Nullbasic, a Potent Anti-HIV Tat Mutant, Induces CRM1-Dependent Disruption of HIV Rev Trafficking

Induces CRM1-Dependent Disruption of HIV Rev Trafficking. PLoS ONE 7(12): e51466. doi:10.1371/journal.pone.0051466 Nullbasic, a Potent Anti-HIV Tat Mutant, Induces CRM1- Dependent Disruption of HIV Rev Trafficking Min-Hsuan Lin 0 Haran Sivakumaran 0 Ann Apolloni 0 Ting Wei 0 David A. Jans 0 David Harrich 0 Luis Menendez-Arias, Centro de Biologa Molecular Severo Ochoa (CSIC-UAM), Spain 0 1 Queensland Institute of Medical Research, Molecular Virology Laboratory , Herston, Brisbane , Australia , 2 School of Chemistry and Molecular Biosciences, University of Queensland , St. Lucia, Queensland , Australia , 3 Department of Biochemistry and Molecular Biology, Monash University , Clayton, Victoria , Australia Nullbasic, a mutant of the HIV-1 Tat protein, has anti-HIV-1 activity through mechanisms that include inhibition of Rev function and redistribution of the HIV-1 Rev protein from the nucleolus to the nucleoplasm and cytoplasm. Here we investigate the mechanism of this effect for the first time, establishing that redistribution of Rev by Nullbasic is not due to direct interaction between the two proteins. Rather, Nullbasic affects subcellular localization of cellular proteins that regulate Rev trafficking. In particular, Nullbasic induced redistribution of exportin 1 (CRM1), nucleophosmin (B23) and nucleolin (C23) from the nucleolus to the nucleus when Rev was coexpressed, but never in its absence. Inhibition of the Rev:CRM1 interaction by leptomycin B or a non-interacting RevM10 mutant completely blocked redistribution of Rev by Nullbasic. Finally, Nullbasic did not inhibit importin b- or transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of Rev was due to increased export by CRM1. Overall, our data support the conclusion that CRM1dependent subcellular redistribution of Rev from the nucleolus by Nullbasic is not through general perturbation of either nuclear import or export. Rather, Nullbasic appears to interact with and disrupt specific components of a Rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation. - Funding: This work in the DH Laboratory was sponsored by the National Health and Medical Research Council (NHMRC) Project grant and the Australian Centre for HIV and Hepatitis Research Centre (ACH2). DH is funded by the Australian Research Council Future Fellowship. MHL is sponsored by a University of Queensland Research Scholarship and a University of Queensland International Research Tuition Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Both the Human immunodeficiency virus type-1 (HIV-1) Tat and Rev proteins are encoded by two exons arranged in alternative reading frames on fully spliced viral mRNA [1]. Tat and Rev are similar in size; Tat is typically 101 amino acids long and Rev is typically 116 amino acids long, and both have RNA binding domains composed of arginine and, in the case of Tat, lysine residues which bind to different HIV-1 RNA stem loop structures. Tat binds to an RNA structure in the 59 untranslated region (UTR) of all viral transcripts called the Trans-Activation Response element (TAR), while Rev binds to an intronic region retained by incompletely spliced transcripts called the Rev Response Element (RRE). The RNA binding domains of both proteins also function as a nuclear/nucleolar localization signal (NLS/NoLS), although recent evidence implies that Tat may passively enter the nucleus by diffusing through nuclear pores [2]. Both proteins are localized primarily in the nucleus; Tat is observed throughout the nucleoplasm with nucleolar accumulation, whereas the nucleocytoplasmic shuttling Rev concentrates in the nucleolus in addition to localizing to the nucleoplasm and, to a lesser extent, to the cytoplasm. Trafficking of Rev in cells has been studied extensively (Fig. 1) [3,4]. In the nucleolus, Rev promotes the nuclear export of various HIV-1 mRNAs by directly binding to singly-spliced and unspliced viral transcripts via the RRE contained therein (Fig. 1, step 1). Exportin 1 (also called CRM1 and XPO1) binds to Rev through a nuclear export signal (NES; HIV-1NL4-3 Rev amino acids 73 to 84, LQLPPLERLTLD) [5,6,7], which leads to colocalization of Rev and CRM1 in the nucleolus and subsequent export of the Rev:mRNA complex from the nucleus to the cytoplasm (Fig. 1, step 2). Many other cellular proteins can contribute to Rev nuclear export, including hRIP/Rab, eIF5A, DDX3, DDX1, RNA helicase A, and PIMT that act through Rev, and Matrin 3 and Sam68 that bind to viral mRNA [3,8,9,10,11,12]. The Rev:mRNA complex disassembles in the cytoplasm (Fig. 1, step 3) allowing Rev to recycle back to the nucleus using the transportin 1 or importin b nuclear import pathways (Fig. 1, step 4) [3]. Once Rev enters the nucleus, nucleophosmin (...truncated)