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News and Publications
Targeted Adenovirus-Based Vectors in Gene Therapy
D.J. Von Seggern, S.K. Fleck, M.A. Friedlander, P. Ghazal, G.R. Nemerow
Targeted gene delivery is a major goal for treating inherited and chronic diseases. Adenovirus-based vectors are currently being used in preclinical and clinical trials for gene therapy. However, these vectors cannot yet be targeted to desired cell types in vivo because the cell receptor recognized by the fiber protein of adenovirus type 5 is widely distributed. We have generated an adenovirus type 5 vector that lacks the gene for the fiber protein; this vector can be propagated in a packaging cell line that expresses the wild-type fiber protein. Cell lines are also being generated that express a fiber protein derived from a different viral serotype (adenovirus type 3) that confers a distinct cell-binding specificity. Genetic modifications of the fiber protein of adenovirus type 5 are also in progress to abrogate attachment to the cell receptor for the protein and to insert epitopes that provide a new cell-binding specificity.
Studies have also been started as part of a recently established ocular gene therapy program to treat inherited or acquired diseases of the eye involving macular degeneration. Adenovirus vectors will be used to deliver a normal rds/peripherin gene to murine photoreceptors in an attempt to prevent macular degeneration and loss of visual function in mice deficient in rds/peripherin. Tissue-specific or regulatable promoters will be used to optimize transgene expression.
Structural Analysis of  v Integrin Interactions With Human Adenoviruses
P. Mathias, C. Chiu,* P.L. Stewart,* G.R. Nemerow
* University of California, Los Angeles, CA
Although  v integrins serve as coreceptors for entry of adenovirus into cells, little is known of the precise interactions of these molecules with host cell--derived or viral ligands. To gain further insights into the structure and function of v integrins, we have produced a soluble form of the integrin vß5 that retains the ability to bind vitronectin as well as multiple adenovirus serotypes. Using cryoelectron microscopy and image reconstruction, we have analyzed the binding of soluble integrin molecules to different adenovirus serotypes. Integrins were visualized bound to adenovirus type 12, a serotype containing a relatively short (19 amino acid) arginine--glycine--aspartic acid domain. These structural studies also revealed the site of integrin binding and intermolecular associations of bound receptors. Further studies are in progress to characterize the kinetics and stoichiometry of integrin binding to different adenovirus serotypes to gain a more complete understanding of integrin associations with viral ligands.
Regulation of v Integrin Expression on B Lymphocytes Transformed by Epstein-Barr Virus
S. Huang, D.G. Stupack, G.R. Nemerow
Although v integrins have an important role in several biological processes, comparatively little is known of how integrin expression is regulated in specific cell types. Infection of human B lymphocytes by Epstein-Barr virus (EBV) upregulates expression of v integrins. Experiments have revealed that the EBV-encoded latent gene products LMP-1 and EBNA-2 promote activation of the v integrin promoter in EBV-transformed B cells. Studies are under way to define the signaling pathways that lead to activation of the promoter and to determine the transcription factors and DNA promoter sequences that regulate integrin expression. These studies should provide further insights into the control of v integrin expression and also of the viral and host cell factors involved in EBV-induced B-cell metastasis.
Integrin-Mediated Signaling Events Involved in Adenovirus Endocytosis
E. Li, D.G. Stupack, K. Wang, D.A. Cheresh, G.M. Bokoch, G.R. Nemerow
Adenovirus type 2 particles enter cells via clathrin-mediated endocytosis. Internalization of virus via v integrins also requires activation of phosphatidylinoside-3-OH kinase. In recent studies, we showed that entry of virus is associated with reorganization of the actin cytoskeleton. Viral particles induce polymerization of cortical actin filaments and formation of membrane ruffles and microspikes at the cell surface, processes that require activation of phosphatidylinoside-3-OH kinase. Entry of virus is also blocked by treatment of cells with cytochalasin D, indicating that an intact actin cytoskeleton is required for internalization of virus. In further studies, we showed that the Rho family of small GTPases, Rac and Cdc42, act downstream of the kinase to promote actin assembly and viral entry. Studies are in progress to determine the upstream signaling molecules and the downstream effector proteins that mediate adenovirus internalization.
PUBLICATIONS
Li, E., Stupack, D.G., Klemke, R., Cheresh, D.A. Adenovirus endocytosis via v integrins requires phosphatidylinoside-3-OH kinase. J. Virol. 72:2055, 1998.
Von Seggern, D., Kehler, J., Endo, R.I., Nemerow, G.R. Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein. J. Gen. Virol. 79:1461, 1998.
Wang, K., Huang, S., Kapoor-Munshi, A., Nemerow, G.R. Adenovirus internalization and infection requires dynamin. J. Virol. 72:3455, 1998.
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