Journal of Molecular Biology
Volume 274, Issue 5, 19 December 1997, Pages 693-707
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Regular article
Interactions between HIV rev and nuclear import and export factors: the rev nuclear localisation signal mediates specific binding to human importin-β1

https://doi.org/10.1006/jmbi.1997.1420Get rights and content

Abstract

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can “shuttle” between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-β. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-β did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-α/β heterodimer, the binding of Rev to importin-β was actually blocked by importin-α receptor. Our findings suggest that Rev and importin-α bind (via an arginine-rich sequence) to a similar region on importin-β. In addition, we show that the complex between Rev and importin-β can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.

Introduction

Human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for viral replication Feinberg et al 1986, Sodroski et al 1986. Rev functions to export unspliced or singly spliced HIV precursor mRNAs from the nucleus, thereby facilitating synthesis of the Env and Gag/Pol structural proteins Feinberg et al 1986, Malim et al 1989a, Felber et al 1989. Rev binds directly to the HIV pre-mRNA in vitro Daly et al 1989, Zapp and Green 1989, Heaphy et al 1990, Malim et al 1989a, targetting a 351 nucleotide HIV env gene sequence rich in secondary structure, termed the Rev-response element (RRE:Rosen et al 1988, Malim et al 1989a). Rev has an inherent tendency to oligomerise, and the high-affinity interaction with a binding site at the apex of the central RRE stem acts as a nucleating event that directs the sequential binding of Rev molecules along the RRE central stem Heaphy et al 1991, Olsen et al 1990, Malim and Cullen 1991, Wingfield et al 1991, Zapp et al 1991, Mann et al 1994, Zemmel et al 1996. Several lines of evidence indicate that formation of this multimeric Rev-RRE complex is required in vivo for the export of RRE-containing pre-mRNA Malim and Cullen 1991, Zapp et al 1991, McDonald et al 1992, Mann et al 1994.

The Rev protein itself can shuttle between the nucleus and cytoplasm Meyer and Malim 1994, Kalland et al 1994, Richard et al 1994, Wolff et al 1995. This bi-directional nuclear transit requires two signal sequences: a nuclear localisation signal (NLS) that overlaps the RNA-binding domain Kubota et al 1989, Cochrane et al 1990, Venkatesh et al 1990, Bohnlein et al 1991, and an export signal (NES) that enables Rev to shuttle Meyer and Malim 1994, Meyer et al 1996, Stauber et al 1995, Wolff et al 1995. The Rev NLS is an arginine-rich sequence (35-RQARRNRRRRWRER QRQ-51) and confers nuclear entry when fused as a peptide to β-galactosidase Cochrane et al 1990, Kubota et al 1989, Venkatesh et al 1990. Single and double amino acid changes in the NLS can prevent both nuclear import (Malim et al., 1989b), and RNA binding (Hammerschmid et al., 1994). Rev is often localised to the nucleolus (Cochrane et al., 1990), and this may reflect the ability of Rev to bind the nucleolar shuttling protein, B23 (Szebeni et al., 1997). Although the B23 protein was found to stimulate import of Rev, it appears to be neither necessary nor sufficient for Rev import (Szebeni et al., 1997). The primary nuclear import pathway of Rev therefore remains to be defined, but it is expected to resemble that of other NLS-containing proteins, and to involve a direct interaction between the Rev NLS and the nuclear import receptor, importin-α (Görlich & Mattaj, 1996).

The Rev NES is a specific and transferable signal sequence, and when a minimal NES peptide 73-LQLPPLERLTLD-84 (Wen et al., 1995) or 75-LPPLERLTL-83 (Fischer et al., 1995) is linked to heterologous proteins it confers rapid nuclear export Fischer et al 1995, Wen et al 1995, Meyer et al 1996. Mutations in any of the three hydrophobic leucine residues of the NES prevent export of both Rev and its RRE-containing pre-mRNA cargo Malim et al 1989b, Meyer and Malim 1994, Wolff et al 1995. Recently, a search for human cellular co-factors that bind the NES and facilitate Rev export has identified two candidates. The eIF-5A translation initiation factor was identified by cross-linking to a Rev NES peptide Ruhl et al 1993, Bevec et al 1996, and human RIP/Rab is a nuclear protein with a nucleoporin-like XXFG repeat region that was detected by the yeast two-hybrid assay Bogerd et al 1995, Fritz et al 1995. Rev also interacts with the FG dipeptide repeat regions of different mammalian and yeast nucleoporin proteins when examined by the yeast two-hybrid system Stutz et al 1995, Stutz et al 1996, Fritz and Green 1996. The hRIP/Rab and eIF-5A proteins were proposed to bind directly to the Rev NES Bogerd et al 1995, Bevec et al 1996, however no systematic study has yet confirmed their NES-dependent binding to Rev, or more importantly in the context of RNA export, to the multimeric Rev-RRE complex.

The aim of this study was to test for sequence-specific interactions between Rev and candidate nuclear import and export factors, in order to begin defining the pathways by which Rev shuttles between the nucleus and cytoplasm. Well-defined mutations in the Rev transport signals were used to control for the specificity of binding. We first established, using a new panel of antibodies, that the Rev NES is accessible for protein interaction even when Rev is bound in multiple copies on an RRE substrate. However, unlike previous reports Bogerd et al 1995, Bevec et al 1996, we detected no direct NES-dependent interaction between Rev and the candidate export co-factors, eIF-5A and hRIP/Rab. By contrast, the Rev NLS is shown to mediate a direct, highly specific and regulated interaction with the nuclear import receptor, importin-β. The Rev NLS thus differs from other NLS sequences, which bind importin-β only indirectly as a complex with importin-α. We present evidence that Rev binds to a site on importin-β that overlaps the binding site of importin-α, and show that Ran-GTP can dissociate the Rev-importin-β complex. Our findings reveal that the NLS is made accessible to bind importin-β only after Rev disengages from the RNA, and provide new insight into the mechanism by which Rev is imported and released into the nucleus.

Section snippets

When Rev binds the RRE, the Rev NLS becomes masked while the NES remains accessible

The aim of this study was to identify proteins that interact with the previously characterised NLS and NES of HIV-1 Rev (sequences shown inFigure 1). Toward this goal, we first characterised several new monoclonal antibodies raised against purified Rev, and mapped the epitopes to which they bind, using a series of overlapping Rev peptides. Of the three antibodies described here, one binds a region that closely overlaps the Rev NLS (antibody 2) and another binds to the NES (antibody 4), as

Rev contains a specific nuclear export signal

The identification of nuclear export signals, first in Rev and then in different cellular proteins, has revealed that certain proteins are both imported and exported from the nucleus by energy-dependent pathways. The Rev export signal comprises a series of closely spaced hydrophobic leucine residues, which appear to be critical as their mutation can block Rev export and inactivate Rev function Malim et al 1989b, Meyer and Malim 1994, Wolff et al 1995. The Rev NES, when transferred as a peptide

Antibody characterisation

Several new monoclonal antibodies (all type IgG1) were raised against purified Rev protein and affinity purified on Protein A-Sepharose columns, and kindly supplied by Tony Lowe and Jonathan Karn (MRC Centre, Cambridge). Epitopes were mapped by immunoblotting or by competition gel mobility shift assays, using a set of 11 overlapping peptides (each 20 amino acid residues long) that span the entire coding region of Rev (obtained from the MRC Aids Directed Programme Reagents Project). Antibodies

Acknowledgements

This study would not have been possible without the generous supply of lab space, materials and support made freely available by Dr Jonathan Karn (to B. R. H.) and Dr Daniela Rhodes (to P. P.) at the MRC Laboratory of Molecular Biology in Cambridge. We thank Drs Jonathan Karn, Daniela Rhodes, Michael Gait, Rodney Zemmel and Murray Stewart for helpful discussions throughout this project, and members of the Karn/Rhodes labs for encouragement and interest in this project. Our sincere thanks to

References (69)

  • M.H. Malim et al.

    HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RREimplications for HIV-1 latency

    Cell

    (1991)
  • M.H. Malim et al.

    Functional dissection of the HIV-1 Rev trans-activatorderivation of a trans-dominant repressor of Rev function

    Cell

    (1989)
  • D.A. Mann et al.

    A molecular rheostatco-operative Rev binding to Stem I of the Rev-response element modulates human immunodeficiency virus type-1 late gene expression

    J. Mol. Biol.

    (1994)
  • F. Melchior et al.

    Mechanisms of nuclear protein import

    Curr. Opin. Cell Biol.

    (1995)
  • S.G. Nadler et al.

    Differential expression and sequence-specific interaction of karyopherin α with nuclear localization sequences

    J. Biol. Chem.

    (1997)
  • P. Percipalle et al.

    Molecular interactions between the importin α/β heterodimer and proteins involved in vertebrate nuclear protein import

    J. Mol. Biol.

    (1997)
  • V.W. Pollard et al.

    A novel receptor-mediated nuclear protein import pathway

    Cell

    (1996)
  • M.G. Prieve et al.

    The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp-1-nuclear localization sequence receptor proteins

    J. Biol. Chem.

    (1996)
  • M. Rexach et al.

    Protein import into nucleiassociation and dissociation reactions involving transport, substrate, transport factors, and nucleoporins

    Cell

    (1995)
  • N. Richard et al.

    HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm

    Virology

    (1994)
  • K. Rittner et al.

    The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription

    J. Mol. Biol.

    (1995)
  • M.P. Rout et al.

    A distinct nuclear import pathway used by ribosomal proteins

    Cell

    (1997)
  • X.-P. Shi et al.

    The subcellular distribution of eukaryotic translation initiation factor eIF-5A, in cultured cells

    Exp. Cell Res.

    (1996)
  • Z. Smit-McBride et al.

    Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

    J. Biol. Chem.

    (1989)
  • R. Stauber et al.

    Analysis of trafficking of Rev and transdominant Rev proteins in living cells using Green Fluorescent Protein fusionstransdominant Rev blocks the export of Rev from the nucleus to the cytoplasm

    Virology

    (1995)
  • F. Stutz et al.

    Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast

    Cell

    (1995)
  • L.K. Venkatesh et al.

    Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization

    Virology

    (1990)
  • W. Wen et al.

    Identification of a signal for rapid export of proteins from the nucleus

    Cell

    (1995)
  • B. Wolff et al.

    Nucleocytoplasmic transport of the rev protein of human immunodeficiency virus type 2 is dependent on the activation domain of the protein

    Exp. Cell Res.

    (1995)
  • R.W. Zemmel et al.

    Flexible regions of RNA structure facilitate co-operative Rev assembly on the Rev-response element

    J. Mol. Biol.

    (1996)
  • J.D. Aitchison et al.

    Kap104pa karyopherin involved in the nuclear transport of messenger RNA binding proteins

    Science

    (1996)
  • D. Bevec et al.

    Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A

    Science

    (1996)
  • E. Böhnlein et al.

    Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domainnew insights into the domain structure of Rev and Rex

    J. Virol.

    (1991)
  • A.W. Cochrane et al.

    Identification of sequences important in the nucleolar localization of human immunodeficiency virus revrelevance of nucleolar localization to function

    J. Virol.

    (1990)
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