The Role of Karyopherin Transportin 3 in Rous Sarcoma Virus Assembly

Open Access
Stake, Matthew Steven
Graduate Program:
Cell and Molecular Biology
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 08, 2013
Committee Members:
  • Leslie Joan Parent, Thesis Advisor
  • Rous Sarcoma Virus
  • TNPO3
  • Nuclear Import
Studies of the oncogenic avian retrovirus Rous sarcoma virus (RSV) have provided numerous insights into complex cellular processes. The multidomain Gag polyprotein of RSV assembles nascent viral particles by first binding the viral genome and then trafficking to the plasma membrane where budding occurs. Gag trafficks through the nucleus during assembly, and nuclear trafficking of Gag is required for efficient packaging of the viral genome. Gag is directed into the nucleus by two distinct nuclear localization signals (NLSs) that bind three different host nuclear import factors. One NLS in the nucleocapsid (NC) domain contains a canonical stretch of basic residues that mediates nuclear entry through the Importin !/" pathway. The other NLS resides in the matrix (MA) domain and does not contain sequence similarity to any previously described NLS. This NLS interacts with the non-canonical import factors Importin 11 and Transportin 3 (TNPO3). The role of each NLS and import factor in virus assembly is not clearly defined. Here, the contribution of TNPO3 to Gag nuclear import is examined. In cells, the C-terminal cargo-binding domain of TNPO3 binds serine-arginine rich regions of splicing factors to import the splicing factor into the nucleus. Quantitative microscopy data presented here demonstrate that overexpression of TNPO3 facilitates the entry of Gag into the nuclei of transfected cells. GST pull down assays revealed that Gag and TNPO3 engage in a novel, direct protein-protein interaction. The MA domain does not contain a serine- arginine-rich domain characteristic of TNPO3 cargoes, and the cargo-binding domain of TNPO3 is dispensable for this interaction. Additional quantitative imaging experiments presented here show that TNPO3 cooperates with Imp" but not Imp11 to mediate Gag nuclear import. Future experiments will address the roles of each NLS and individual import factors in Gag nuclear trafficking and virus replication, as well as the relationships between them. Overall, the complex signals regulating nuclear trafficking of RSV Gag provide a unique tool for examining the interplay and mechanisms of host nuclear import pathways.