While adenoviral vectors continue to be employed in phase 1-III clinical trials for the treatment of cancers and cardiovascular diseases, we still have an incomplete understanding of the virus entry pathway. During the previous funding period, researchers in the Nemerow and Reddy laboratories at the Scripps Research Institute solved the crystal structure of adenovirus at near atomic resolution thereby revealing the locations, folds and interactions of several viral capsid proteins. Additional investigations in the Nemerow and Stewart laboratories (Vanderbilt) discovered that specific host factors either destabilize (alpha v integrins) the virus capsid or restrict (defensins) capsid disassembly. Although this information has shed further light on the mechanisms involved in virus entry into cells, numerous gaps remain in our knowledge of virus disassembly and membrane penetration events. In this proposal, a team of experienced investigators with expertise in molecular virology, structural biology, and biophysical techniques will investigate the precise mechanisms involved in capsid disassembly and membrane penetration by protein VI. X-ray diffraction and cryoEM structural analyses of several key capsid proteins including proteins VI, V and hexon will be undertaken. Advanced imaging of adenovirus by atomic force microscopy will be employed to measure the tensile strength and mechanical properties of the viral capsid. Native and denatured mass spectrometry will be used to determine the copy number of protein VI in the virion as well as its association states with other capsid proteins. Protein VI mutagenesis studies will analyze the role of protein VI in capsid assembly/disassembly and endosome penetration. The long-term goal of these studies is to obtain a more complete picture of host cell invasion by adenovirus. Such knowledge should bolster the emerging knowledge of how non-enveloped viruses breach host cell membranes as well as provide a foundation for the development of safer and more efficient viral vectors.

Public Health Relevance

The use of viral vectors, including adenovirus, is stymied by a fundamental lack of knowledge of their structure and cell entry mechanisms. Inevitably, this situation negatively impacts implementation of effective vaccine, antiviral and gene delivery strategies. The specific goals of this proposal seek to remedy this deficiency by undertaking a comprehensive analysis of two key events in the adenovirus life cycle, capsid disassembly and membrane penetration.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
4R01HL054352-22
Application #
9095427
Study Section
Virology - A Study Section (VIRA)
Program Officer
Sheridan, John T
Project Start
1995-01-20
Project End
2017-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
22
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Nemerow, Glen R; Stewart, Phoebe L (2016) Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity. Viruses 8:
Moyer, Crystal L; Besser, Eli S; Nemerow, Glen R (2016) A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly. J Virol 90:521-32
Cassany, Aurélia; Ragues, Jessica; Guan, Tinglu et al. (2015) Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214. J Virol 89:1719-30
Kremer, Eric J; Nemerow, Glen R (2015) Adenovirus tales: from the cell surface to the nuclear pore complex. PLoS Pathog 11:e1004821
Wiethoff, Christopher M; Nemerow, Glen R (2015) Adenovirus membrane penetration: Tickling the tail of a sleeping dragon. Virology 479-480:591-9
Pérez-Vargas, Jimena; Vaughan, Robert C; Houser, Carolyn et al. (2014) Isolation and characterization of the DNA and protein binding activities of adenovirus core protein V. J Virol 88:9287-96
Reddy, Vijay S; Nemerow, Glen R (2014) Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection. Proc Natl Acad Sci U S A 111:11715-20
Sanchez-Cespedes, Javier; Moyer, Crystal L; Whitby, Landon R et al. (2014) Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative. Antiviral Res 108:65-73
Benevento, Marco; Di Palma, Serena; Snijder, Joost et al. (2014) Adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and quantitative proteomics. J Biol Chem 289:11421-30
Snijder, Joost; Benevento, Marco; Moyer, Crystal L et al. (2014) The cleaved N-terminus of pVI binds peripentonal hexons in mature adenovirus. J Mol Biol 426:1971-9

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