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

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.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

“…As expected, the localization of MYC-Rev expressed alone was predominantly nucleolar of Nullbasic-mCherry and MYC-Rev had no apparent effect on DDX3 and DDX17 subcellular distribution [see Additional file 3]. As previous observed [35,37], coexpression of Nullbasic-mCherry with MYC -Rev induced Rev mislocalization from the nucleolus to the nucleoplasm and cytoplasm ( Figure 4A, row 4). These data thus show that Nullbasic associates with DDX1 leading to the alteration of DDX1 subcellular distribution.…”

“…In this study, we focused on nuclear Nullbasic interactors as our previous studies indicated that Nullbasic disrupted the nucleolar colocalization of HIV-1 Rev with CRM1, B23 and C23 but did not affect importin β-and transportin 1-mediated Rev nuclear import pathways [37], suggesting that Nullbasic interferes with specific components of a Rev nuclear complex. The experimental strategy used to identify protein complexes associated with Nullbasic is illustrated in Figure 1A.…”

“…cell from the nucleolus to the nucleoplasm and cytoplasm but also certain cellular proteins, including CRM1, B23 and C23, in a Rev-dependent manner [37]. To investigate whether alteration of Rev subcellular localization induced by Nullbasic also affects the subcellular distributions of DDX1, we visualized its subcellular localization in HeLa cells using fluorescence microscopy.…”

“…Rev is a 116 amino acid protein and can be divided into three discrete function domains. The Rev RNA-binding domain (RBD, amino acids [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] is an arginine-rich motif that serves as the nucleolar localization signal (NLS), which can be recognized by the cellular importin β-like import receptors and nucleophosmin (NPM), also known as B23 [2][3][4][5]. This region also specifically interacts with a stem loop RNA sequence called Rev response element (RRE), which is located within the env gene among unspliced and singly spliced HIV-1 mRNAs [6,7].…”

“…It inhibits Tat-mediated transactivation activity, suppresses HIV-1 reverse transcription and reduces steady state levels of unspliced and singlyspliced viral mRNA by the inhibition of Rev activity [35,36]. With respect to Rev, Nullbasic also alters its subcellular localization as well as the subcellular localizations of cellular proteins including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic interferes with a component of the Rev nucleocytoplasmic transport complex required for Rev trafficking and function [35,37]. Here we employed a proteomics approach to identify cellular proteins that interact with Nullbasic.…”