Scientific Report 2005

Immunology

Structural Analyses of an Adenoviral Vector Targeted to Hematopoietic Cells

S. Saban,* R.R. Nepomuceno, G.R. Nemerow, P.L. Stewart*

* Vanderbilt University Medical Center, Nashville, Tennessee

Despite recent advances in the uses of adenovirus vectors for vaccines and gene delivery, we still lack basic knowledge of the structure of intact adenovirus particles. In recent studies, we used electron cryomicroscopy and image reconstruction to determine the 3-dimensional structure of an adenovirus vector, Ad35F, at 9-Å resolution. This viral vector recognizes the receptor CD46, a member of the complement regulatory protein family, thereby allowing improved gene delivery to human hematopoietic cells that express this receptor.

Important new advances in data acquisition and image processing resulted in major improvements in resolution compared with the resolution in earlier studies. For example, electron cryomicroscopy density was observed for hexon residues missing from the crystal structure that included hypervariable regions and the epitope of a neutralizing antibody. On the inner capsid surface, density was revealed at the base of the hexons and below the penton base that most likely correspond to minor adenovirus proteins, including protein VI.

On the basis of the new structural information, we proposed a new model for Ad35F. In particular, the model presents 2 possible orientations for protein IX, either binding on the capsid surface or extending away from the capsid, consistent with the use of the C terminus of protein IX for the insertion of exogenous ligands to redirect adenovirus vectors to alternative receptors. These studies increase our knowledge of adenovirus capsid assembly and antibody neutralization and thus may promote further improvements in gene delivery to hematopoietic cell types.

Targeting a General Biochemical Pathway in Viral Infections Via Cyclic D,L-α-Peptides

W.S. Horne, C.M. Wiethoff, C. Cui, K.M. Wilcoxen,
M. Amorin, M.R. Ghadiri, G.R. Nemerow

Diverse human viruses have coevolved to exploit the acidification of endosomal compartments to gain entry into host cells. Recently, we used a supramolecular approach to selectively target and inhibit viral infections through this central pathway. We used a high-throughput screen with an adenovirus vector encoding green fluorescent protein to select an 8-residue cyclic D,L-α-peptide from a directed combinatorial library that specifically inhibited the development of low pH inside endocytic vesicles, thereby arresting escape of virus from these compartments. The peptide had no adverse effect on cell viability and was only able to exert its inhibitory activity when added to cells in the presence of the virus. Confocal fluorescence microscopic studies with labeled adenovirus particles indicated that the peptide did not hinder viral attachment or entry but rather extinguished the pH gradient inside cell endosomes. Influenza virus that uses a mode of entry similar to that of adenovirus was also inhibited by the peptide. Our results suggest that self-assembling cyclic peptides may provide a broad-spectrum and alternative approach to the design of antiviral drugs.

Inhibiting Expression of Proinflammatory Cytokines With Adenoviruses

M. Iacobelli-Martinez, R.R. Nepomuceno, J. Connolly,* G.R. Nemerow

* Isis Pharmaceuticals, Inc., Carlsbad, California

Most adenovirus serotypes bind to host cells via the coxsackievirus-adenovirus receptor, but subgroup B and subgroup D (adenovirus 37) viruses recognize the receptor CD46. Interestingly, diverse microbial pathogens that use CD46 for infection downregulate the expression of IL-12, a cytokine involved in both the innate and the adaptive immune responses. We determined whether adenovirus serotypes that use CD46 alter the expression of proinflammatory cytokines.

We found that subgroup B adenoviruses type 16 and 35 and subgroup D adenovirus type 37, but not subgroup C adenoviruses type 2 or 5, significantly reduced expression of IL-12 by peripheral blood mononuclear cells stimulated by IFN-γ and lipopolysaccharide. IL-12 mRNA, as well as mRNA encoding other mediators, such as IL-1α, IL-β, the receptor for IL-1α, and IL-6, were also downregulated upon interaction with adenoviruses that use CD46. Analysis of transcription factor activity required for cytokine expression indicated that adenoviruses that used CD46 preferentially inhibited the DNA-binding activity of the transcription factor CCAAT/enhancer-binding protein β (C/EBP-β). Expression of C/EBP-β protein induced by IFN-γ was also impaired by adenoviruses that use CD46, consistent with the reduced DNA-binding activity of C/EBP-β. Interference with IFN-γ signaling events by adenoviruses that use CD46, but not adenoviruses that use the coxsackievirus-adenovirus receptor, revealed a potentially critical difference in the host immune response against adenovirus vectors, a situation that has implications for gene delivery and vaccine development.

Publications

Horne, W.S., Wiethoff, C.M., Cui, C., Wilcoxen, K.M., Amorin, M., Ghadiri, M.R., Nemerow, G.R. Antiviral cyclic D,L-α-peptides: targeting a general biochemical pathway in virus infections. Bioorg. Med. Chem. 13:5145, 2005.

Hsu, C., Boysen, M., Gritton, L.D., Frosst, P.D., Nemerow, G.R., Von Seggern, D.J. In vitro dendritic cell infection by pseudotyped adenoviral vectors does not correlate with their in vivo immunogenicity. Virology 332:1, 2005.

Iacobelli-Martinez, M., Nepomuceno, R.R., Connolly, J., Nemerow, G.R. CD46-utilizing adenoviruses inhibit C/EBPβ-dependent expression of proinflammatory cytokines. J. Virol. 79:11259, 2005.

Saban, S.D., Nepomuceno, R.R., Gritton, L.D., Nemerow, G.R., Stewart, P.L. CryoEM structure at 9Å resolution of an adenovirus vector targeted to hematopoietic cells. J. Mol. Biol. 349:526, 2005.

Wiethoff, C.M., Wodrich, H., Gerace, L., Nemerow, G.R. Adenovirus protein VI mediates membrane disruption following capsid disassembly. J. Virol. 79:1992, 2005.