During the morphogenesis of several animal and bacterial viruses, a DNA-free protein capsid is assembled and this capsid subsequently packages the viral DNA. During packaging, some bacteriophages cut their genomes from a larger (concatemeric) DNA. Our long-range objective is to understand viral DNA packaging at the molecular level. To accomplish this objective, we will use bacteriophages T3, T7 and P22 as model systems and will work toward the accomplishment of the following: a) isolation of capsid-DNA complexes in the DNA packaging pathway in vivo and in vitro: attempts will be made to isolate capsid-DNA complexes that have in the past been too unstable or short-lived to be observed; electrophoresis in density gradients and velocity sedimentation will be used for isolation; variation of isolation buffers, chemical cross-linking of intermediates and genetic perturbation of intermediates will be used. b) Determination of the temporal order of capsid-DNA complexes in the DNA packaging pathway: kinetic labeling experiments will be performed and attempts to have isolated capsid-DNA complexes re-enter the DNA packaging pathway in vitro will be made. c) Determination of the proteins and nucleotide sequences participating in capsid-DNA binding: immunoelectron microscopy, DNA-protein cross-linking and restriction endonuclease cleavage analysis will be used. d) Determination of the source(s) of energy for DNA packaging: a procedure for packaging DNA in vitro at high efficiency in a chemically defined mixture will be developed and will be used to test the effects of possible sources of energy. e) Determination of the arrangement of partially and totally packaged DNA: new procedures for utilizating DNA-reactive reagents will be used. f) Determination of DNA concatemer cutting mechanisms: restriction endonuclease cleavage analysis of the nature of the cut will be made and isolation of the nuclease will be attempted. g) Further characterization of capsids and DNA participating in DNA packaging: agarose gel electrophoresis, isoelectric focusing, protein-protein cross-linking and electron microscopy will be used. h) Further development of our procedures for isolating and characterizing viral assembly intermediates. The data obtained are expected to be useful in developing procedures of antiviral chemotherapy. Procedures in h) are expected to be useful in detecting, isolating and characterizing disease-causing viruses.
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