Suzanne B. Sandmeyer
Biological Chemistry, School of Medicine
Microbiology & Molecular Genetics, School of Medicine
Phone: (949) 824-7571
Email: sbsandme@uci.edu
http://www.ucihs.uci.edu/microbio/
www.ucihs.uci.edu/biochem/faculty/sandmeyer.html
http://www.faculty.uci.edu/profile.cfm?faculty_id=2247
Suzanne Sandmeyer
Dr. Sandmeyer’s laboratory studies Ty3, a retrovirus-like element in S. cerevisiae as a model system for understanding the interactions of retroviruses and their host cells. Retroviruses are not only of interest as the causative agents of acquired immunodeficiency syndrome and some cancers, but also for their potential applications in gene therapy. The sophisticated approaches available through yeast genomics make it an appealing system for the identification of candidate host functions that can later be tested in human cells.
The Ty3 element is comprised of GAG3 and POL3 open reading frames which encode capsid (CA), spacer (SP), and nucleocapsid (NC), and protease, reverse transcriptase, and integrase (IN), respectively. Ty3 expression results in production of Gag3 and Gag3-Pol3 polyproteins which form viruslike particles (VLPs) within which proteolytic processing results in maturation of particle proteins. Reverse transcription of Ty3 cDNA occurs and is followed by integration into the yeast genome at sites of pol III transcription initiation.
Genomics of Ty3 transposition: The Sandmeyer laboratory has characterized the basic molecular biology and biochemical components of the Ty3 system and is now using genomic and proteomic approaches to understand the role of the host in its transposition. Genomic screens have implicated approximately 180 genes in Ty3 transposition including ones involved in nuclear export, RNA processing, vesicular trafficking, and nuclear import. Subsequent studies have shown that Ty3 proteins and RNA are localized together with a subset of the identified RNA-related proteins in RNA processing bodies or P bodies. In the past year they undertook extensive mutagenesis of the Ty3 structural domains encoded in Gag3. These experiments showed that the NC protein plays an essential role in targeting RNA to the P body and for consistent association of Gag3 with P bodies and formation of normal particles. Mutations in P body proteins result in reduced levels of Ty3 RNA and protein. The mechanism of this decrease is not known. It is proposed that Ty3 might assemble in association with P bodies so that translation does not interfere with packaging. Clusters of Ty3 and P body proteins are associated with nuclear pores. Genetic studies identified several nucleoporins which support transposition. In vitro studies have shown that GST fusions of these nucleoporin proteins interact with Ty3 Gag3 produced in yeast or bacteria suggesting that nuclear entry is initiated by Gag3 engaging with the nuclear pore. These studies are being pursued to better understand how Ty3 actually uncoats and enters the nucleus.
Structure of Ty3 VLPs: The Ty3 VLP is a roughly spherical particle approximately 45 nm in diameter. The shell is comprised primarily of Gag3 protein. A collaboration with the McPherson laboratory using atomic force microscopy to characterize the particle showed that it has features of icosahedral symmetry including pentameric vertices. The CA structure was predicted to be comprised of two bundles of alpha helices, similar to retroviral CA structure. Mutagenesis of the Gag3 protein has shown that the amino-terminal domain of CA is critical for VLP formation. Two hybrid analysis of this domain show that it interacts with itself and with the carboxyl terminal domain of CA.
Targeting of Ty3 integration: Ty3 has the unusual property that it integrates specifically into pol III transcription initiation sites. Current work in the lab has shown that the Ty3 integrase protein interacts with the pol III transcription factor Brf1. The Sandmeyer laboratory is using a Brf1-TBP fusion protein in order to target integration in vitro so that Brf1 can be mutagenized in order to better understand its role in integration. In vitro binding reactions have shown that the Ty3 IN interacts with Brf via its carboxyl-terminal domain. Mutations are being introduced into this domain in order to define the region required for position specific integration.
Selected Publications:
Irwin, B., Aye, M., Baldi, P., Beliakova-Bethell, N., Cheng, H., Dou, Y., Liou, W., and Sandmeyer, S. (2005). Retroviruses and yeast retrotransposons use overlapping sets of host genes. Genome Res 15(5), 641-54.
Kuznetsov, Y. G., Zhang, M., Menees, T. M., McPherson, A., and Sandmeyer, S. (2005). Investigation by atomic force microscopy of the structure of Ty3 retrotransposon particles. J Virol 79(13), 8032-45.
Beliakova-Bethell, N., Beckham, C., Giddings, T. H., Jr., Winey, M., Parker, R., and Sandmeyer, S. (2006). Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components. Rna 12(1), 94-101.
Larsen, L. S., Zhang, M., Beliakova-Bethell, N., Bilanchone, V., Lamsa, A., Nagashima, K., Najdi, R., Kosaka, K., Kovacevic, V., Cheng, J., Baldi, P., Hatfield, G. W., and Sandmeyer, S. (2007). Ty3 capsid mutations reveal early and late functions of the amino-terminal domain. J Virol 81(13), 6957-72.
Larsen, L. S., Beliakova-Bethell, N., Bilanchone, V., Zhang, M., Lamsa, A., Dasilva, R., Hatfield, G. W., Nagashima, K., and Sandmeyer, S. (2008). Ty3 nucleocapsid controls localization of particle assembly. J Virol 82(5), 2501-14. |
