INSIGHTS INTO RETROVIRAL ASSEMBLY FROM MUTATION OF THE ROUS SARCOMA VIRUS p10 NUCLEAR EXPORT SEQUENCE
Open Access
- Author:
- Kenney, Scott Patrick
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 22, 2008
- Committee Members:
- Leslie Joan Parent, Committee Chair/Co-Chair
Sergei A Grigoryev, Committee Member
John Warren Wills, Committee Member
Richard James Courtney, Committee Member
David Joseph Spector, Committee Member - Keywords:
- Retrovirus
retroviral assembly
Gag
p10
nuclear export
protein dimerization
FRET
FLIP
CRM1 - Abstract:
- Assembly of retroviruses is mediated by a polyprotein termed Gag, which is both necessary and sufficient to drive particle assembly. Gag functional domains are conserved among retroviruses, and each Gag protein encodes a matrix, capsid, and nucleocapsid protein that serves specific functions during particle assembly. Numerous protein-interaction partners and conformational changes occur during the course of assembly to mediate each step of the process. In this dissertation, I present my work on the subcellular trafficking patterns of the Gag protein of the simple avian retrovirus Rous sarcoma virus (RSV) to gain insights into the role of nuclear transport mechanisms in the assembly of retroviruses. RSV Gag undergoes transient nuclear localization in which nuclear export is mediated through the host cellular factor CRM1. The nuclear trafficking step divides the assembly process into early (pre nuclear) and late (post nuclear) events. Utilizing a complementation approach, I found that Gag proteins interact early in the assembly process, either prior to nuclear import or within the nucleus. Under normal conditions, higher-ordered protein complex formation is prevented from occurring in the nucleus because of efficient nuclear export. However, in the overexpression system, Gag multimers form in the nucleus when Gag is capable of interacting with RNA. Through the use of CRM1-dependent and CRM1-independent nuclear export signal substitutions, I showed that CRM1-dependent export signals could substitute for the Gag nuclear export signal and virus assembly, although infectivity was impaired. In the course of these experiments, I made several novel observations. In addition to the CRM1-mediated nuclear export pathway, RSV Gag appears to be exported from the nucleus in a CRM1-independent manner. Furthermore, substitution of the p10 NES with heterologous NESs increased genomic RNA packaging, implicating the nuclear export sequence in Gag as a determinant for RNA incorporation. Finally, I showed that nuclear export activity of the Gag polyprotein can be genetically separated from the role of this region in virus replication. Together, these findings confirm that the region of Gag which regulates nuclear trafficking plays multiple overlapping roles in the viral lifecycle.