Many important viruses and nucleoprotein cores of enveloped animal viruses (e.g. retroviruses) are unstable and pleomorphic and principles governing their structure are generally unknown due to the lack of crystallographic studies. Crystallography has been most successful with robust particles because homogeneous preparations are readily produced and these are likely to form well ordered crystals. Particles stabilized predominantly by protein-RNA interactions are unstable when exposed to even mild physical or chemical denaturants. For this reason simple viruses of this type are often the most accessible for assembly studies because subunit tertiary structure is not disturbed when the virion is disassembled. Cowpea chlorotic mottle virus (CCMV) is typical of this class. It was the first icosahedral virus reassembled in vitro to form infectious particles and the literature on the assembly of CCMV and the related alfalfa mosaic virus (ALMV) exceeds 100 papers and numerous reviews. With support from an NSF grant it was discovered that CCMV particles are highly sensitive to ultracentrifugation and this lead to the development of a new preparation procedure and the production of the first good quality CCMV crystals. CCMV is the type member of a super group of viruses known as tricorna viruses. The capsids of all members of this group are relatively unstable but there is a clear trend of reduced importance of protein-protein interactions from CCMV (T=3) to cucumber mosaic virus (CMV) to ALMV, which forms bacilliform particles in vivo from subunit hexamers and pentamers, to tobacco streak virus (TSV), which forms spheroidal particles in vivo with subunits that will not assemble when released from RNA. Previous studies indicate that structures of all the subunits in this super group are likely to be beta- barrels. The long term goal of this proposal is to solve and superimpose the 4 different subunit structures and identify the amino acid determinants of capsid stability and morphology. Towards this end the structure of CCMV was solved at 3.2 angstrom resolution. 180 canonical virus beta-barrels (190AA) form pentamers and true hexamers that associate through interwoven C terminal polypeptides to form a new type of T=3 particle. This association of morphological units is strikingly similar to the quaternary structure of the DNA tumor virus SV40. The structure of CCMV will be refined at 3.2 angstrom, the T=3 structure of cucumber mosaic virus will be extended from its current 5.5 angstrom resolution to at least 4.0 angstrom resolution; the structure of the T=1 reassembly product of ALMV subunits will be determined at 4.0 angstrom resolution, and the structure of the TSV dimeric subunits will be determined at 2.6 angstrom resolution. Crystals diffracting to the resolutions indicated are in hand for all these viruses. Heterogeneous assembly products and disassembly intermediates of these viruses will be analyzed by cryoEM and protein modeling. Systems for E coli expression and in vitro assembly of expressed CCMV capsid protein developed by collaborator Mark Young will be used to confirm determinants of capsid stability and morphology with site directed mutants (i.e. reverse genetics).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054076-04
Application #
2734794
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1995-07-01
Project End
1999-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
4
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
Doerschuk, Peter C; Gong, Yunye; Xu, Nan et al. (2016) Virus particle dynamics derived from CryoEM studies. Curr Opin Virol 18:57-63
Routh, Andrew; Chang, Max W; Okulicz, Jason F et al. (2015) CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data. Methods 91:40-47
McNulty, Reginald; Lokareddy, Ravi Kumar; Roy, Ankoor et al. (2015) Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22. J Mol Biol 427:3285-3299
Routh, Andrew; Head, Steven R; Ordoukhanian, Phillip et al. (2015) ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs. J Mol Biol 427:2610-6
Campbell, Melody G; Kearney, Bradley M; Cheng, Anchi et al. (2014) Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV. J Struct Biol 188:183-7
Routh, Andrew; Johnson, John E (2014) Discovery of functional genomic motifs in viruses with ViReMa-a Virus Recombination Mapper-for analysis of next-generation sequencing data. Nucleic Acids Res 42:e11
Kearney, Bradley M; Johnson, John E (2014) Assembly and maturation of a T = 4 quasi-equivalent virus is guided by electrostatic and mechanical forces. Viruses 6:3348-62
Tang, Jinghua; Kearney, Bradley M; Wang, Qiu et al. (2014) Dynamic and geometric analyses of Nudaurelia capensis ? virus maturation reveal the energy landscape of particle transitions. J Mol Recognit 27:230-7
Domitrovic, Tatiana; Movahed, Navid; Bothner, Brian et al. (2013) Virus assembly and maturation: auto-regulation through allosteric molecular switches. J Mol Biol 425:1488-96
Wang, Qiu; Matsui, Tsutomu; Domitrovic, Tatiana et al. (2013) Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps. J Struct Biol 181:195-206

Showing the most recent 10 out of 52 publications