JVI Accepts, published online ahead of print on 18 April 2007

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J. Virol. doi:10.1128/JVI.02207-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Ty3 CAPSID MUTATIONS REVEAL EARLY AND LATE FUNCTIONS FOR THE AMINO-TERMINAL DOMAIN

Liza S.Z. Larsen, Min Zhang, Nadejda Beliakova-Bethell, Virginia Bilanchone, Anne Lamsa, Kunio Nagashima, Rani Najdi, Kathryn Kosaka, Vuk Kovacevic, Jianlin Cheng, Pierre Baldi, G. Wesley Hatfield, and Suzanne Sandmeyer^*

Departments of Microbiology and Molecular Genetics and Biological Chemistry, University of California, Irvine, CA 92697; School of Information and Computer Science and Institute for Genomics and, Bioinformatics; Image Analysis Laboratory, NCI-Frederick, SAIC Frederick, Inc.

^* To whom correspondence should be addressed. Email: sbsandme{at}uci.edu.

The Ty3 retrotransposon assembles into 50 nm virus-like particles which occur in large intracellular clusters in the case of wt Ty3. Within these particles, maturation of Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, similar to retroviral CA, Ty3 CA contains a high amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles.

Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, sedimentation and fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced formation of fluorescent Ty3 protein foci. In at least one MHR mutant, Ty3 protein concentrated in foci, but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P-bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore not necessary for assembly, but correlated with production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are first concentrated, assembly occurs associated with P-bodies, and particle morphogenesis concludes with a post reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

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