Purification of the pets Factor

• Published in: The Journal of biological chemistry Vol. 271; no. 32; pp. 19599 - 19605
• Main Authors: Fu, Glenn K., Markovitz, David M.
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 09.08.1996
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.32.19599

The peri-ets (pets) site is a TG-rich element found immediately adjacent to two binding sites for the ets family member Elf-1 in the human immunodeficiency virus type 2 (HIV-2) enhancer. Enhancer activation in response to T cell stimulation by phorbol myristate acetate, phytohemagglutinin, soluble or cross-linked antibodies to the T cell receptor, or antigen is mediated through this site in conjunction with its two adjacent Elf-1 binding sites, PuB1 and PuB2, and a κB site. Site-specific mutation of the pets element significantly reduces inducible activation of this enhancer but does not affect its transactivation by HIV-2 tat or other viral transactivators. Similar TG-rich sequences adjacent to ets-binding sites have also been found to be functionally important in the human T-cell leukemia virus type I and murine Moloney leukemia virus enhancers. As the cellular factor binding to the pets site plays a significant role in regulating the HIV-2 enhancer in both T cells and monocytes, we have purified this protein from bovine spleens and demonstrate that it is 43 kDa in size. In addition, using glycerol gradient centrifugation, Southwestern blotting, electrophoretic mobility shift assays employing purified protein eluted from a gel, and a new in solution UV cross-linking competitive assay, we show that the dominant protein binding to the pets site is 43 kDa in size. These results indicate that a nuclear protein of 43 kDa binds specifically to the pets site of the HIV-2 enhancer and may mediate transcriptional activation of this important human pathogen in response to T cell stimulation. As retroviruses generally expropriate important human regulatory proteins for their own use, the 43-kDa pets factor is also likely to play a significant role in signal transduction in T cells and in other cellular processes.