No abstract

In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic ␣-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.

Abstract. Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins otv/33 and otv/35. While both o~v integrins promote virus internalization, olv/35 is involved in the subsequent event of membrane permeabilization. Cells transfected with the r5 or/33 subunit, expressing either otvfl5 and otv/33, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing otvfl5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing avfl3. Adenovirus-mediated gene delivery was also more efficient in cells expressing txvfl5. These results suggest that the interaction of oLv/35 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virusmediated membrane permeabilization and suggest a novel biological role for integrin avfl5 in the infectious pathway of a human adenovirus. crucial step in virus infection of host cells is penetration/permeabilization of the cell plasma membrane, a past-internalization event required for delivery of the viral genome into the cytoplasm. Although a substantial amount of knowledge exists on cell entry by enveloped viruses, the mechanism(s) by which nonenveloped viruses penetrate cells is not well understood. Adenovirus, a nonenveloped DNA virus that is a major cause of respiratory and gastrointestinal infections of children (3,14), has proved useful for studying cell entry by nonenveloped viruses.Of the over 40 different serotypes of human adenovirus, the majority of cell interaction studies have been performed with serotype 2 (human adenovirus type 2; Ad2) t. Initial attachment of Ad2 to host cells is mediated by the fiber protein (13, 17), an elongated 62-kD protein that is present on each of the 12 vertices of the virion capsid (28). The fiber receptor, which is broadly distributed on a variety of cells, has not yet been identified. After Ad2 attachment to the fiber receptor, virus particles are rapidly internalized into clathrin-coated vesicles by receptor-mediated endocytosis (4, 33). The fiber protein is dissociated from the virion particle early in the entry pathway (12). Ad2 internalization is mediated by either of two secondary host cell receptors, integrins olv/33 and o~v/35 (38).

Defensins are naturally occurring antimicrobial peptides that disrupt bacterial membranes and prevent bacterial invasion of the host. Emerging studies indicate that certain defensins also block virus infection; however, the mechanism(s) involved are poorly understood. We demonstrate that human alpha-defensins inhibit adenovirus infection at low micromolar concentrations, and this requires direct association of the defensin with the virus. Moreover, defensins inhibit virus disassembly at the vertex region, thereby restricting the release of an internal capsid protein, pVI, which is required for endosomal membrane penetration during cell entry. As a consequence, defensins hamper the release of adenovirus particles from endocytic vesicles, resulting in virion accumulation in early endosomes and lysosomes. Thus, defensins possess remarkably distinct modes of activity against bacteria and viruses, and their function may provide insights for the development of new antiviral strategies.

The structure of adenovirus was determined to a resolution of 6 Å by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that ␣-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed ␣-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly ␣-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in ϳ1.5 copies per hexon trimer and is predicted to have two long ␣-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long ␣-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long ␣-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal ␣-helix from protein IX.Adenovirus (Ad) is a common etiologic agent of respiratory, gastrointestinal, and ocular infections. Ad vectors also have substantial potential for diverse gene therapy approaches to treat cancer and other chronic diseases, as well as promise for vaccine delivery (33). The lack of an atomic resolution structure of an intact Ad virion has hindered further development of practical applications, as well as limited our understanding of Ad cell entry. The mature Ad virion has an icosahedral protein capsid composed of three major capsid proteins (hexon, penton base, and fiber) and four additional capsid proteins (proteins IIIa, VI, VIII, and IX). The capsid is ϳ926 Å in diameter, not including the fibers that vary in length from 120 to 315 Å depending on the serotype. The double-stranded DNA Ad genome is packaged inside of the capsid, together with four additional proteins (V, VII, mu, and terminal protein) and multiple copies of the Ad protease. Cryoelectron microscopy (cryoEM) structures of Ad and Ad vectors (12, 37), Ad/receptor (7), and Ad/antibody (46) complexes, combined with crystal structures for hexon (36), penton base (53), and fiber (45), have provided a wealth of structural information on Ad, but significant gaps remain in our knowledge of the virus capsid structure. In particular, the structure and locations of

Fas/APO-1(CD95) ligation activates programmed cell death, a cellular process that plays an important role in the maturation of the host immune response. We show that activation of a specific MAP kinase kinase (MKK), MKK6b, is necessary and sufficient for Fas-induced apoptosis of Jurkat T cells. MKK6b activation occurs downstream of an interleukin-1 converting enzyme-like (ICE-like) protease(s), while execution of the apoptotic pathway by MKK6b requires both ICE- and CPP32-like proteases. Surprisingly, the p38 MAP kinase protein, a known substrate of MKK6b, does not participate in Fas/MKK6b-mediated apoptosis. These findings indicate a divergence of the MKK6b signaling pathways, one of which activates p38 and leads to regulation of gene expression, and one of which activates the ICE/Ced-3 family of proteases and leads to cell death. These studies represent a demonstration of an apoptotic pathway that is comprised of both the ICE/Ced-3 family of proteases and MAP kinase kinase 6.

Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here we report the X-ray structure at 3.5 Å resolution of the 150 megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a significant advance towards understanding the assembly and cell entry mechanisms of a large dsDNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.Human adenoviruses (HAdV) are non-enveloped dsDNA viruses that are associated with acute infections (1-3). While these infections are generally self-limiting, the re-emergence of certain HAdV types has also been linked to potentially fatal respiratory infections in both civilian and military populations (4). Severe disseminated diseases also occur in patients receiving bone marrow-derived stem cells (5,6). In addition to their disease associations, replication-defective or conditionally replicating HAdVs continue to be evaluated in ~25% of approved Phase I-III clinical trials for vaccine and therapeutic gene transfer (7,8). However, the lack of accurate details of the virus structure limits the re-engineering of HAdV vectors and prevents a better understanding of the virus life cycle. High resolution HAdV structure determination presents a challenge because of the large size (910 Å ave. dia., 150 MDa) and complexity (pseudo-T=25) of the virus. The crystal structures of the major HAdV capsid proteins; the fiber (9), penton base (PB) (10) and hexon (11) have been solved. The hexon and penton base crystal structures were subsequently used to derive pseudo-atomic models of the HAdV capsid at moderately high resolution (7-10 Å) (12-14) by cryoelectron microscopy (cryoEM). CryoEM structural analyses provided considerable insight into HAdV organization; however, they did not furnish detailed information on the interactions between the major and accessory (cement) proteins (IIIa,VI,VIII, and IX).We report here the crystal structure of a recombinant HAdV-5 vector, designated Ad35F, that is equipped with a short and flexible fiber protein derived from . Details of the crystallization (16), diffraction data statistics (Table S1) and structure determination of Ad35F at near atomic resolution (3.5 Å) are described in the Methods (17).The architecture of the HAdV capsid is shown in Fig. 1a, b. The hexon is the most abundant protein in the capsid with 720 subunits arranged as 240 trimers on a pseudo-T=25 icosahedral * "This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any ma...