Structural and biophysical characterization of Nodamura virus
Abstract
The correlation between virus structure and viral stability and assembly is poorly understood. The following studies were undertaken to gain insight into this relationship. The nodaviruses are a useful model system for studying virus structure and assembly because of their simple architecture and genome. They are assembled as a T = 3 provirion with 180 copies of coat protein and the bipartite RNA genome. Provirions mature by autoproteolytic cleavage of the coat protein. Maturation imparts stability to the capsid and is required for infectivity. The type member of the family, Nodamura virus (NOV), has a broader host range, including mammals and insects, and is less stable to denaturants and high salt than other nodaviruses. Monoclinic crystals of NOV contained four virus particles per unit cell, arranged with pseudo-rhombohedral symmetry. The pseudo symmetry generated novel difficulties in the structure determination. The 4.2 A resolution structure of NOV has several unique features. Most notable was weak density corresponding to 24 base pairs of ordered RNA extending $\sim$70 A in a groove between icosahedral threefold axes. The "peptide in the groove," at the ends of the groove, has many interactions with ordered RNA; this sequence is more basic in NOV than in other nodaviruses. Other changes in the NOV structure include surface loops which correlate with differences in host range. A mechanism was proposed for autoproteolytic maturation in the nodavirus family. Aspartic acid 75, buried at the cleavage site during capsid assembly, activates the scissile bond by a mechanism which resembles general acid catalyzed hydrolysis; mutation of ASP75 prevents maturation. Analysis of cleavage kinetics suggests that maturation is driven by the instability of the provirion; the rate is proportional to the extent of cleavage. Capsid dissociation by urea was observed by turbidity and electron microscopy. Disassembly was an equilibrium process. A formalism developed to describe equilibrium assembly of polyhedral protein complexes allowed further analysis of these studies. The presence of disassembly intermediates, in some conditions, suggests that capsid assembly involves generation of intersubunit strain. Disruption of the capsid breaks some associative interactions between subunits but allows relaxation of this strain energy.
Degree
Ph.D.
Advisors
Johnson, Purdue University.
Subject Area
Biophysics
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