B.A. in ACS Certified Biochemistry from Ohio Wesleyan University
Rous sarcoma virus (RSV) is an oncogenic alpha retrovirus that infects chickens. Alpha retroviruses are good model systems as they are the simplest of all the retroviral genera, and understanding mechanisms in simple systems serve as the foundation for determining mechanisms in more complex retroviruses like HIV-1. RSV is unique in that it was the first virus discovered that could cause cancer, and it has been a critical model for studying viral oncogenesis and the retroviral life cycle since its discovery in 1911. I am examining RNA/Protein and Protein/Protein interactions in both the RSV and HIV-1 systems (and plan to expand to HTLV-1). One goal of this work is to better understand which mechanisms are conserved and which differ across the retroviral genera.
Retroviruses use specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNALys3 serves as the primer, whereas in Rous sarcoma virus (RSV), tRNATrp is used. The HIV-1 Gag protein is thought to chaperone tRNALys placement onto the genomic RNA primer binding site (PBS) in vivo, and it has also been demonstrated that Gag exhibits nucleic acid chaperone activity in vitro 1; 2; 3. The nucleocapsid (NCp7) domain of HIV-1 Gag is critical for this activity. By comparison to HIV-1, little is known about the chaperone activity of RSV Gag, therefore I am working to determine the mechanism by which RSV Gag anneals tRNATrp to the primer binding site in vitro. More specifically, I examine the kinetics of Gag chaperone activity using gel shift RNA annealing assays and fluorescence anisotropy binding experiments.
Another unanswered question in the life cycle of RSV, is “How is the tRNA primer packaged into the budding virion?” Studies in HIV-1 have shown tRNALys is packaged into virions via interactions with human lysyl-tRNA synthetase (LysRS), HIV-1 Gag and the reverse transcriptase (RT) domain of Gag-Pol 4; 5; 6; 7; 8. Similar to HIV-1 the cognate aminoacyl tRNA synthetase for tRNATrp, tryptophanyl-tRNA synthetase (TrpRS) is packaged into RSV. It has also been shown that RSV RT and Gag-Pol are required for tRNATrp recruitment and annealing, respectively9; 10; 11. Based on these data, we hypothesize that interactions between tRNATrp, chicken TrpRS and RSV Gag and/or RT play a role in specific tRNA primer recruitment into RSV. Affinity pull-down assays and western blots combined with fluorescence anisotropy binding assays are being used to explore this hypothesis.
1. Feng, Y. X., Campbell, S., Harvin, D., Ehresmann, B., Ehresmann, C. & Rein, A. (1999). The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site. J Virol 73, 4251-6.
2. Wu, T., Datta, S. A., Mitra, M., Gorelick, R. J., Rein, A. & Levin, J. G. (2010). Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications. Virology 405, 556-67.
3. Jones, C. P., Datta, S. A., Rein, A., Rouzina, I. & Musier-Forsyth, K. (2011). Matrix domain modulates HIV-1 Gag’s nucleic acid chaperone activity via inositol phosphate binding. J Virol 85, 1594-603.
4. Saadatmand, J., Guo, F., Cen, S., Niu, M. & Kleiman, L. (2008). Interactions of reverse transcriptase sequences in Pol with Gag and LysRS in the HIV-1 tRNALys3 packaging/annealing complex. Virology 380, 109-17.
5. Cen, S., Khorchid, A., Javanbakht, H., Gabor, J., Stello, T., Shiba, K., Musier-Forsyth, K. & Kleiman, L. (2001). Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1. J Virol 75, 5043-8.
6. Mak, J., Jiang, M., Wainberg, M. A., Hammarskjold, M. L., Rekosh, D. & Kleiman, L. (1994). Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol 68, 2065-72.
7. Javanbakht, H., Halwani, R., Cen, S., Saadatmand, J., Musier-Forsyth, K., Gottlinger, H. & Kleiman, L. (2003). The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly. J Biol Chem 278, 27644-51.
8. Kovaleski, B. J., Kennedy, R., Hong, M. K., Datta, S. A., Kleiman, L., Rein, A. & Musier-Forsyth, K. (2006). In vitro characterization of the interaction between HIV-1 Gag and human lysyl-tRNA synthetase. J Biol Chem 281, 19449-56.
9. Panet, A., Haseltine, W. A., Baltimore, D., Peters, G., Harada, F. & Dahlberg, J. E. (1975). Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase). Proc Natl Acad Sci U S A 72, 2535-9.
10. Sawyer, R. C. & Hanafusa, H. (1979). Comparison of the small RNAs of polymerase-deficient and polymerase-positive Rous sarcoma virus and another species of avian retrovirus. J Virol 29, 863-71.
11. Fu, W., Ortiz-Conde, B. A., Gorelick, R. J., Hughes, S. H. & Rein, A. (1997). Placement of tRNA primer on the primer-binding site requires pol gene expression in avian but not murine retroviruses. J Virol 71, 6940-6.