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.