huang, and G. R. Nemerow, Virology 279:78-89, 2001). Consistent with this hypothesis, our structural results show that the Ad37 fiber is straight and rigid. Modeling of the interaction between Ad37f and host cell receptors indicates that fiber flexibility or rigidity, as well as length, can affect receptor usage and cellular tropism.

Integrin alpha(v)beta1 is an adenovirus coreceptor.

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

Production of functional antibodies generated in a nonlytic insect cell expression system.

A monoclonal antibody directed against the type 2 adenovirus (Ad2) penton base protein was cloned and expressed in Spodoptera frugiperda (Sf9) cells using a nonlytic vector system. The coding sequences for the immunoglobulin light and heavy chains were placed under the control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected Sf9 cells continuously secreted the antibody which retained the ability to recognize both native and recombinant Ad2 penton base proteins. Bifunctional penton base antibodies were also generated by fusing a gene for a growth factor or a cytokine at the 3' end of the Ig constant heavy chain domain. The quantity and activity of recombinant antibodies generated in the nonlytic insect cell system could be determined relatively quickly compared to other expression systems. Moreover, these recombinant proteins were not subjected to proteolytic degradation as frequently occurs during baculovirus-mediated cell lysis and the levels of recombinant antibodies produced in the nonlytic system were comparable to those reported for cytolytic baculovirus vectors.

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.

A 50-kDa membrane protein mediates sialic acid-independent binding and infection of conjunctival cells by adenovirus type 37.

The ocular tropism of adenovirus type 37 (Ad37) does not correlate with the wide distribution of the 46-kDa coxsackievirus and adenovirus receptor (CAR), the major receptor for most adenovirus serotypes. We previously found that Ad37 infects and binds well to conjunctival cells (Chang C), but poorly to lung epithelial (A549) cells that express CAR and hypothesized that this serotype uses a distinct receptor that is selectively expressed on conjunctival cells. To test this, we produced particles of a fiber-deleted Ad5 vector containing the Ad37 fiber protein. The "pseudotyped" vector infected Chang C cells better than A549 cells using a CAR-independent pathway. Ad37 binding was calcium-dependent and was abolished by protease digestion of cell surface proteins. Using a virus overlay protein blot assay (VOPBA), we detected calcium-dependent Ad37 binding to 50- and 60-kDa membrane proteins on permissive Chang C cells. In contrast, calcium-dependent binding was detected with only the 60-kDa protein on nonpermissive A549 cells. Ad19p, a closely related serotype that failed to bind to conjunctival cells, recognized the 60-kDa, but not the 50-kDa, protein. Ad37 has been reported to use sialic acid instead of CAR as a cell receptor on A549 cells. Pretreatment of Chang C cells with neuraminidase abolished Ad37 binding to only the 60-kDa protein, suggesting that sialic acid mediates Ad37 binding to the 60-kDa protein. The pseudotyped Ad37 vector was also able to infect neuraminidase-treated Chang C cells. Thus, subgroup D adenoviral binding to the 50-kDa protein is calcium-dependent and cell type- and serotype-specific, whereas binding to the 60-kDa protein is not necessary for infection of conjunctival cells. Together, these data suggest that the 50-kDa protein is the major receptor for Ad37 on conjunctival cells.

Signaling antibodies complexed with adenovirus circumvent CAR and integrin interactions and improve gene delivery.

Current adenoviral (Ad) vectors cannot be targeted to specific cell types due to the widespread distribution of the Ad receptor (CAR). Moreover, CAR and/or internalization receptors (alphav integrins) are absent or present at low levels on some cell types, rendering them resistant to Ad-mediated gene delivery. To address these problems, we have developed a novel vector targeting approach that takes advantage of the common cell signaling pathways initiated by ligation of alphav integrins and growth factor receptors. Recombinant growth factor/cytokines (TNF-alpha, IGF-1, EGF) which trigger phosphatidylinositol-3-OH kinase (PI3K) activation, a signaling molecule involved in adenovirus internalization, were fused to a monoclonal antibody specific for the viral penton base. Ad vectors complexed with these bifunctional mAbs increased gene delivery 10 to 50-fold to human melanoma cells lacking alphav integrins. The bifunctional mAbs also enhanced gene delivery by fiberless adenovirus particles which cannot bind to CAR. Improved gene delivery correlated with increased virus internalization and attachment as well as PI3K activity. The use of bifunctional mAbs to trigger specific cell signaling pathways offers a widely applicable method for bypassing the normal Ad receptors in gene delivery and potentially increasing the selectivity of gene transfer.

Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry.

The Crk-associated substrate, p130(CAS), has been implicated in the regulation of the actin cytoskeleton following ligation of cell integrins with the extracellular matrix. Integrin-mediated cell adhesion involves p130(CAS) association with focal adhesion kinase (p125(FAK)). Internalization/cell entry of type 2 and type 5 adenoviruses (Ad) is also mediated by alpha(v) integrins. However, expression of dominant negative forms of p125(FAK) does not alter virus entry, and Ad entry occurs normally in p125(FAK)-deficient fibroblasts. We now provide evidence that Ad internalization, a process which is mediated by alpha(v) integrins, also requires p130(CAS) and phosphatidylinositol-3-OH kinase (PI 3-kinase). Ad induces p130(CAS) phosphorylation and inhibition of p130(CAS) phosphorylation by tyrphostin and genistein, or expression of the substrate domain deleted p130(CAS) blocks Ad internalization. p130(CAS) was also found to associate with the p85 subunit of PI 3-kinase through its proline-rich domain during virus internalization and expression of p130(CAS) containing a deleted proline-rich domain (PRD) inhibited adenovirus cell entry. We showed further that the RPLPSPP motif in the proline-rich region of p130(CAS) interacts with the SH3 domain of p85/PI 3-kinase. These studies reveal the molecular basis by which p130(CAS) coordinates the signaling pathways involved in integrin-mediated Ad endocytosis.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration.

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor alpha, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IkappaB kinase signaling cascade.

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity.

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins.

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5.

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70.

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.

Adenovirus vector pseudotyping in fiber-expressing cell lines: improved transduction of Epstein-Barr virus-transformed B cells.

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.betagal.DeltaF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.betagal.DeltaF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.

Role of alpha(v) integrins in adenovirus cell entry and gene delivery.

Adenoviruses (Ad) are a significant cause of acute infections in humans; however, replication-defective forms of this virus are currently under investigation for human gene therapy. Approximately 20 to 25% of all the gene therapy trials (phases I to III) conducted over the past 10 years involve the use of Ad gene delivery for treatment inherited or acquired diseases. At present, the most promising applications involve the use of Ad vectors to irradicate certain nonmetastatic tumors and to promote angiogenesis in order to alleviate cardiovascular disease. While specific problems of using Ad vectors remain to be overcome (as is true for almost all viral and nonviral delivery methods), a distinct advantage of Ad is the extensive knowledge of its macromolecular structure, genome organization, sequence, and mode of replication. Moreover, significant information has also been acquired on the interaction of Ad particles with distinct host cell receptors, events which strongly affect virus tropism. This review provides an overview of the structure and function of Ad attachment (coxsackievirus and Ad receptor [CAR]) and internalization (alpha(v) integrins) receptors and discusses their precise role in virus infection and gene delivery. Recent structure studies of integrin-Ad complexes by cryoelectron microscopy are also highlighted. Finally, unanswered questions arising from the current state of knowledge of Ad-receptor interactions are presented in the context of improving Ad vectors for future human gene therapy applications.