Project: Research project

Project Details


During the morphogenesis of double-stranded DNA bacteriophages, a DNA-free
protein capsid (procapsid) is assembled and subsequently packages the
bacteriophage DNA. Procapsids consist of a multimolecular outer shell and
two classes of internal proteins that assist, by unknown mechanisms,
assembly of the outer shell. Previous studies of the structure and
assembly of viruses suggest that assembly of outer procapsid shells
kinetically controlled by induction of changes in the conformation in
unassembled subunits. By this hypothesis, unassembled subunits in a
nonassociating conformation switch to a conformation appropriate for entry
into the assembly pathway after contact with a partially assembled
precursor of the procapsid (conformational switching). However, details of
the extent to which conformational switching occurs are not known for any
viral capsid. The long-range goal of the research proposed here is to
determine the mechanisms by which procapsids assemble from unassembled
proteins. Emphasis is to be placed on determining how internal proteins
assist assembly of the procapsid's outer shell and on determining the
extent to which assembly is controlled by conformational switching. To
reach this goal, the specific aims are to accomplish the following, using
the related bacteriophages, T7 and T3, as models: (a) increase
understanding of the structure of procapsids; electron microscopy,
immuno-electron microscopy, protein-protein crosslinking, controlled
proteolysis (based on our previous determination of tertiary structure by
this procedure) and laser-Raman spectroscopy will be used, (b) detect and
isolate multimolecular potential procapsid precursors that are produced
during assembly of procapsids "in vivo" and "in vitro"; for detection and
isolation, procedures of gel electrophoresis, density gradient
electrophoresis and centrifugation, several recently developed in the
laboratory of the PI, will be used, (c) characterize the multimolecular
particles isolated using the above procedures and also our newly-developed
procedures of gel sieving, (d) determine which of the multimolecular
particles isolated are in the assembly pathway of the procapsid and
determine the temporal sequence of these particles in the pathway;
procedures of kinetic labeling-gel electrophoresis-autoradiography will be
used whenever possible, (e) continue our development of gel sieving for
determining the shape of a multimolecular particle. The information
obtained will assist in understanding mechanisms of the assembly of
multimolecular complexes in normal and diseased cells. Some of the
procedures developed should have use in diagnostic virology.
Effective start/end date9/1/858/31/89


  • National Institutes of Health


  • Medicine(all)
  • Immunology and Microbiology(all)


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