Our long-term goals are to understand the catalytic mechanism and biological specificity of virus-encoded enzymes that modify RNA ends. We are studying viral mRNA capping and viral tRNA repair as paradigms of RNA transactions that enable virus replication. The studies of capping provide fresh insights to the evolution of a uniquely eukaryotic mRNA processing event and are opening up new approaches to antiviral drug discovery targeted at viral mRNA cap formation. The current research plan is focused on the RNA triphosphatase and RNA (guanine-N7) methyltransferase components of the poxvirus mRNA capping apparatus and includes specific aims to: (a) interrogate whether the viral capping activities are essential for poxvirus replication in cell culture;(b) map the active site of the cap methyltransferase and determine its structure with substrates and inhibitors bound;(c) evaluate a sinefungin-based transition state analog as a specific inhibitor of poxvirus cap methylation;(d) determine the structure of the viral RNA triphosphatase and identify novel inhibitors via high-throughput screening. Our studies of tRNA repair are illuminating the evolutionary transitions from RNA-world to DNA-world enzymology. This proposal focuses on bacteriophage T4 Pnkp, a bifunctional 5'kinase/3'phosphatase that heals broken tRNA ends. Pnkp functions in vivo to antagonize a host antiviral response that blocks viral protein synthesis through tRNA depletion.
Our aim i s to elucidate the mechanism of the 3'-phosphatase domain by capturing structures of the enzyme at different steps along the reaction pathway. Relevance: Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed. The outbreak of human monkeypox infections in the US in 2003 highlighted the risks of re-emergence of human poxvirus disease. mRNA capping enzyme is an attractive therapeutic target for smallpox because the active site and catalytic mechanism of the poxvirus RNA triphosphatase are completely different from that of the human RNA triphosphatase. Poxvirus cap methyltransferase is also distinguished from the human counterpart by its reliance on a unique virus-encoded regulatory subunit.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042498-21
Application #
7798068
Study Section
Virology - A Study Section (VIRA)
Program Officer
Bender, Michael T
Project Start
1989-07-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
21
Fiscal Year
2010
Total Cost
$497,234
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Bacusmo, Jo Marie; Orsini, Silvia S; Hu, Jennifer et al. (2017) The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans. RNA Biol :1-10
Remus, Barbara S; Goldgur, Yehuda; Shuman, Stewart (2017) Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase. Nucleic Acids Res 45:12945-12953
Remus, Barbara S; Schwer, Beate; Shuman, Stewart (2016) Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis. RNA 22:1500-9
Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart (2015) Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase. Proc Natl Acad Sci U S A 112:13868-73
Unciuleac, Mihaela-Carmen; Shuman, Stewart (2015) Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi. RNA 21:824-32
Kyrieleis, Otto J P; Chang, Jonathan; de la Peña, Marcos et al. (2014) Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus. Structure 22:452-65
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoac Nucleic Acids Res 42:1152-61
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide. J Bacteriol 196:4285-92
Remus, Barbara S; Jacewicz, Agata; Shuman, Stewart (2014) Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase. RNA 20:1697-705
Schmier, Brad J; Shuman, Stewart (2014) Effects of 3'-OH and 5'-PO4 base mispairs and damaged base lesions on the fidelity of nick sealing by Deinococcus radiodurans RNA ligase. J Bacteriol 196:1704-12

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