The proposed project involves the use of low energy (0.5 keV - 5.0 keV) Ar+ beams to study the structure of bacteriophage lambda, the large (50S) subunit of E. coli ribosomes and equine herpes virus type 1 (EHV-1). The experimental strategy is to employ ion beams with limited (5.0nm - 10nm) penetrating power to etch or erode the particle surface progressively from the outside toward the inside. At various stages of the etching process, virions will be examined in the electron microscope to identify internal structural features revealed when more external layers are eroded away. Biochemical analyses will also be carried out with irradiated particles to identify exposed proteins or regions of the nucleic acid that have been damaged during etching. Studies of lambda phage will be devoted to clarifying the way DNA is arranged in the mature phage head. The goal is to test our recent suggestion that the first DNA to enter the prohead (the left end on the standard genetic map) should be found at the center of the overall DNA condensate with the last-packaged DNA at the periphery. Experimentally, this will involve etching intact phage and then examining the DNA (by Southern hybridization to sequence-specific probes) to identify the sites of damage. The proposed analysis of E. coli 50S ribosomal subunits is intended to identify those proteins and regions of the 23S RNA nearest the particle surface. Intact 50S subunits will be etched for various periods and the remaining protein components analyzed by two- dimensional SDS-polyacrylamide gel electrophoresis. Sites of damage to the 23S RNA will be identified by Southern hybridization experiments involving chemically-synthesized DNA probes corresponding to specific regions of the rRNA sequence. Etching experiments with intact EHV-1 are designed to clarify the structure of the tegument layer and its polypeptide composition. Analyses of EHV-1 nucleocapsids should illuminate the arrangement of DNA in these structures and allow one to determine the polypeptide composition of the core found in the center of the DNA-containing torroid.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034036-08
Application #
3284453
Study Section
Virology Study Section (VR)
Project Start
1984-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Newcomb, W W; Trus, B L; Booy, F P et al. (1993) Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes. J Mol Biol 232:499-511
Mendelson, E C; Newcomb, W W; Brown, J C (1992) Ar+ plasma-induced damage to DNA in bacteriophage lambda: implications for the arrangement of DNA in the phage head. J Virol 66:2226-31
Trus, B L; Newcomb, W W; Booy, F P et al. (1992) Distinct monoclonal antibodies separately label the hexons or the pentons of herpes simplex virus capsid. Proc Natl Acad Sci U S A 89:11508-12
Booy, F P; Newcomb, W W; Trus, B L et al. (1991) Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus. Cell 64:1007-15
Baker, T S; Newcomb, W W; Olson, N H et al. (1991) Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys J 60:1445-56
Newcomb, W W; Brown, J C (1991) Structure of the herpes simplex virus capsid: effects of extraction with guanidine hydrochloride and partial reconstitution of extracted capsids. J Virol 65:613-20
Baker, T S; Newcomb, W W; Booy, F P et al. (1990) Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy. J Virol 64:563-73
Newcomb, W W; Brown, J C (1989) Use of Ar+ plasma etching to localize structural proteins in the capsid of herpes simplex virus type 1. J Virol 63:4697-702
Newcomb, W W; Brown, J C; Booy, F P et al. (1989) Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron microscopic study. J Virol 63:3777-83
Salerno, J C; Bolgiano, B; Ingledew, W J (1989) Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction. FEBS Lett 247:101-5

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