We propose to continue our studies of insect viruses employing crystallography, molecular genetics, electron microscopy and computational and membrane chemistry to refine and extend models for virus assembly, maturation, cell attachment and genome delivery. Viruses with T=3 and T=4 quasi-equivalent capsids, as well as a picorna- like P=3 capsids will be used for the study. Crystallographic, biophysical and molecular genetic investigations of T=3 nodaviruses show that the capsid protein is modular in its function. The N-terminal portion controls the generic neutralization and condensation of RNA, while the C-terminal portion determines the specificity of RNA packaging. Autocatalytic maturation cleavage of the capsid protein, required for infectivity and creating a covalently independent, C- terminal module, is controlled by an internal helical domain, the quaternary structure of the capsid and the binding of divalent metal ions near the particle surface. Portions of the capsid protein and ordered duplex regions of the RNA genome function as molecular switches that lead to T=3 quasi-equivalence. Computational studies indicate that the particles assemble from a stable trimer intermediate and that there is conformational polymorphism between the formation of hexamers and pentamers of trimers that facilitate quasi-equivalent capsid formation. Biophysical investigations lead to a dynamic model for RNA uncoating in which the independent helical bundle, created during maturation, is associated with the 5' end of the RNA and is released from the particle when the virus binds to the receptor and enters the endosome. The model connotes that the helical bundle serves as conduit for membrane translocation of the 5' region of the viral RNA allowing the ribosome to initiate translation and drag the RNA through the membrane and into the cytosol. The hypothesis was strengthened when we demonstrated through structural and functional studies that a T=4 insect virus undergoes a maturation cleavage like the T=3 virus and that a pentameric helical bundle near the C-terminus was cut from the remainder of the coat protein. The cleavage site and helical bundle are nearly identical in the two different virus particles. We now propose to investigate issues of assembly through crystallographic studies of mutant virus-like particles generated in baculovirus expression systems for both virus families and to extend our computational studies of assembly. We will also identify and clone the soluble portion of the receptor for nodaviruses and attempt to make a virus receptor complex for cryoEM investigation. We will investigate the in vitro interaction of the virus particles with membranes and determine the structure of a picorna- like insect virus to see if there is evidence for functional and evolutionary similarity between T=3 ans P=3 particles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034220-15
Application #
2406513
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1984-12-01
Project End
2002-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Walukiewicz, Hanna E; Banerjee, Manidipa; Schneemann, Anette et al. (2008) Rescue of maturation-defective flock house virus infectivity with noninfectious, mature, viruslike particles. J Virol 82:2025-7
Walukiewicz, Hanna E; Johnson, John E; Schneemann, Anette (2006) Morphological changes in the T=3 capsid of Flock House virus during cell entry. J Virol 80:615-22
Johnson, Karyn N; Tang, Liang; Johnson, John E et al. (2004) Heterologous RNA encapsidated in Pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions. J Virol 78:11371-8
Tihova, Mariana; Dryden, Kelly A; Le, Thuc-vy L et al. (2004) Nodavirus coat protein imposes dodecahedral RNA structure independent of nucleotide sequence and length. J Virol 78:2897-905
Lin, Tianwei; Cavarelli, Jean; Johnson, John E (2003) Evidence for assembly-dependent folding of protein and RNA in an icosahedral virus. Virology 314:26-33
Tang, Liang; Johnson, John E (2002) Structural biology of viruses by the combination of electron cryomicroscopy and X-ray crystallography. Biochemistry 41:11517-24

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