Packaging of viral genomes is a fundamental process in biology. In many DNA viruses this reaction is powered by a large (M.W. ~1.5MDa) genome-packaging motor, which is formed by a terminase holoenzyme assembled to a dodecameric portal protein. This macromolecular complex functions like a chemical motor, which hydrolyzes ATP to translocate a copy of the viral genome inside a preformed capsid, at rates as high as 2000 bases per second. In addition to being the fastest and most powerful engine in nature, the genome-packaging motor is also an intriguing molecular machine, which is poorly characterized both in structure and catalytic mechanisms. In this grant, we will use a combination of crystallographic and biochemical techniques to characterize the structure of the bacteriophage P22 genome-packaging motor. Building upon the structure of P22 portal protein, which was recently determined in my laboratory, our work will focus on the chemistry of P22 terminase that is a functional ATPase in the motor. The structural characterization of P22 terminase in complex with portal protein will provide a structural framework to decipher how, within the packaging motor, terminase couples ATP hydrolysis to translocation of viral genomes. In addition, since terminases are highly conserved in herpesviruses, the work proposed in this grant will also provide a logical framework to begin structural characterization of packaging motors in pathogenic human viruses.
Specific aims of our work are: 1.) to determine the structure of the P22 terminase holoenzyme assembled at the portal protein vertex;2.) to define how small terminase subunit stimulates large terminase ATPase activity during genome-packaging.

Public Health Relevance

Viral genome-translocating motors are multisubunit ATPases that burn ATP to power packaging of viral genomes inside a preformed capsid. In this grant, we propose to study the structure and activity of the bacteriophage P22 genome-translocating motor. Our work will determine how one of smallest motors in nature couples chemical energy to genome translocation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM100888-03
Application #
8711498
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2012-08-01
Project End
2016-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
3
Fiscal Year
2014
Total Cost
$294,500
Indirect Cost
$104,500
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Bhardwaj, Anshul; Casjens, Sherwood R; Cingolani, Gino (2014) Exploring the atomic structure and conformational flexibility of a 320?Å long engineered viral fiber using X-ray crystallography. Acta Crystallogr D Biol Crystallogr 70:342-53
Sankhala, Rajeshwer Singh; Lokareddy, Ravi Kumar; Cingolani, Gino (2014) Structure of human PIR1, an atypical dual-specificity phosphatase. Biochemistry 53:862-71
Bhardwaj, Anshul; Olia, Adam S; Cingolani, Gino (2014) Architecture of viral genome-delivery molecular machines. Curr Opin Struct Biol 25:1-8
Leavitt, Justin C; Gogokhia, Lasha; Gilcrease, Eddie B et al. (2013) The tip of the tail needle affects the rate of DNA delivery by bacteriophage P22. PLoS One 8:e70936