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Chromatin Tethering and Nuclear Retention of the Retroviral Integration Machinery by the Murine Leukemia Virus P12 Protein

by
Jonathon Brzezinski
B.S., University of Wisconsin - Madison, 2009


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

Robert Wood Johnson Research Tower
Room V-14 - Basement
Piscataway

Friday, April 29, 2016
10:00 a.m.


Abstract

Murine leukemia virus is a great model to study basic retroviral mechanisms, encoding

only three genes, which are translated and cleaved into eight functional proteins. This dissertation focuses on the definition of new functions for the p12 protein, encoded within the gag structural gene of MLV. Functional complementation was used to rescue a p12 early mutant with alternative viral chromatin binding motifs, establishing that p12 performed a chromatin binding function for MLV. Confocal microscopy of p12-GFP fusion constructs was used to confirm the chromatin binding of the p12 protein constructs via fluorescence recover after photobleaching.

Using point mutants and N-terminal truncations of these p12-GFP fusion proteins, we discovered the presence of a motif that was responsible for repressing/regulating the chromatin binding motif of p12. Phosphorylations detected in p12 at the chromatin binding motif and its repression motif were dispensable for viral fitness and chromatin binding, after viral generation of secondary suppressor mutants activating constitutive chromatin binding. This suggested phosphorylation was used for regulation of wild type p12 chromatin binding. Overall, the residues and post-translational modifications critical for p12 chromatin binding and its regulation were defined in this study.

Lastly, analysis of p12 chimeric viral constructs found that p12 chromatin binding does not select the integration site of MLV. We also found that p12 binding too tightly to the nuclear chromatin inhibited integration of the viral DNA. Altogether, these studies helped to elucidate MLV p12ís role in tethering the viral preintegration complex to mitotic chromatin, facilitating its nuclear retention after mitosis, and then releasing the chromatin to allow for viral integration.


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