Multiple Importins Function as Nuclear Transport Receptors for the Rev Protein of Human Immunodeficiency Virus Type 1

Abstract

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin ␤, but not on the adapter protein importin ␣. We now show that, similar to importin ␣, Rev is able to dissociate RanGTP from recycling importin ␤, a reaction that leads to the formation of a novel import complex. Besides importin ␤, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin ␤-binding domain of importin ␣, Rev interacts with an N-terminal fragment of importin ␤. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin ␤ and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.The machinery for transport of macromolecules across the nuclear envelope consists of the nuclear pore complex, which is embedded between the inner and outer nuclear membrane (1), and a large variety of soluble transport factors (for reviews see Refs. 2-4). The transport cargoes are characterized by recognition sequences for soluble transport receptors: nuclear export sequences mediate the interaction of proteins with exportins, allowing transport out of the nucleus, whereas nuclear localization signals (NLSs) 2 bind to importins, promoting transport of proteins into the nucleus. Importins and exportins, collectively also referred to as karyopherins, belong to the importin ␤ superfamily of transport receptors. They interact not only with their transport cargo but also with proteins of the nuclear pore complex, as well as with the small GTP-binding protein Ran, a factor that plays an important role in the assembly or disassembly of transport complexes (3). Importin ␤, the prototype of this family, was originally identified as the transport receptor that is involved in nuclear import of proteins containing a "classic" NLS, i.e. a short stretch of amino acids enriched in basic residues, forming either a single or a bipartite transport motif. These NLSs do not bind to importin ␤ directly but via an adapter protein, importin ␣. Importin ␣ contains a characteristic importin ␤-binding-(IBB) domain. A ternary complex containing the import cargo, importin ␣, and importin ␤ assembles in the cytoplasm, translocates across the nuclear pore complex, and dissociates in the nucleus upon binding of RanGTP to importin ␤. The import receptor then recycles back to the cytoplasm in a complex with RanGTP. A dedicated exportin, CAS, is used for export of importin ␣ out of the nucleus (5). Hundreds of proteins...

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1

“…Indeed, even though it is assumed that each epitope can accommodate substantial sequence diversity, the limits are still vague. The fact that some characterized Trn1 cargos lack a PY-NLS suggests that the actual variability is larger than currently conceived (12)(13)(14)(15). We believe that more bimodular NLSs, such as in ADAR1, will be found in the future and that our study will help identifying them.…”

“…These principles can be described as follows: a peptide segment of 15-30 residues with intrinsic structural disorder, an overall basic character, and some weakly conserved sequence motifs, including a relatively conserved proline-tyrosine (PY) dipeptide, leading to the name PY-NLS for this class of signals (8). However, many characterized Trn1 cargoes do not contain such a PY-NLS (4,(12)(13)(14)(15). Therefore, Trn1 interacts not only with the well-characterized PY-NLSs, but at least with another class of NLS, the recognition of which is far less understood.…”

The nuclear pore component Nup358 promotes transportin-dependent nuclear import

“…Recently, we reported that importin-β, importin-5, importin-7 and transportin function as import receptors for HIV-1 Rev in vitro (Arnold et al, 2006a). In the previous study, we used an excess of import receptors for the import of GST-tagged Rev.…”

“…The NLS of HIV-1 Rev is enriched in the basic amino acid arginine and can interact directly with importin-β (Henderson and Percipalle, 1997;Truant and Cullen, 1999). Recently, we demonstrated that additional import receptors, namely importin-5, importin-7 and transportin, bind to the same NLS and mediate the import of HIV-1 Rev in vitro (Arnold et al, 2006a). The identity of the endogenous Rev import receptor, however, remains to be elucidated.…”

Repression of classical nuclear export by S-nitrosylation of CRM1

“…We employed a live-cell-based nuclear export system by evaluating the nuclear export capability of Rev, a nucleocytoplasmic shuttling HIV protein with both a basic aminoacid-rich NLS recognized by multiple importins (Arnold et al, 2006) and a classical NES recognized by CRM1 (Askjaer et al, 1998). We used expression vectors derived from Rev-GFP in which the endogenous Rev NES was mutationally inactivated (Henderson and Eleftheriou, 2000).…”