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 ( ts 1) particles that fail to escape the early endosome. Wild-type Ad but not ts 1 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 α-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.

SUMMARY: 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 (α _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.

Human Adenovirus Structures: Human adenoviruses may be a common cause of acute infections in humans, but they can also be used as vectors for vaccine and therapeutic gene transfer. Rational engineering of safe adenovirus vectors has been hampered by a lack of high-resolution structural information. Two papers now describe the structure of human adenovirus using complementary techniques. Reddy et al. (p. 1071 ; see the Perspective by Harrison ) have determined the crystal structure at 3.5 angstrom resolution, while Liu et al. (p. 1038 ; see the Perspective by Harrison ) solved the structure to 3.6 angstrom resolution by electron microscopy. Together the structures provide insights into viral assembly, stabilization, and cell entry mechanisms.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α _v integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α _v integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α _v integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

Adenovirus (Ad) endocytosis via α_vintegrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated withClostridium difficiletoxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor αvβ3 or αvβ5 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 αvβ1. Function-blocking antibodies directed against αv or β1, but not β3, β5, or α5, integrin subunits block Ad infection and viral endocytosis. Therefore, αvβ1 serves as a coreceptor for Ad infection, and the lack of β3 and/or β5 but the relatively high expression of αvβ1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.