The arsenal of weapons for treating virus infections is relatively meager. Although vaccines for some viruses can be effective, for other viruses, antiviral agents are needed. Among potential targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes, essential for the synthesis of infectious virus, are required to process virus-specific precursor proteins involved in the maturation, assembly and replication of such pathogenic human viruses as adenovirus, poliovirus, hepatitis C virus, and human immunodeficiency virus. Inhibition of the proteinase aborts the virus infection. Our model system for the development of new antiviral agents is human adenovirus.
One specific aim i s to understand at the biochemical and structural levels how the activity of the adenovirus proteinase (AVP) is regulated. Two major questions are being addressed: 1) How is AVP activated inside the virion? AVP is synthesized in an inactive form that requires cofactors that restrict its activity in both space and time. One cofactor is pVIc, an 11 amino acid viral peptide;another is the viral DNA;actin is a cellular cofactor. The cofactors increase the kcat/Km for substrate hydrolysis. We determined the crystal structure of AVP-pVIc to a resolution of 1.6 ? and of AVP to 0.98 ?. The fold of the protein is unique;AVP represents the first member of a new class of cysteine proteinases. Our current model is that AVP is activated by pVIc via a contiguous series of conformational changes over a 53 amino acid long branched pathway;this will be investigated by mutational analysis coupled with binding and activity assays. 2) How can 70 molecules of AVP-pVIc process virion precursor proteins at 3200 sites to render a virus particle infectious, this occurring in young virions where the rate of diffusion of enzymes and substrates is nearly zero? We have an insight into this conundrum from single molecule experiments which show AVP-pVIc complexes robustly sliding along tens of thousands of base pairs of viral DNA via one-dimensional diffusion. This is a way to locate the precursor proteins and may represent a new paradigm for virion maturation. Sliding activity will be characterized, and the concept of a """"""""molecular sled"""""""" will be explored. The second specific aim is to use the information obtained via the first specific aim to identify drug targets in AVP, in its cofactors, and in its substrates and to use structure-based drug design to discover compounds that bind to these targets. A novel aspect of our work is in identifying drug targets other than those in the active site. Our work has shown that more than 25% of the surface of AVP is a legitimate drug target. By DOCKing, we already have identified some non-active site compounds that inhibit enzyme activity and are pursuing others. They will then be tested as antiviral agents.

Public Health Relevance

Although vaccines for some viruses can be effective, for other viruses, antiviral agents are needed. We are studying virus-coded proteinases as targets for antiviral agents. Several novel classes of drug targets have been uncovered, and we are now identifying compounds that bind to them and act as anti-viral agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041599-13
Application #
7849010
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
1997-07-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
13
Fiscal Year
2010
Total Cost
$786,554
Indirect Cost
Name
Brookhaven National Laboratory
Department
Type
DUNS #
027579460
City
Upton
State
NY
Country
United States
Zip Code
11973
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Mangel, Walter F; McGrath, William J; Xiong, Kan et al. (2016) Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA. Nat Commun 7:10202
Geertsema, Hylkje J; Schulte, Aartje C; Spenkelink, Lisanne M et al. (2015) Single-molecule imaging at high fluorophore concentrations by local activation of dye. Biophys J 108:949-56
Pérez-Berná, Ana J; Mangel, Walter F; McGrath, William J et al. (2014) Processing of the l1 52/55k protein by the adenovirus protease: a new substrate and new insights into virion maturation. J Virol 88:1513-24
Mangel, Walter F; San Martín, Carmen (2014) Structure, function and dynamics in adenovirus maturation. Viruses 6:4536-70
Graziano, Vito; McGrath, William J; Suomalainen, Maarit et al. (2013) Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: I. binding to DNA AND to hexon of the precursor to protein VI, pVI, of human adenovirus. J Biol Chem 288:2059-67
Graziano, Vito; Luo, Guobin; Blainey, Paul C et al. (2013) Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: II. adenovirus proteinase is activated in an unusual one-dimensional biochemical reaction. J Biol Chem 288:2068-80
McGrath, William J; Graziano, Vito; Zabrocka, Katarzyna et al. (2013) First generation inhibitors of the adenovirus proteinase. FEBS Lett 587:2332-9
Baniecki, Mary Lynn; McGrath, William J; Mangel, Walter F (2013) Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: III. atomic resolution structure of the nascent form of the adenovirus proteinase. J Biol Chem 288:2081-91
Blainey, Paul C; Graziano, Vito; Pérez-Berná, Ana J et al. (2013) Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: IV. viral proteinase slides along DNA to locate and process its substrates. J Biol Chem 288:2092-102

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