Abstract
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 β- 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 CRM1-dependent 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.
Highlights
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)
Summary
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]. The RNA binding domains of both proteins function as a nuclear/nucleolar localization signal (NLS/NoLS), 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. 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)
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