This grant application proposes continuation of research toward two main objectives: 1) understanding the mechanism of terminase catalyzed viral DNA translocation into the procapsid, and 2) determining the host resistance mechanism leading to the evolution of the CTS- (capsid targeting sequence) initiated set of polymorphic internal proteins injected by the T-even phages. The objectives will be furthered by development of a new phage-derived technique for assessing protein interactions. As part of the first objective, the DNA packaging mechanism will be investigated by focusing on terminase and portal dodecamer structure and function. A multimeric activated ATPase form of the terminase large subunit can be produced by incubation with the terminase small subunit and with antibody against denatured terminase large subunit. Together with portal or functional portaI-GFP (green fluorescent protein) fusion, the reconstituted packasome motor (terminase + portal + DNA) will be characterized. Work incorporating portaI-GFP and HOC-portal fusions into functional proheads and phage suggests that the portal dodecamer of the prohead can be immobilized without blocking DNA packaging. Continuation of this work can determine whether a favored portal rotation model for DNA packaging is valid. Preliminary work reveals that NMR can solve the structure of the small terminase subunit. T4 display has successfully identified a new terminase interactant, T4 late ( factor gp55, required for replication-coupled T4 late transcription. Recent work has established a dependence of DNA packaging on this component; we believe the interaction serves to load the large terminase subunit onto DNA. This interaction could also serve to tightly couple packaging to DNA repair at the late replication-dependent transcription stage of T4 development. Reconstitution of active in vitro T4 DNA packaging will employ isolated gene 55 defective in vivo concatemers as well as T4 transcription assay plasmids. These DNAs will be activated for packaging by loading late transcription-replication components. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus of the T-even phages to challenge a corresponding set of resistance genes in natural hosts of these viruses. We intend to determine the host resistance mechanism by analysis of two resistance gene(s), IBEGs and IBEGd, cloned from one pathogenic strain of E. coli. These two cloned genes are necessary and sufficient for phage T4 IPI- and other T-even phage resistance, and the two his-tag modified genes also confer resistance, thus facilitating study of the mechanism at the protein level.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
2R21AI011676-28A1
Application #
6681705
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (01))
Program Officer
Park, Eun-Chung
Project Start
1977-07-01
Project End
2004-08-31
Budget Start
2003-09-15
Budget End
2004-08-31
Support Year
28
Fiscal Year
2003
Total Cost
$371,250
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Thomas, Julie A; Benítez Quintana, Andrea Denisse; Bosch, Martine A et al. (2016) Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly. J Virol 90:10284-10298
Mullaney, Julienne M; Black, Lindsay W (2014) Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins. Methods Mol Biol 1108:69-85
Thomas, Julie A; Black, Lindsay W (2013) Mutational analysis of the Pseudomonas aeruginosa myovirus KZ morphogenetic protease gp175. J Virol 87:8713-25
Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A et al. (2013) The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging. Virology 446:293-302
Wu, Weimin; Thomas, Julie A; Cheng, Naiqian et al. (2012) Bubblegrams reveal the inner body of bacteriophage ?KZ. Science 335:182
Black, Lindsay W; Rao, Venigalla B (2012) Structure, assembly, and DNA packaging of the bacteriophage T4 head. Adv Virus Res 82:119-53
Thomas, Julie A; Weintraub, Susan T; Wu, Weimin et al. (2012) Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giant Pseudomonas aeruginosa phage ?KZ. Mol Microbiol 84:324-39
Dixit, Aparna Banerjee; Ray, Krishanu; Black, Lindsay W (2012) Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation. Proc Natl Acad Sci U S A 109:20419-24
Dixit, Aparna; Ray, Krishanu; Lakowicz, Joseph R et al. (2011) Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates. J Biol Chem 286:18878-89

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