A Conserved Calcineurin-binding Motif in Human T Lymphotropic Virus Type 1 p12I Functions to Modulate Nuclear Factor of Activated T Cell Activation

doi:10.1074/jbc.M210210200

A Conserved Calcineurin-binding Motif in Human T Lymphotropic Virus Type 1 p12^I Functions to Modulate Nuclear Factor of Activated T Cell Activation*

Abstract

The PXIXIT calcineurin binding motif or highly related sequences are found in a variety of calcineurin-binding proteins in yeast, mammalian cells, and viruses. The accessory protein p12^I encoded in the HTLV-1 pX ORF I promotes T cell activation during the early stages of HTLV-1 infection by activating nuclear factor of activated T cells (NFAT) through calcium release from the endoplasmic reticulum. We identified in p12^I, a conserved motif, which is highly homologous with the PXIXIT calcineurin-binding motif of NFAT. Both immunoprecipitation and calmodulin agarose bead pull-down assays indicated that wild type p12^I and mutants of p12^I that contained the motif-bound calcineurin. In addition, an alanine substitution p12^I mutant (p12^I AXAXAA) had greatly reduced binding affinity for calcineurin. We then tested whether p12^I binding to calcineurin affected NFAT activity. p12^I competed with NFAT for calcineurin binding in calmodulin bead pull-down experiments. Furthermore, the p12^I AXAXAA mutant enhanced NFAT nuclear translocation compared with wild type p12^I and increased NFAT transcriptional activity 2-fold greater than wild type p12^I. Similar to NFAT, endogenous calcineurin phosphatase activity was increased in Jurkat T cells expressing p12^Iindependent of its calcineurin binding property. Thus, the reduced binding of p12^I to calcineurin allows enhanced nuclear translocation and transcription mediated by NFAT. Herein, we are the first to identify a retroviral protein that binds calcineurin. Our data suggest that HTLV-1 p12^I modulates NFAT activation to promote early virus infection of T lymphocytes, providing a novel mechanism for retrovirus-mediated cell activation.

Footnotes

↵* This work was supported by National Institutes of Health Grants RR-14324, AI-01474, and CA-92009 (to M. D. L.) and NCI, National Institutes of Health, Grant CA-70529 (to the Ohio State University Comprehensive Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Howard Hughes Medical Institute, Molecular Pathogenesis Program, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.
↵** To whom correspondence should be addressed: Center for Retrovirus Research and Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Tel.: 614-292-4489; Fax: 614-292-6473; E-mail: Lairmore.1@osu.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M210210200
↵2 W. Ding, S.-J. Kim, A. M. Nair, B. Michael, K. Boris-Lawrie, A. Tripp, G. Feuer, and M. D. Lairmore, submitted for publication.
Abbreviations:

HTLV-1

human T lymphotropic virus type 1

ORF

open reading frame

NFAT

nuclear factor of activated T cells

GFP

green fluorescent protein

STLV-1

simian T cell leukemia virus type 1

HA

hemagglutinin

BAPTA-AM

glycineN,N′-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl-, bis(ace-tyloxy)methyl ester
- Received October 4, 2002.
- Revision received February 12, 2003.
The American Society for Biochemistry and Molecular Biology, Inc.