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Understanding Integration Targeting Mechanisms of Murine Leukemia Virus (MLV) on a Local and Global Scale

by
Sriram Aiyer
M. Sc., University of Madras - 2007

Thesis Advisor: Monica Roth, Ph.D.
Graduate Program in Biochemistry

RWJMS Research Tower
Room V-14
Piscataway

Friday, April 24, 2015
3:30 p.m.


Abstract

The murine leukemia virus (MLV) integrase1-408 (IN) protein has three domains - the N terminal domain, the catalytic core domain (CCD) and the C-terminal domain (CTD). Of the three domains, the CTD is the least conserved amongst all retroviruses. We have successfully expressed and purified the CTD329-408 of MLV IN and determined the NMR solution structure of this domain. We have also generated an MLV IN CCD structural homology model using the SWISS-MODEL, MMM-tree and I-TASSER servers.
Target-site selection by retroviral IN proteins profoundly affects viral pathogenesis in primarily two ways, namely, local sequence recognition and global targeting. In order to identify determinants of local sequence specificity, we made use of the crystal structures of the prototype foamy virus (PFV) IN that have shed light on the IN residues that are involved in binding target DNA (tDNA). Using the PFV IN target capture complex structure together with our MLV IN domain structures, we predicted residues within the CCD 2 helical region and the CTD 1-2 loop that bind tDNA. Viable viruses with substitutions at the IN CCD 2 helical region and the CTD 1-2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicated that the CCD 2 helical region, in particular MLV IN P187, interacts with the sequences distal to the scissile bonds whereas the CTD 1-2 loop binds to residues proximal to it.
On a global level, we report alterations to the MLV IN protein that successfully result in decreasing its integration frequency in genomic features such as transcription start sites (TSS) and CpG islands. Host BET proteins Brd2, 3 and 4 were identified to interact with MLV IN primarily through the BET protein ET domain. We have shown that the C-terminal tail peptide (TP) region within the CTD of MLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. Viruses bearing MLV IN C-terminal truncations can thus provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy.


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