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HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs Christian C. Fritz and Michael R. Green
Background: The structural proteins of human immunodeficiency virus type 1 Address: Howard Hughes Medical Institute, (HIV-1) are encoded by intron-containing mRNAs that normally are retained in Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation the nucleus. A viral regulatory protein, Rev, specifically induces the accumulation Street, Suite 309, Worcester, Massachusetts of these transcripts in the cytoplasm. Rev is an RNA-binding protein that also 01605, USA. contains an ‘effector’ domain. The Rev effector domain has recently been shown to function as an autonomous nuclear export signal (NES) that, when fused to a Correspondence: Michael R. Green E-mail: [email protected] foreign protein, will cause its rapid nuclear export. We and others have recently reported the cloning of a human protein (hRIP/Rab), that specifically interacts Received: 16 April 1996 with the effector domain of Rev. Revised: 16 May 1996 Accepted: 16 May 1996 Results: Here we show that the NESs contained within two cellular proteins, PKI Current Biology 1996, Vol 6 No 7:848–854 and IB, which are not involved in RNA metabolism, also interact with hRIP. Fusion of these cellular sequences to the Rev RNA-binding domain reconstitutes © Current Biology Ltd ISSN 0960-9822 a functional Rev protein. In addition to hRIP, these NESs also bind to several nuclear pore proteins located on the nucleoplasmic, membrane, and cytoplasmic side of the nuclear pore complex (NPC). We show that this protein export pathway is highly conserved by demonstrating that mammalian NESs also function in yeast.
Conclusions: Our results indicate that the HIV-1 Rev protein evolved to take advantage of a cellular protein export pathway in order to allow the nucleocytoplasmic transport of unspliced viral RNA. Our data suggest a model in which the export substrate is translocated through the NPC by sequential interactions with different nucleoporins. Finally, our experiments suggest a mechanism by which IB can downregulate nuclear NFB activity by causing its rapid export from the nucleus.
Background virus has evolved a mechanism that selectively allows In the eukaryotic cell, the nuclear genome is separated unspliced viral, but not cellular, mRNAs to accumulate in from the cytoplasm by a double membrane. Although this the cytoplasm. This pathway is mediated by the viral Rev arrangement allows the cell an additional level of regula- protein, an RNA-binding protein that binds specifically to tion, a steady stream of macromolecules must be trans- an RNA element present in all unspliced viral transcripts. ported into and out of the nucleus. In recent years, much Mutational analysis has identified a domain at the carboxyl has been learned about protein import into the nucleus. terminus of Rev — the ‘effector domain’ — which is dis- Proteins destined for the interior of the nucleus contain tinct from the RNA-binding domain and is essential for the short signal sequences, termed nuclear localization signals RNA-bound Rev protein to function (for review see [5,6]). (NLSs), which bind to a dimeric complex of cytoplasmic This effector domain has recently been identified as a receptor proteins (importins/karyopherins) that mediate nuclear export sequence (NES) which, in a manner analo- docking at and translocation through the nuclear pore gous to NLSs, induces rapid nuclear export when fused to a complex (NPC; for review see [1–3]). foreign protein [7]. The human cellular protein hRIP/Rab specifically interacts with the Rev effector domain, and has Much less is currently known about the equally important the properties expected of a Rev cellular co-factor [8,9]. process that exports RNAs and proteins from the nucleus [3,4]. An important model system for the study of mRNA Taylor and coworkers [10] have independently identified nuclear transport is the post-transcriptional regulation of an autonomous NES as part of the thermostable inhibitor human immunodeficiency virus type 1 (HIV-1) gene protein, PKI, of protein kinase A (PKA). Intriguingly, both expression. The structural proteins of this virus are the Rev NES and the PKI NES contain several leucine encoded by large, intron-containing transcripts, and the residues that have been shown to be essential for their Research Paper Nuclear export of HIV RNAs by conserved cellular pathway Fritz and Green 849
function. This raises the possibility that these two signals, Figure 1 which serve disparate functions, use the same nuclear export pathway. To address this question, we have tested (a) whether the PKI NES, like the Rev NES, can bind to ∆hRIP hRIP. Here, we report that the PKI NES can interact with hRIP hRIP and, like the Rev NES, also interacts with several subunits of the NPC. We also show, by domain-swap LexA experiments, that the PKI NES can functionally replace the Rev effector domain. These results suggest that the LexA–Rev effector domain of Rev evolved to mimic a cellular NES, allowing the virus to use a cellular protein export pathway LexA–Rex for the transport of unspliced viral transcripts.
Results LexA–PKI The PKI NES and a putative NES in IB interact with hRIP A short sequence within the cellular inhibitor of PKA has LexA–IκB been identified as an autonomous NES [10]; a related sequence is present at the carboxyl terminus of the IB␣ protein [10]. Although these sequences do not display a (b) high degree of identity with each other or with the Rev Export Binding to effector domain, all three sequences are rich in leucine hRIP residues and, in the case of Rev and PKI, it has been PKI wild-type NELALKLAGLDIKT + + shown that some of these leucines are essential for func- PKI P3 --A-A-A------tion (see Fig. 1c). These similarities raise the possibility PKI P12 ------A------that all three leucine-rich elements belong to the same class of NES and may interact with a similar set of cellular proteins mediating NES-directed export from the nucleus. (c) Rev (73): LQLPPLERLTLDCN To test the hypothesis that the leucine-rich sequences PKI (36): ELALKLAGLDINKT present in PKI and IB are NESs that use the same κ nuclear export pathway as HIV-1 Rev, we first asked I B (264): RIQQQLGQLTLENL whether these sequences interact with hRIP/Rab [8,9]. Rex (82): LSAQLYSSLSLDSP Using a yeast two-hybrid assay, we determined that both the PKI and the IB sequences interact with wild-type but not mutant hRIP (Fig. 1a). Also shown in Figure 1a is The PKI NES and the putative NES in IB interact specifically with the previously demonstrated interaction of hRIP with the hRIP. (a) Two-hybrid interactions between LexA–NES fusion proteins and hRIP derivatives tagged with a transcriptional activation domain. human T-cell leukemia virus type 1 (HTLV-I) Rex (b) Comparison between export activity and hRIP binding for PKI protein, which contains an effector domain functionally mutants, whose substituted residues are shown. The data for export equivalent to that of HIV Rev [11,12]. are from [10]. Binding to hRIP was determined in a two-hybrid assay. (c) Sequence alignment of leucine-rich NESs. Asterisks denote residues in which a single substitution abolishes NES function [10,13]. The PKI NES has been analyzed in detail by site-directed mutagenesis [10]. To test the significance of the inter- action between hRIP and the PKI NES, we analyzed function and hRIP binding (for example, Rev mutants several substitution mutants that lacked NES activity and M27, M28 and M29) [8,13]. Individual amino-terminal found a perfect correlation between NES activity and leucines are not essential, and their positions can vary in hRIP binding (Fig. 1b). Alignment of the Rev, PKI, IB relation to the core; however, deletion of all upstream and Rex sequences shows that the only feature in common leucine residues also results in a non-functional NES (for between these sequences is a set of characteristically example, Rev ⌬9/19, [13]). In the case of IkB, our align- spaced leucine or isoleucine residues (Fig. 1c). Using our ment shows that a single isoleucine seems to fulfil the binding data together with previously published functional function of these amino-terminal hydrophobic residues. data [10,13], it is possible to distinguish between an almost invariable core of the sequence LxxLxL (amino-acid single IB and PKI contain NESs that can functionally replace the letter code, where ‘x’ denotes any residue) and more irreg- Rev effector domain ularly spaced amino-terminal leucines (dotted squares in We next asked whether the leucine-rich sequences present Fig. 1c). Core leucine residues are characterized by the fact in PKI and IB could functionally substitute for the Rev that single point mutations at these positions abolish NES effector domain. Hybrid proteins were constructed, fusing 850 Current Biology 1996, Vol 6 No 7
the Rev RNA-binding domain (RBD) in-frame to amino Figure 2 acids 35–47 of PKI (RBD–PKI) and to amino acids 263–281 of IB␣ (RBD–IB). These fusion proteins were tested for Rev activity using a reporter construct that con- κB tains the chloramphenicol acetyl transferase (CAT) coding sequence and a Rev-binding site in an intron, allowing the Vector Rev wild-typeRev M10 RBD–PKI RBD–PKI P12RBD-I translation of CAT only from messages that reach the cyto- plasm unspliced [14]. Both fusion proteins were fully func- tional for Rev-mediated RNA export (Fig. 2, compare lanes 4 and 6 with lane 2), whereas a fusion protein con- taining the single point mutant PKI P12 (see Fig. 1b), which does not bind to hRIP, displayed no Rev function (Fig. 2, lane 5). These results not only show that IB con- tains a functional NES, but also that both cellular NESs can functionally replace the Rev effector domain for the export of pre-mRNA from the nucleus. 1234 56 Cellular and viral NESs interact with multiple components of the NPC PKI and IB contain NESs that can functionally substitute for the Rev The region of hRIP that interacts with the Rev NES con- effector domain. CV1 cells were transfected with 0.5 g of the CAT tains repeats of the two amino acids phenylalanine and reporter CM128 [14] and 1 g of the indicated Rev derivatives using glycine (FG repeats), which are similar to the repeat the calcium-phosphate technique. RBD-fusions: the PKI, the PKI regions of a class of nuclear pore proteins [15]. The FG mutant P12, or the IB NES were fused at amino acid 77 to the Rev RNA-binding domain (RBD). repeat region of hRIP is responsible for interaction with the Rev effector domain [8]. Stutz et al. [16] have shown that the Rev NES can interact with several nucleoporins, Rev and the yeast nucleoporin Nup159, as compared with although the positions of these nucleoporins within the that between and the other NESs, might explain why NPC are unknown. It is not clear, therefore, whether Rev works poorly in Saccharomyces cerevisiae [22]. there is a functional redundancy between FG repeat-con- taining proteins in nuclear export, or whether an NES To investigate the specificity of these interactions, we interacts sequentially with different nucleoporins while tested the interaction of these nucleoporins with a panel being transported through the NPC. of Rev effector-domain mutants, which have been charac- terized for Rev function [13]. As described previously for We analyzed the interaction of NESs with the repeat hRIP [8], there was a perfect correlation between Rev regions of several nucleoporins that have been localized function (wild-type, M20 and M25) and binding to the within the NPC by electron microscopy (see Fig. 5): the repeat regions of the nucleoporins tested (Fig. 3b). Rev mammalian proteins Nup153 and Nup98, which reside on mutants that lacked activity (M10, M22, and the two point the nucleoplasmic side of the NPC [17,18]; the rat protein mutants, M27 and M29, which eliminate single leucine Pom121, which is a pore membrane protein [19]; and two residues at positions 78 and 83 of the Rev NES, respec- proteins that are located at the cytoplasmic face of the tively; see Fig. 1c) did not interact with the repeat regions, NPC, yeast Nup159 and human Nup214 [20,21]. The FG not even with those of Nup153 and Nup214 (which inter- repeat regions of these proteins were fused to a transcrip- acted very strongly with functional Rev effector domains). tional activation domain, and transformed together with the indicated LexA fusion proteins into a yeast strain con- The PKI NES is also functional in yeast taining an integrated lacZ reporter gene. Interaction of Many cellular processes are conserved between yeast and the respective fusion proteins resulted in the develop- humans. Rev has been reported to function at a low level ment of blue dye on medium containing the colour indi- in yeast [22]; consistent with this study, we found that the cator X-Gal. This matrix of two-hybrid interactions Rev NES interacted very weakly with yeast Nup159 com- showed that all FG repeat-containing nucleoporins that pared with the other mammalian NESs tested (Fig. 3a). To we tested interacted with each of the NESs, albeit with ask whether the NES within PKI was functional in yeast, different intensities (Fig. 3a). Interestingly, most of the we constructed two fusion proteins with the LexA DNA- nucleoporins appeared to interact more strongly with binding domain and a transcriptional activation region (Fig. NESs than hRIP. Note that the yeast strain containing 4a): one fusion protein also contained the PKI NES; the the LexA–Rev and Nup159 fusion proteins turned blue second also contained the P12 mutant version of the PKI after prolonged incubation on medium containing X-Gal NES, which does not bind hRIP and has no NES activity (see also Fig. 3b). This very weak interaction between (see Figs 1b and 2). If the PKI NES were functional in Research Paper Nuclear export of HIV RNAs by conserved cellular pathway Fritz and Green 851
Figure 3 Figure 4
(a) (a)
hRIP Nup153 Nup98 Pom121Nup159 Nup214 Construct #1: LexA (1–202) PKI (35–47) AD
LexA Construct #2: LexA (1–202) PKI (35–47)P12 AD
LexA–Rev
LexA–Rex (b) 150 LexA–PKI 100 LexA–IκB -Gal activity
β 50
(b) 0 LexA #1 #2 hRIP Nup153 Nup98 Pom121Nup159 Nup214
The PKI NES is functional in S. cerevisiae. (a) Constructs used. AD; Rev wild - type (+) acidic transcriptional activation domain from the pseudorabies virus immediate early protein, residues 1–34 [38]. (b) Transcriptional Rev M10 (–) activation by LexA and LexA fusion proteins in S. cerevisiae. A yeast strain containing an integrated lexAop-lacZ construct was Rev M20 (+) transformed with plasmids encoding the indicated constructs and liquid -galactosidase (-Gal) assays were performed in triplicate. Rev M22 (–) -galactosidase activity is in nmoles minute–1 mg–1 protein; error bars represent the standard deviation. Rev M25 (+)
Rev M27 (–) Discussion Rev M29 (–) The recent identification of a cellular co-factor for HIV Rev, hRIP/Rab [8,9], raises the question as to its normal cellular function. Here, we report that the NESs of two cellular proteins, PKI and IB, also interact specifically NESs interact with multiple nucleoporins. (a) Two-hybrid interactions with hRIP and can functionally substitute for the Rev between LexA–NES fusion proteins and repeat regions of the indicated effector domain. These results imply that hRIP is part of a nucleoporins tagged with a transcriptional activation domain. (b) cellular pathway for protein export from the nucleus. Curi- Interaction of nucleoporin repeat regions with a panel of mutants in the Rev NES. Two-hybrid interactions between activation-domain-tagged ously, both natural substrates for hRIP identified so far nucleoporins and LexA–Rev fusions are shown. Signs in parentheses appear to play a negative regulatory role in signal transduc- indicate whether these mutants retain Rev function (from [13]). tion. Although the role of PKI has not been elucidated fully [23], this protein can enter the nucleus independently and can then inhibit the nuclear functions of the cAMP yeast, one would expect the fusion protein containing the dependent protein kinase (PKC) by two mechanisms: first, wild-type NES to be exported constantly to the cyto- binding by PKI inhibits the activity of the catalytic subunit plasm, whereas the other fusion protein would remain in (C) of the kinase [24]; and second, upon binding, the NES the nucleus. In the steady-state, this should result in a on PKI becomes exposed and triggers the rapid export of higher transcriptional activation of a reporter gene under the PKI–C complex from the nucleus [25]. LexA control by the fusion protein containing the mutant PKI NES. As shown in Figure 4b, we found these predic- A new function for IB tions to be the case; in fact, the fusion protein with the IB negatively regulates members of the Rel family of wild-type PKI NES was exported from the yeast nucleus transcription factors. Apart from its previously recognized so efficiently that we detected very little reporter-gene ability to mask the NLS of NFB, our results suggest that activation. IB might work similarly to PKI. IB can enter the 852 Current Biology 1996, Vol 6 No 7
Figure 5
A working model for protein export through α the nuclear pore. Export substrate is shown in pink, and import substrate in orange. ␣ and  hRIP refer to the subunits of the NLS receptor NES NLS (karyopherin/importin).
Nup153
Nup98
Pom121
Nucleus
Membrane Cytoplasm
β Nup214 α NES NLS
nucleus independently [26,27] and, by binding to NFB, Our results also explain the observation by Wu et al. [33] can dissociate NFB from its binding site on DNA [28]. By that overexpression of IB␣ inhibited the function of analogy to PKI, binding would then expose the leucine- HIV-1 Rev and HTLV-I Rex. These authors concluded rich region on IB, which we have shown is a functional that an essential Rev or Rex cofactor might be under NES. This would result in the net expulsion of the NFB control. Although this might still be the case, our IB–NFB complex from the nucleus, creating a classical data suggest that the inhibition of Rev and Rex results negative feedback loop: treatment of cells with inducer from competition for a common nuclear-export pathway. (tumour necrosis factor (TNF), phorbol ester (PMA), inter- leukin-1 (IL-1), ultraviolet light and so on) leads to nuclear A working model for nuclear export accumulation of NFB via rapid degradation of IB; IB Our experiments provide insight into how nuclear export synthesis is, in turn, strongly induced by nuclear NFB of proteins containing NESs might occur. We have found [29], and this newly synthesized IB accumulates in the NES interactions with nucleoporins that are located on nucleus [30] where it induces the rapid export of NFB to the nucleoplasmic side, the pore membrane domain, and the cytoplasm. This model is consistent with several obser- the cytoplasmic side of the NPC. Thus, the most vations in the literature. First, in the pre-B cell line 70Z/3, straightforward model is that a NES-containing protein the activation of NFB in response to PMA has been moves through the NPC by sequential interactions with shown to be only transient and inhibition of protein syn- more peripheral nucleoporins (Fig. 5). This is strikingly thesis leads to a superinduction [31]. Second, in HeLa cells similar to the model proposed for the nuclear import of briefly exposed to TNF and then maintained in its proteins containing NLSs [34,35]. In this model, the FG- absence, Arenzana-Seisdedos et al. [30] found a substantial repeat regions of several nucleoporins spaced throughout decrease in nuclear NFB content, which was again the NPC channel provide the stationary phase along dependent on new protein synthesis. Third, in fibroblasts which the NLS–karyopherin/importin complex is translo- from IB␣ knockout mice, the failure to clear NFB from cated from the cytoplasm to the nucleus. Intriguingly, an the nucleus after a short pulse of TNF directly proves the overlapping set of repeat-containing proteins, including involvement of IB␣ in this process [32]. Nup98, Nup153 and Nup214, appear to be involved in Research Paper Nuclear export of HIV RNAs by conserved cellular pathway Fritz and Green 853
the binding of both import [34] and export (this study) fusion was constructed by polymerase chain reaction (PCR) amplifica- substrates. In this context, the nucleoplasmic, NES- tion of the sequence encoding amino acids 263–281 of IB␣ [33], with oligonucleotides creating EcoRI (5′) and BamHI (3′) sites flanking binding hRIP is analogous to the cytoplasmic, NLS- the fragment. This fragment was inserted between the EcoRI and binding karyopherin␣/importin␣. However, whereas BamHI sites of pEG202 [36]. karyopherin␣/importin␣ is a true ‘adaptor’ that shuttles import substrates into the nucleus, hRIP does not appear For the construction of Rev wild-type and M10 eukaryotic expression vectors, EcoRI fragments obtained by PCR from pcRev and pcRevM10 to shuttle (our unpublished observations), and itself [13] were cloned into the EcoRI site of pcDNAI (Invitrogen). For the resembles the stationary phase by containing FG repeats. construction of RBD–NES fusion expression vectors, fragments encod- ing amino acids 35–47 of PKI␣ and PKI␣P12 or amino acids 263–281 For both protein import and RNA export, a major question of IB␣ were excised by EcoRI/Klenow/XhoI treatment from the LexA remains over the driving force that transports substrates vectors and inserted into BglII/Klenow/XhoI-treated pcDNAI-RevM10, creating fusions of the NES regions at amino acid 77 of Rev. through the 100 nm-long NPC. One attractive hypothesis is that the affinity of a substrate for the stationary phase For the analysis of nucleoporin repeat regions in the two-hybrid assay, gradually increases from one side of the NPC to the other. the following regions (amino acids) of the nucleoporin coding Although our data do not generally support such a simple sequences were amplified by PCR and cloned into pJG4-5 [36] using EcoRI (5′) and XhoI (3′) sites encoded by the oligonucleotides: Nup98, model, it is interesting to note that the nucleoporins on the 1–517; Nup153, 647–1468; Pom121, 796–1199; Nup159, nucleoplasmic side (Nup153 and Nup98) interact more 451–738; and Nup214, 1611–2093. The oligonucleotides for strongly with NESs than hRIP, and that the strongest Nup214 were made according to the sequence in the genebank interaction we observe is with Nup214 on the cytoplasmic (Accession number, D14689) that differs in the carboxyl terminus from the sequence in [37]. side. Another important question is how the repeat regions of Nup98, Nup153 and Nup214 can be involved simulta- The LexA–PKI–AD fusions were constructed by cloning a blunt-ended neously in binding NLS-containing complexes for import fragment encoding the activation domain of the pseudorabies virus and NES-containing proteins for export. immediate early protein [38] (amino acids 1–34) into XhoI/Klenow- treated LexA–PKI fusion vectors described above.
A nuclear export pathway conserved from yeast to man Yeast two-hybrid and liquid -galactosidase assays Although we show that the protein-export pathway medi- Plasmids pairs encoding LexA-fusion proteins (pEG202 derivatives) ated by leucine-rich NESs is functional in yeast, no natural and activation-domain-tagged proteins (pJG4-5 derivatives) were trans- yeast substrates have been identified as yet; database formed simultaneously into the yeast strain CFY1, a derivative of searches are hampered by the degenerate nature of the EGY48 containing an integrated version of pSH18-34 [36]. Transfor- mations were carried out using the lithium-acetate method according to NES consensus sequence (Fig. 1c). However, several yeast [39]. Individual colonies were restreaked onto nitrocellulose filters on proteins are known to shuttle between the nucleus and glucose, His–,Trp– plates. After 24 h at 30 °C, the nitrocellulose filters cytoplasm. Furthermore, the yeast nuclear envelope does were placed onto His–,Trp– plates containing 2 % galactose, 1 % raffi- nose, 0.1 M KPO pH 7.0, and 0.2 mg ml–1 X-Gal. Plates were pho- not break down during mitosis, which necessitates the 4 tographed after two or three days at 30 °C. Liquid -galactosidase regulated import and export of certain cell-cycle regulators. assays were done by the ‘glass bead’ method according to [40]. In any case, our results indicate that yeast genetics can now be used to elucidate the components and mechanisms of Transfection and CAT assays this nuclear-export pathway. Transfections were done using the calcium-phosphate technique according to [41]. Transfection mixes included 1 g of Rev derivative, 0.5 g CAT reporter CM128, 2 g RSV-Gal and 15 g pGEM3 as Conclusions carrier DNA. Precipitates were left on the cells for 20 h, and cells were We show that the pathway used by the HIV-1 Rev protein harvested 48 h after the removal of the calcium-phosphate precipitate. for the export of unspliced HIV RNAs is a cellular protein- CAT assays were done using standard protocols [40], the amount of export pathway. Cellular substrates for this pathway extract used was adjusted to -galactosidase activities to adjust for transfection efficiencies. include PKI, the inhibitor protein for PKA, and IB, the inhibitor of Rel family transcription factors. Our results Acknowledgements suggest a model whereby translocation of these proteins We thank R. Brent for yeast strains and plasmids, G. Blobel J. Sukegawa, A. through the nuclear pore is mediated by sequential inter- Radu, E. Hallberg, B. Cullen and G. Grosveld for plasmids, Thu Dang and actions with different nuclear pore proteins. We show this John Keilty for expert technical assistance. This work was supported by a grant from the National Institutes of Health to MRG. M.R.G. is an Investiga- pathway to be functional in yeast, and predict the tor of the Howard Hughes Medical Institute. existence of yeast proteins that contain leucine-rich NESs. References Materials and methods 1. Sweet DJ, Gerace L: Taking from the cytoplasm and giving to the Plasmid constructs pore: soluble transport factors in nuclear protein import. Trends Cell Biol 1995, 5:444–447. The LexA–Rev, LexA–Rev mutants, LexA–Rex, hRIP and ⌬hRIP con- 2. Moore MS, Blobel G: A G protein involved in nucleocytoplasmic structs were as described [8]. For the LexA–PKI constructs, comple- transport: the role of Ran. Trends Biochem Sci 1994, 19:211–216. mentary oligonucleotides were synthesized encoding amino acids 3. Görlich D, Mattaj IW: Nucleocytoplasmic transport. Science 1996, 35–47 of PKI␣ [10] flanked by BamHI and XhoI sites. These 271:1513–1518. BamHI–XhoI fragments were cloned into pEG202 [36]. The LexA–IB 4. Izaurralde E, Mattaj IW: RNA export. Cell 1995, 81:153–159. 854 Current Biology 1996, Vol 6 No 7
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