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Identification and characterization of the post-translational modifications of the HTLV types 1 and 2 regulatory protein Rex

Kesic, Matthew John
http://rave.ohiolink.edu/etdc/view?acc_num=osu1241104210

Abstract Details

2009, Doctor of Philosophy, Ohio State University, Molecular, Cellular, and Developmental Biology.
Human T-cell Leukemia Virus (HTLV) types 1-4 are classified as complex retroviruses and are members of the genus Deltaretrovirus. HTLV-1 and HTLV-2 are the most prevalent worldwide with approximately 10-20 million people infected, whereas HTLV-3 and HTLV-4 were discovered recently in a very limited number of individuals in Africa. Of the HTLV isolates, only HTLV-1 has clearly been linked to the development of adult T-cell leukemia/lymphoma (ATL/ATLL), an aggressive CD4+ T-lymphocyte malignancy and the neurodegenerative disease, HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Disease association with HTLV-2 is less clear, with only a few cases of leukemia and neurological diseases reported. The difference in pathology between the two related viruses has yet to be elucidated, but likely results from the activities of the regulatory and accessory proteins. We report molecular studies regarding the role phosphorylation plays in Rex function. In Chapter 2, we provide further characterization of the Rex-2 carboxy terminal phosphorylation domain (CTPD). Our studies identified the ability of the CTPD to regulate Rex-2 function and its importance for HTLV-2-mediated cellular proliferation and immortalization in vitro and its contributions to virus infection and persistence in a rabbit model of infection. Introduction of a phosphomimetic amino acid (negative charge) into the Rex-2 c-terminus or deletion of certain c-terminal amino acids can maintain Rex-2 in a highly functional state. Studies also found that this domain plays an important role in Rex-2 protein stability. Thus, our data provides evidence that the Rex-2 c-terminus contains a functional inhibitory domain that is regulated via phosphorylation. Cells harboring HTLV Rex-2 CPTD mutant proviruses display enhanced infection in vitro and increased virion production. Furthermore, these mutants promoted HTLV-2- induced proliferation of human primary T-cells and displayed increased replication in inoculated rabbits as measured by significantly stronger antibody responses as compared to wtHTLV-2 infected animals. Overall, our study demonstrated the importance of Rex-2 post-translational modifications and their role in regulating protein stability and function. Chapters 3 and 4 focus on identifying phosphorylation sites within Rex-1 and Rex-2 in vivo. We conducted a phosphoryl mapping of mammalian-expressed Rex-1 and Rex-2 proteins using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved 100% physical coverage of both Rex-1 and Rex-2 sequences. In Chapter 3, we report the identification of six novel phosphorylation events in Rex-2 at Thr-19, Ser-117, Ser-125, Ser-151, Ser-153, and Thr-164. We evaluated the functional significance of these novel phosphorylation sites and found that only phosphorylation within the CPTD at Ser-151, Ser-153, and Thr-164 are critical for Rex-2 function in vivo. Indirect immunofluorescence revealed that Ser-151 is the only site important for proper subcellular localization of Rex-2. Chapter 4 reports the identification of five novel phosphorylation sites within Rex-1 at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed two previously identified residues Ser-70 and Thr-174. The functional significance of these phosphorylation sites was evaluated. We determined that phosphorylation of Ser-97 and Thr-174 is critical for Rex-1 function, but do not play a role in subcellular localization of the Rex-1 protein.
Patrick Green, PhD (Advisor)
Michael Lairmore, DVM,PhD (Committee Member)
Mary Jo Burkhard, DVM,PhD (Committee Member)
Harold Fisk, PhD (Committee Member)
178 p.
Virology
Rex; HTLV; phosphorylation

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Citations

  • Kesic, M. J. (2009). Identification and characterization of the post-translational modifications of the HTLV types 1 and 2 regulatory protein Rex [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1241104210

    APA Style (7th edition)

  • Kesic, Matthew. Identification and characterization of the post-translational modifications of the HTLV types 1 and 2 regulatory protein Rex. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1241104210.

    MLA Style (8th edition)

  • Kesic, Matthew. "Identification and characterization of the post-translational modifications of the HTLV types 1 and 2 regulatory protein Rex." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1241104210

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.