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Journal of Virology, September 2009, p. 8859-8868, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00908-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Site-Specific Phosphorylation Regulates Human T-Cell Leukemia Virus Type 2 Rex Function In Vivo {triangledown}

Matthew Kesic,1,2 Michael Ward,5 O. John Semmes,5 and Patrick L. Green1,2,3,4*

Center for Retrovirus Research,1 Departments of Veterinary Biosciences,2 Molecular Virology, Immunology, and Medical Genetics,3 Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, Ohio 43210,4 Department of Microbiology and Molecular Cell Biology and Center for Biomedical Proteomics, Eastern Virginia Medical School, Norfolk, Virginia 235075

Received 6 May 2009/ Accepted 12 June 2009

Human T-cell leukemia virus type 2 (HTLV-2) Rex is a transacting regulatory protein required for efficient cytoplasmic expression of the unspliced and incompletely spliced viral mRNA transcripts encoding the structural and enzymatic proteins. Previously, it was demonstrated that phosphorylation of Rex-2, predominantly on serine residues, is correlated with an altered conformation, as observed by a gel mobility shift and the detection of two related protein species (p24Rex and p26Rex). Rex-2 phosphorylation is required for specific binding to its viral-mRNA target sequence and inhibition of mRNA splicing and may be linked to subcellular compartmentalization. Thus, the phosphorylation-induced structural state of Rex in the infected cell may be a switch that determines whether HTLV exists in a latent or productive state. We conducted a phosphoryl and functional mapping of both structural forms of mammalian-cell-expressed Rex 2 using affinity purification, liquid chromatography-tandem mass spectrometry, and site-directed substitutional mutational analysis. We identified two phosphorylation sites in p24Rex at Ser-117 and Thr-164. We also identified six phosphorylation sites in p26Rex at Thr-19, Ser-117, Ser-125, Ser-151, Ser-153, and Thr-164. We evaluated the functional significance of these phosphorylation events and found that phosphorylation on Thr-164, Ser-151, and Ser-153 is critical for Rex-2 function in vivo and that phosphorylation of Ser-151 is correlated with nuclear/nucleolar subcellular localization. Overall, this work is the first to completely map the phosphorylation sites in Rex-2 and provides important insight into the phosphorylation continuum that tightly regulates Rex-2 structure, cellular localization, and function.

* Corresponding author. Mailing address: The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210. Phone: (614) 688-4899. Fax: (614) 292-6473. E-mail: green.466{at}osu.edu

{triangledown} Published ahead of print on 24 June 2009.

Journal of Virology, September 2009, p. 8859-8868, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00908-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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