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ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein
BBRC Biochemical and Biophysical Research Communications 321 (2004) 517–523 www.elsevier.com/locate/ybbrc
ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein Lixin Liu, Guifang Chen, Xin Ji, Guangxia Gao * Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Received 15 June 2004
Abstract The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus. 2004 Elsevier Inc. All rights reserved. Keywords: ZAP; CRM1-dependent; Shuttling; NES; NLS
The zinc finger antiviral protein (ZAP) is a recently identified host factor that inhibits the infection of Moloney murine leukemia virus (MMLV) [1]. Screening of a cDNA library for host factors that inhibit the infection of MMLV led to the identification of NZAP, the N-terminal 254 amino acids of ZAP [1]. Overexpression of NZAP as a fusion with selection marker Zeo, NZAPZeo, rendered the cells resistant to MMLV infection by 30-fold. The full-length ZAP displayed the same antiviral activity as NZAP-Zeo. However, NZAP-myc, NZAP tagged with the myc-epitope, displayed very weak inhibitory activity, if any ([1], data not shown). The mechanism underlying the seemingly discrepancy between NZAP-Zeo and NZAP-myc remained elusive. Analysis for the step at which MMLV infection is inhibited revealed that ZAP specifically eliminates the viral mRNA in the cytoplasm, but not in the nucleus [1].
*
Corresponding author. Fax: +86-10-62653562. E-mail address: [email protected] (G. Gao).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.174
In addition to inhibiting MMLV infection, ZAP also inhibits the replication of multiple members of alphaviruses, including Sindbis virus (SIN), Smliki Forest virus (SFV), Ross River virus (RRV), and Venezuelen equine encephalitis virus (VEE) [2]. But expression of ZAP does not induce a broad spectrum antiviral state as some viruses, including herpes simplex virus type 1 (HSV-1) and yellow fever virus (YFV), grow normally in the cells expressing ZAP or NZAP-Zeo [2]. Alphaviruses are RNA viruses that replicate entirely in an RNA state in the cytoplasm [3]. ZAP targets SIN at a stage after binding and penetration but before the production of new genomic viral RNA. These results suggest that a common mechanism may exist in the cytoplasm by which ZAP prevents the accumulation of the viral mRNA of MMLV and SIN. In the N-terminus of ZAP there are four CCCH-type zinc finger motifs [1]. Similar motifs are also found in tristetraprolin (TTP) and its family members [4–7]. TTP specifically modulates the stability of mRNAs containing the AU-rich element (ARE) [8]. TTP directly binds to ARE through the zinc finger motifs [9] and
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recruits the exosome, an exoribonuclease complex, to degrade the ARE-containing mRNAs [10–12]. The similarity between ZAP and TTP in the CCCH zinc finger motifs suggests they may share a common mechanism. In the present study, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the cytoplasm and the nucleus in a CRM1-dependent manner. One NES and two NLS motifs are identified. The different antiviral activity between NZAP-Zeo and NZAP-myc seems to be accounted for by the different subcellular localization: NZAP-Zeo is localized in the cytoplasm whereas NZAP-myc is localized predominantly in the nucleus.
Materials and methods Plasmid construction. The plasmids pZAP-myc and pNZAP-myc have been described previously [1]. pNZAP-Zeo-myc expresses NZAP-Zeo fused to the myc-tag at the C-terminus. To generate NZAPZeo-myc, the Zeo coding sequence was PCR amplified from pBabeNZAP-Zeo [3] using forward primer 5 0 -GACAGAAGCAAAAGCAG AGAC-3 0 (right before the NotI site) and reverse primer 5 0 -ATAT AGTCTAGAGGGTCCTGCTCCTCGGCCACGAA-3 0 bearing an XbaI site (underlined). The PCR product was cloned into pNZAP-myc using the NotI and XbaI sites to generate pNZAP-Zeo-myc. The plasmids pEGFPC1 and pEGFPN1 are commercially available from Clontech. The C-terminal deletion mutants were created by cloning PCR-derived ZAP fragments into pEGFP-N1 using the KpnI and BamHI sites. In the forward primer, a KpnI site was built; in the reverse primers, a BamHI site was built. The primers are listed below. The restriction sites built in the primers are underlined. Forward primer 5 0 -CGGGGTACCATGGCAGATCCCGGGGTA-3 0 . Reverse primers ZAP/1–675: 5 0 -CGCGGATCCGCGGCCGCTGGCTTTTCCTCATAT GAG-3 0 ; ZAP/1–575: 5 0 -CGCGGATCCGCGGCCGCGATGTAGCGTAGGAG ATACTCA-3 0 ; ZAP/1–475: 5 0 -CGCGGATCCGCGGCCGCCTCCTTCTTGCCCTT GACCTCC-3 0 ; ZAP/1–275: 5 0 -CGCGGATCCGCGGCCGCCTCACAGATGTGGAG TCTC-3 0 ; ZAP/1–175: 5 0 -CGCGGATCCGCGGCCCGACGGCAGACGCGGG CT-3 0 ; ZAP/1–75: 5 0 -CGCGGATCCGCGGCCCGACGGCAGACGCGGG CT-3 0 . The N-terminal deletion mutants were created by cloning PCRderived ZAP fragments into pEGFP-C1 using the KpnI and BamHI sites built in the primers (underlined). The primers are listed below. Reverse primer 5 0 -CGCGGATCCGCGGCCGCCCTCTGGACC-3 0 . Forward primers ZAP/179–776: 5 0 -CGGGGTACCACCCGGGGCAACTGC-3 0 ; ZAP/279–776: 5 0 -CGGGGTACCAGCTGTAAAGATTCC-3 0 ; ZAP/379–776: 5 0 -CGGGGTACCGTCGGGTCCCATTTTTA-3 0 ; ZAP/479–776: 5 0 -CGGGGTACCTCAGAGGATGGGAATCTAGAT -3 0 ; ZAP/429–776: 5 0 -CGGGGTACCCAGGATCTGCAGACCACA-3 0 ; and ZAP/398–776: 5 0 -CGGGGTACCTCGGTCTCAGGAATTCCA-3 0 .
The ZAP/179–776 NES mutants were generated by replacing the KpnI–BamHI fragment of ZAP/179–776 with two PCR-derived fragments. The 5 0 fragment was generated using a common forward primer (NES-SP) bearing a KpnI site and a mutation specific reverse primer (RP) bearing silent mutations creating a restriction site; the 3 0 fragment was generated using a common reverse primer (NES-RP) bearing a BamHI site and a mutation specific forward primer (FP) bearing silent mutations creating the same restriction site as created in the 5 0 fragment. The codon for alanine was built in the mutation specific primers. The primer sequences are listed below. The restriction sites built in the primers are underlined; the codons for alanine are in boldface type. NES-FP: 5 0 -CGGGGTACCACCCGGGGCAACTGC-3 0 ; L284A-RP: 5 0 -TCCCCGCGGAATCTTTACAGCTGGTGAC-3 0 ; L284A-FP: 5 0 -TCCCCGCGGAGGATGTGTGTCTGTGGAT-3 0 ; V287A-RP: 5 0 -CGACGCGTCCTCCAGGGAATCTTT-3 0 V287A-FP: 5 0 -CGACGCGTCTGTGGATGTCACCCAG-3 0 V289A-RP: 5 0 -TCCCCGCGGACACATCCTCCAGGGAATC-3 0 ; V289A-FP: 5 0 -TCCCCGCGGATGTCACCCAGAAGTTC-3 0 ; V291A-RP: 5 0 -AAAAGTACTTGAACTTCTGGGTGGCATCCACA GACACA-3 0 ; V291A-FP: 5 0 -AAAAGTACTTGGGGACGCATGACCGT-3 0 ; and NES-RP: 5 0 -CGCGGATCCCTCTGGACCTCTTCT-3 0 ; The same strategy was used to construct ZAP/68–179-NLS1, ZAPNLS1, ZAP-pNES, and ZAP-NES mutants. The primer sequences are listed below. Primers for constructing ZAP/68–179-NLS1m ZAP/68–179-NLS1-FP: 5 0 -TGCTCCGGAATGGCAGATCCCGGG TA-3 0 ; ZAP/68–179-NLS1m-RP: 5 0 -AAACTGCAGCAGTAGTAGCCACT ACAGACCG-3 0 ; ZAP/68–179-NLS1m-FP: 5 0 -AAACTGCAGCTGTCTGCGCTGCTA AGTACCAGAGACCCTGCGAC-3 0 ; ZAP/68–179-NLS1-RP: 5 0 -CGGGGTACCGAAGTGCTCACAGAT GTG-3 0 . Primers for constructing ZAP-NLS1m ZAP-NLS1-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGG GTATGCTGT-3 0 ; ZAP-NLS1m-RP: 5 0 -AAACTGCAGCAGTAGTAGCCACTACAGA CCG-3 0 ; ZAP-NLS1m-FP: 5 0 -AAACTGCAGCTGTCTGCGCTGCTAAGTA CCAGAGACCCTGCGAC-3 0 ; ZAP-NLS1-RP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGAC C-3 0 . Primers for constructing ZAP-pNESm pNES-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGGGTAT GCTGT-3 0 ; pNESm-RP: 5 0 -GGCGCGCGCCTCACCCAGCAGTTCCTCCAG-3 0 ; pNESm-FP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGACC-3 0 ; pNES-RP: 5 0 -GAGGCGCGCGCCCCCGAGGCGCAGCTC-3 0 . Primers for constructing ZAP-NESm ZAP-NES-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGGG TATGCTGT-3 0 ; ZAP-NESm-RP: 5 0 -CGACGCGTCCTCCAGGGAATCTTT-3 0 ; ZAP-NESm-FP: 5 0 -CGACGCGTCTGTGGATGTCACCCAG-3 0 ; ZAP-NES-RP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGACC30. The ZAP/398–776-NLS2 mutant was generated by replacing the KpnI–BamHI fragment with a PCR derived fragment using primers ZAP-NLS2m-FP and ZAP-NLS2m-RP. The KpnI site and the codon for alanine are built in the forward primer. The primer sequences are listed below. ZAP-NLS2m-FP 5 0 -CGGGGTACCTCGGTCTCAGGAATTCCAGCGGAGGATTCA CA-3 0 ; ZAP-NLS2m-RP 5 0 CGCGGATCCCGGCCGCCCTGTGACC-3 0 .
L. Liu et al. / Biochemical and Biophysical Research Communications 321 (2004) 517–523 ZAP-NLS2m was generated by replacing the 33 bp EcoRI fragment of ZAP/398-776-NLS2m with a 1.2 kb EcoRI fragment from pEGFPN1-ZAP. Replacement of the 33 bp fragment with the 1.2 kb fragment from ZAP-NLS1m created ZAP-NLSdm. ZAP-NESdm was generated by replacing the 405 bp NheI fragment of ZAP-NESm with the corresponding fragment from ZAP-pNESm. Cell culture and transfection. HeLa cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37 C with 5% CO2. Transient transfection was performed using Lipofectamine 2000 (Boehringer–Mannheim) following manufacturerÕs instruction. Eight hours posttransfection, the cells were re-plated onto coverslips. 24 h posttransfection, the cells were fixed for further processing. For LMB treatment, the cells were cultured in fresh medium containing 5 or 10 ng/ml LMB for 6 h before fixation. Immunofluorescence staining and PI staining. The HeLa cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS three times for 5 min each, permeabilized with 0.4% Triton X-100 in PBS for 10 min, and then washed with PBS three times for 10 min each. Subsequently, the samples were blocked in staining buffer for 5 min, washed once with PBS, and then incubated with 9E10 anti-myc antibody (Santa Cruz) at room temperature for 1 h. The coverslips were washed three times with PBS and incubated with FITC-conjugated anti-mouse IgG (Sigma) at room temperature for 45 min. The samples were then washed three times for 10 min each and mounted with Vectashield (Vector Laboratories). The subcellular localizations of the stained proteins were photographed using a Laser Confocal Microscope (Leica PCS SP2) with the 100· Plan-NeoFluor oil objective. In some cases, the cells were also stained with propidium iodide (PI) to mark the nuclei. The cells were fixed and permeabilized as described above. The samples were treated with 10 U/ml RNase A at 37 C in a humidified chamber for 30 min to remove the cellular RNA. The samples were then washed three times with PBS for a total of 5 min and incubated with PI (Sigma) at a final concentration of 2.5 lg/ml for 1 h. The samples were washed three times with PBS for 10 min each time, mounted, and photographed as described above.
Results ZAP is localized in the cytoplasm at steady state and shuttles between the nucleus and the cytoplasm To analyze the subcellular localization of ZAP, GFP was fused to the C-terminus of ZAP for convenient visualization. The subcellular localization of ZAP in HeLa cells at steady state was analyzed by confocal microscopy. The results are shown in Fig. 1A. GFP alone was expressed in both nucleus and cytoplasm (Fig. 1A, I), whereas ZAP-GFP was localized predominantly in the cytoplasm (Fig. 1A, II). When the cells were treated with leptomycin B (LMB), a specific inhibitor of the nuclear export receptor CRM1 [13–15], a large fraction of ZAP shifted to the nucleus (Fig. 1A, III). These data indicate that ZAP shuttles between the nucleus and the cytoplasm and this shuttling activity is CRM1-dependent. To test whether fusion of GFP to ZAP affected the localization of ZAP, myc-tagged ZAP was expressed in HeLa cells and analyzed for subcellular localization. ZAP-myc displayed the same localization pattern as ZAP-GFP (Fig. 1A, IV), indicating that fusion with GFP does not change the localization of
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ZAP. In the following experiments, GFP was used as a tag for convenient visualization of the subcellular localization. The subcellular localization of NZAP-Zeo-myc and NZAP-myc In an attempt to understand the difference between NZAP-Zeo and NZAP-myc in the activity to inhibit the expression of retroviral vectors, we analyzed the subcellular localization of the two proteins in HeLa cells. For side-by-side comparison with NZAP-myc and for immunofluorescence detection, NZAP-Zeo was tagged with the myc epitope. As shown in Fig. 1B, NZAPZeo-myc is predominantly cytoplasmic. In contrast, NZAP-myc is predominantly nuclear. The different subcellular localization seems to explain the different activities between NZAP-myc and NZAP-Zeo. Mapping the NLS and NES All the shuttling proteins contain specific sequences for translocation. Typically, a short positively charged nuclear localization sequence (NLS) is responsible for importing the protein into the nucleus and a nuclear export sequence (NES) is responsible for exporting the protein out of the nucleus [16]. The well-characterized classical NLS either contains a cluster of basic amino acids or is bipartite, with two basic amino acids in one cluster and three or more basic amino acids in the other cluster [17–19]. The NLS-containing proteins are imported to the nucleus by the heterodimeric carrier importin a/b [8,20,21]. Importin a recognizes NLS and importin b mediates the interaction with the nuclear pore [22]. The classical NES is composed of a short stretch of leucine-rich sequence [18,23]. This NES element directly interacts with the nuclear pore complex, which comprises the nuclear export receptor CRM1 and the FG-repeats of nucleoporin Can/Nup 214 [24– 27]. This export activity can be blocked by LMB, an inhibitor of CRM1 [13–15]. To map the NLS and NES motifs in ZAP, a series of C-terminal deletion mutants were constructed, tagged with GFP at the C-terminus, and analyzed for subcellular localization in HeLa cells. The results are shown in Fig. 2. Deletion up to amino acid 325 (ZAP/1–325) did not change the cytoplasmic localization of ZAP (Fig. 2, I–V). However, ZAP/1–275 is predominantly nuclear (Fig. 2, VI), suggesting that there is a NES domain between amino acids 276 and 325. The nuclear localization of ZAP/1–275 also suggested that there is a NLS domain in this fragment. The truncation mutants ZAP/1–175 and ZAP/1–75 displayed the same nuclear localization as ZAP/1–275 (Fig. 2, VII and VIII), indicating that the NLS domain is localized between amino acids 1 and 75.
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Mapping the second NLS
Fig. 1. The subcellular localization of ZAP. HeLa cells were transfected with plasmids expressing the indicated proteins. Twelve hours posttransfection, the cells were replated on 24-well chamber multiwell slides. The cells expressing GFP or ZAP-GFP were grown for an additional 12 h and then fixed with 4% paraformaldehyde and permeabilized with Triton X-100; the cells expressing myc-tagged proteins were grown for an additional 24 h and then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and probed with 9E10 anti-myc antibody and FITC-conjugated anti-mouse IgG. For LMB treatment, and 10 ng/ml LMB was added to the cells 6 h before fixation. The cells were visualized by confocal microscopy.
In the course of mapping the NLS and NES motifs, we found that LMB treatment of ZAP/179–776, which does not include the NLS identified above (referred to as NLS1), caused change of the localization from the cytoplasm to the nucleus (Fig. 3A). This result suggested that there is another NLS (referred to as NLS2) domain between amino acids 179 and 776. To map NLS2, a series of N-terminal deletion mutants were constructed. In these mutants, GFP was fused to the N-terminus of ZAP. The subcellular localizations of the deletion mutants are shown in Fig. 3B. The nuclear localization of ZAP/379–776 (Fig. 3B, III) is consistent with the above result that there is a NES domain between amino acids 276 and 325. The cytoplasmic localization of ZAP/479– 776 indicated that NLS2 is localized between amino acids 379 and 479 (Fig. 3B, IV). That ZAP/429–776 (Fig. 3B, V) is cytoplasmic whereas ZAP/398–776 is nuclear (Fig. 3B, VI) further mapped NLS2 to between amino acids 398 and 429.
Fig. 2. C-terminal deletion mutagenesis to map NLS1 and NES. Top panel: confocal microscopy of the subcellular localization in HeLa cells of ZAP deletion mutants with GFP fused to the C-terminus. Bottom panel: schematic representation of the mutants, showing the amino acid numbers and GFP (shaded ovals).
Fig. 3. Mapping NLS2. (A) ZAP/179–776 contains a NLS domain. HeLa cells transfected with the plasmid expressing ZAP/179–776 with GFP fused to the N-terminus were treated with 5 ng/ml LMB or 75% methanol. The cells were visualized by confocal microscopy. (B) Deletion mutagenesis to map NLS2. Top panel: confocal microscopy of the subcellular localization in HeLa cells of ZAP deletion mutants with GFP fused to the N-terminus. Bottom panel: schematic representation of the mutants, showing the amino acid numbers and GFP (shaded ovals).
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Fig. 4. Identification of NLS1 and NLS2 by site-directed mutagenesis. HeLa cells were transfected with plasmids expressing the indicated proteins. Twenty-four hours posttransfection the cells were fixed, permeabilized, treated with 10 U/ml RNase A to remove the RNA, and stained with propidium iodide (PI) to mark the nucleus. The cells were visualized by confocal microscopy. (A) The subcellular localization of ZAP/68–179 and the mutant ZAP/68–179-R68A/R70A/R73A/R74A (ZAP/68–179-NLS1m). GFP was fused to the N-terminus of ZAP/68–179. (B) The subcellular localization of ZAP/398–776 and the mutant ZAP/398–776-K405A/K406A (ZAP/398–776-NLS2m). GFP was fused to the N-terminus of ZAP/398776. (C) The subcellular localization of full-length ZAP harboring mutations in NLS1 (ZAP-NLS1m), in NLS2 (ZAP-NLS2m) or in both NLS1 and NLS2 (ZAP-NLSdm) in the absence (top two panels) or presence (bottom panels) of LMB (10 ng/ml). GFP was fused to the C-terminus of ZAP and the mutants.
Fig. 5. Identification of NES by site-directed mutagenesis. HeLa cells were transfected with plasmids expressing the indicated proteins. Twenty-four hours posttransfection the cells were fixed, permeabilized, treated with 10 U/ml RNase A to remove the RNA, and stained with propidium iodide (PI) to mark the nucleus. The cells were visualized by confocal microscopy. (A) The subcellular localization of ZAP/179–776 harboring the L284A, V287A, V289A, V291A or L284A/V287A/V289A/V291A mutations. GFP was fused to the N-terminus of ZAP/179–776. (B) The subcellular localization of full-length ZAP harboring mutations V287A (ZAP-NESm), L29A/L31A (ZAP-pNESm) or both (ZAP-NESdm).
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Identification of NLS1 and NLS2 As mentioned above, the traditional NLS motifs are characterized by a cluster of basic residues. Sequence analysis of ZAP/1–75, in which NLS1 was mapped, suggested that 68-RARVCRRK-75 might meet this criterion. To validate this notion, the basic residues R68, R70, R73, and R74 were substituted with alanines. As predicted, ZAP/68–179 is predominantly nuclear, whereas the mutant ZAP/68–179-R68A/R70A/R73A/ R74A (referred to as ZAP/68–179-NLS1m) is localized in both the nucleus and the cytoplasm (Fig. 4A). The disperse distribution of the mutant could be due to diffusion of the relatively small protein, which is about 35 kDa. In the region between amino acids 398 and 429, where NLS2 was mapped, the only clustered basic residues are K405 and K406. Substitution of these two residues with alanines changed the subcellular localization of ZAP/398–776 from both nuclear and cytoplasmic to predominantly cytoplasmic (Fig. 4B). To further analyze the nuclear localization function of NLS1 and NLS2, the mutations of NLS1 and NLS2 were introduced into full-length ZAP. The mutants were analyzed for the subcellular localization in the absence or presence of LMB. The results are shown in Fig. 4C. Without LMB treatment, all the mutants were localized predominantly in the cytoplasm (Fig. 4C, upper panels). When treated with LMB, the NLS1 mutant (ZAP-NLS1m) and NLS2 mutant (ZAPNLS2m) displayed the same localization pattern as ZAP, both cytoplasmic and nuclear (Fig. 4C, lower panels). In contrast, the double mutant (ZAP-NLSdm) remained predominantly cytoplasmic (Fig. 4C, lower panels). These results indicated that both NLS1 and NLS2 are functional in the full-length ZAP. Identification of the NES The typical CRM1-dependent NES motifs are characterized by a cluster of bulky hydrophobic residues with conserved spacing. Sequence analysis of the fragment between amino acids 276 and 325, where the NES was mapped, identified a potential NES motif: 284-LEDVSVDV-291. To test whether this motif is required to direct the nuclear export, L284, V287, V289, and V291 of ZAP/179–776 were substituted with alanines. To analyze the functions of these residues, they were also individually substituted with alanine. The mutants were analyzed for the subcellular localization and the results are shown in Fig. 5A. Substitution of the leucine with alanine did not change the cytoplasmic localization of ZAP/179–776 (Fig. 5A, L284A). However, substitution of any one of the valines with alanine resulted in the localization in both nucleus and cytoplasm (Fig. 5A, V287A, V289A, and V291A). These results in-
dicated that amino acids V287, V289, and V291 are critical for the nuclear export of ZAP. Sequence analysis revealed that in the N-terminus of ZAP there is a stretch of amino acids (22-LEELLGEIRL-31) that has the typical NES consensus sequence. To test whether this motif (referred to as pNES for pseudo-NES) is a functional NES, I29 and L31 were substituted with alanines in full-length ZAP. For comparison, the above NES mutation V287A was also introduced into full-length ZAP. The subcellular localizations of the mutants are shown in Fig. 5B. Consistent with the above result, the NES mutation rendered ZAP both cytoplasmic and nuclear (Fig. 5B, ZAPNESm). However, the pNES mutant remained predominantly cytoplasmic (Fig. 5B, ZAP-pNESm), suggesting that pNES is not a functional NES. Consistently, the NES and pNES double mutant displayed the same localization pattern as the NES single mutant (Fig. 5B, ZAP-NESdm).
Discussion In this report, we demonstrated that ZAP is a nucleocytoplasmic shuttling protein and is localized predominantly in the cytoplasm at steady state. Two NLS and one functional NES motifs were identified. NLS1 was mapped to 68-RARVCRRK and NLS2 was mapped to K405 and K406. The functional CRM1-dependent NES was mapped to 284-LEDVSVDV, which is similar to but not so characteristic of the classical NES consensus sequences. Mutation of this NES motif or treatment with LMB caused the shift of ZAP from the cytoplasm to the nucleus. Consistent with the location of the NLS and NES motifs, NZAP-myc and NZAP-GFP are localized predominantly in the nucleus. But surprisingly, NZAP-Zeo is localized predominantly in the cytoplasm. A possible explanation is that the NLS motif is not well exposed in NZAP-Zeo. Our previous work demonstrated that ZAP specifically prevents the accumulation of MMLV RNA in the cytoplasm but not in the nucleus [1]. Since the viral mRNA is transcribed in the nucleus and exported into the cytoplasm, the reduced level of viral mRNA in the cytoplasm could be accounted for either by blocked nuclear export of the RNA or by degradation of the RNA in the cytoplasm. The predominant cytoplasmic localization at steady state and the shuttling capability of ZAP could explain either possibility. Many shuttling proteins have been demonstrated to be involved in RNA export or in modulating RNA stability [28–32]. However, the fact that ZAP also specifically inhibits the replication of SIN, which replicates entirely in an RNA state in the cytoplasm, favors the possibility that the RNA is degraded in the cytoplasm. If this were true, one would predict that disruption of the NES motif should abolish
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the activity of ZAP. But this is technically difficult to prove because even substitution of all the leucine and valine residues in the NES motif with alanines caused only partial nuclear localization of ZAP (Fig. 5). A large fraction of the protein remained in the cytoplasm, which is sufficient for ZAPÕs activity. The results presented in this report are consistent with our previous observations that ZAP inhibits the replication of SIN, which replicates in the cytoplasm, and that ZAP specifically eliminates MMLV RNA in the cytoplasm. The nucleocytoplasmic shuttling capability suggests that besides modulating the mRNA level in the cytoplasm ZAP may also function in the nucleus.
Acknowledgments This work is supported by grants to Guangxia Gao from the National Science Foundation (30225002) and the Ministry of Science and Technology (2002CB513001) of China. We thank Drs. Hong Tang and Quan Chen for helpful discussion, and Guangjun Dong and Shiwen Li for technical support during the course of this work.
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ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein Lixin Liu, Guifang Chen, Xin Ji, Guangxia Gao * Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Received 15 June 2004
Abstract The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus. 2004 Elsevier Inc. All rights reserved. Keywords: ZAP; CRM1-dependent; Shuttling; NES; NLS
The zinc finger antiviral protein (ZAP) is a recently identified host factor that inhibits the infection of Moloney murine leukemia virus (MMLV) [1]. Screening of a cDNA library for host factors that inhibit the infection of MMLV led to the identification of NZAP, the N-terminal 254 amino acids of ZAP [1]. Overexpression of NZAP as a fusion with selection marker Zeo, NZAPZeo, rendered the cells resistant to MMLV infection by 30-fold. The full-length ZAP displayed the same antiviral activity as NZAP-Zeo. However, NZAP-myc, NZAP tagged with the myc-epitope, displayed very weak inhibitory activity, if any ([1], data not shown). The mechanism underlying the seemingly discrepancy between NZAP-Zeo and NZAP-myc remained elusive. Analysis for the step at which MMLV infection is inhibited revealed that ZAP specifically eliminates the viral mRNA in the cytoplasm, but not in the nucleus [1].
*
Corresponding author. Fax: +86-10-62653562. E-mail address: [email protected] (G. Gao).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.174
In addition to inhibiting MMLV infection, ZAP also inhibits the replication of multiple members of alphaviruses, including Sindbis virus (SIN), Smliki Forest virus (SFV), Ross River virus (RRV), and Venezuelen equine encephalitis virus (VEE) [2]. But expression of ZAP does not induce a broad spectrum antiviral state as some viruses, including herpes simplex virus type 1 (HSV-1) and yellow fever virus (YFV), grow normally in the cells expressing ZAP or NZAP-Zeo [2]. Alphaviruses are RNA viruses that replicate entirely in an RNA state in the cytoplasm [3]. ZAP targets SIN at a stage after binding and penetration but before the production of new genomic viral RNA. These results suggest that a common mechanism may exist in the cytoplasm by which ZAP prevents the accumulation of the viral mRNA of MMLV and SIN. In the N-terminus of ZAP there are four CCCH-type zinc finger motifs [1]. Similar motifs are also found in tristetraprolin (TTP) and its family members [4–7]. TTP specifically modulates the stability of mRNAs containing the AU-rich element (ARE) [8]. TTP directly binds to ARE through the zinc finger motifs [9] and
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recruits the exosome, an exoribonuclease complex, to degrade the ARE-containing mRNAs [10–12]. The similarity between ZAP and TTP in the CCCH zinc finger motifs suggests they may share a common mechanism. In the present study, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the cytoplasm and the nucleus in a CRM1-dependent manner. One NES and two NLS motifs are identified. The different antiviral activity between NZAP-Zeo and NZAP-myc seems to be accounted for by the different subcellular localization: NZAP-Zeo is localized in the cytoplasm whereas NZAP-myc is localized predominantly in the nucleus.
Materials and methods Plasmid construction. The plasmids pZAP-myc and pNZAP-myc have been described previously [1]. pNZAP-Zeo-myc expresses NZAP-Zeo fused to the myc-tag at the C-terminus. To generate NZAPZeo-myc, the Zeo coding sequence was PCR amplified from pBabeNZAP-Zeo [3] using forward primer 5 0 -GACAGAAGCAAAAGCAG AGAC-3 0 (right before the NotI site) and reverse primer 5 0 -ATAT AGTCTAGAGGGTCCTGCTCCTCGGCCACGAA-3 0 bearing an XbaI site (underlined). The PCR product was cloned into pNZAP-myc using the NotI and XbaI sites to generate pNZAP-Zeo-myc. The plasmids pEGFPC1 and pEGFPN1 are commercially available from Clontech. The C-terminal deletion mutants were created by cloning PCR-derived ZAP fragments into pEGFP-N1 using the KpnI and BamHI sites. In the forward primer, a KpnI site was built; in the reverse primers, a BamHI site was built. The primers are listed below. The restriction sites built in the primers are underlined. Forward primer 5 0 -CGGGGTACCATGGCAGATCCCGGGGTA-3 0 . Reverse primers ZAP/1–675: 5 0 -CGCGGATCCGCGGCCGCTGGCTTTTCCTCATAT GAG-3 0 ; ZAP/1–575: 5 0 -CGCGGATCCGCGGCCGCGATGTAGCGTAGGAG ATACTCA-3 0 ; ZAP/1–475: 5 0 -CGCGGATCCGCGGCCGCCTCCTTCTTGCCCTT GACCTCC-3 0 ; ZAP/1–275: 5 0 -CGCGGATCCGCGGCCGCCTCACAGATGTGGAG TCTC-3 0 ; ZAP/1–175: 5 0 -CGCGGATCCGCGGCCCGACGGCAGACGCGGG CT-3 0 ; ZAP/1–75: 5 0 -CGCGGATCCGCGGCCCGACGGCAGACGCGGG CT-3 0 . The N-terminal deletion mutants were created by cloning PCRderived ZAP fragments into pEGFP-C1 using the KpnI and BamHI sites built in the primers (underlined). The primers are listed below. Reverse primer 5 0 -CGCGGATCCGCGGCCGCCCTCTGGACC-3 0 . Forward primers ZAP/179–776: 5 0 -CGGGGTACCACCCGGGGCAACTGC-3 0 ; ZAP/279–776: 5 0 -CGGGGTACCAGCTGTAAAGATTCC-3 0 ; ZAP/379–776: 5 0 -CGGGGTACCGTCGGGTCCCATTTTTA-3 0 ; ZAP/479–776: 5 0 -CGGGGTACCTCAGAGGATGGGAATCTAGAT -3 0 ; ZAP/429–776: 5 0 -CGGGGTACCCAGGATCTGCAGACCACA-3 0 ; and ZAP/398–776: 5 0 -CGGGGTACCTCGGTCTCAGGAATTCCA-3 0 .
The ZAP/179–776 NES mutants were generated by replacing the KpnI–BamHI fragment of ZAP/179–776 with two PCR-derived fragments. The 5 0 fragment was generated using a common forward primer (NES-SP) bearing a KpnI site and a mutation specific reverse primer (RP) bearing silent mutations creating a restriction site; the 3 0 fragment was generated using a common reverse primer (NES-RP) bearing a BamHI site and a mutation specific forward primer (FP) bearing silent mutations creating the same restriction site as created in the 5 0 fragment. The codon for alanine was built in the mutation specific primers. The primer sequences are listed below. The restriction sites built in the primers are underlined; the codons for alanine are in boldface type. NES-FP: 5 0 -CGGGGTACCACCCGGGGCAACTGC-3 0 ; L284A-RP: 5 0 -TCCCCGCGGAATCTTTACAGCTGGTGAC-3 0 ; L284A-FP: 5 0 -TCCCCGCGGAGGATGTGTGTCTGTGGAT-3 0 ; V287A-RP: 5 0 -CGACGCGTCCTCCAGGGAATCTTT-3 0 V287A-FP: 5 0 -CGACGCGTCTGTGGATGTCACCCAG-3 0 V289A-RP: 5 0 -TCCCCGCGGACACATCCTCCAGGGAATC-3 0 ; V289A-FP: 5 0 -TCCCCGCGGATGTCACCCAGAAGTTC-3 0 ; V291A-RP: 5 0 -AAAAGTACTTGAACTTCTGGGTGGCATCCACA GACACA-3 0 ; V291A-FP: 5 0 -AAAAGTACTTGGGGACGCATGACCGT-3 0 ; and NES-RP: 5 0 -CGCGGATCCCTCTGGACCTCTTCT-3 0 ; The same strategy was used to construct ZAP/68–179-NLS1, ZAPNLS1, ZAP-pNES, and ZAP-NES mutants. The primer sequences are listed below. Primers for constructing ZAP/68–179-NLS1m ZAP/68–179-NLS1-FP: 5 0 -TGCTCCGGAATGGCAGATCCCGGG TA-3 0 ; ZAP/68–179-NLS1m-RP: 5 0 -AAACTGCAGCAGTAGTAGCCACT ACAGACCG-3 0 ; ZAP/68–179-NLS1m-FP: 5 0 -AAACTGCAGCTGTCTGCGCTGCTA AGTACCAGAGACCCTGCGAC-3 0 ; ZAP/68–179-NLS1-RP: 5 0 -CGGGGTACCGAAGTGCTCACAGAT GTG-3 0 . Primers for constructing ZAP-NLS1m ZAP-NLS1-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGG GTATGCTGT-3 0 ; ZAP-NLS1m-RP: 5 0 -AAACTGCAGCAGTAGTAGCCACTACAGA CCG-3 0 ; ZAP-NLS1m-FP: 5 0 -AAACTGCAGCTGTCTGCGCTGCTAAGTA CCAGAGACCCTGCGAC-3 0 ; ZAP-NLS1-RP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGAC C-3 0 . Primers for constructing ZAP-pNESm pNES-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGGGTAT GCTGT-3 0 ; pNESm-RP: 5 0 -GGCGCGCGCCTCACCCAGCAGTTCCTCCAG-3 0 ; pNESm-FP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGACC-3 0 ; pNES-RP: 5 0 -GAGGCGCGCGCCCCCGAGGCGCAGCTC-3 0 . Primers for constructing ZAP-NESm ZAP-NES-FP: 5 0 -ACGCGTCGACACCATGGCAGATCCCGGGG TATGCTGT-3 0 ; ZAP-NESm-RP: 5 0 -CGACGCGTCCTCCAGGGAATCTTT-3 0 ; ZAP-NESm-FP: 5 0 -CGACGCGTCTGTGGATGTCACCCAG-3 0 ; ZAP-NES-RP: 5 0 -CGCGGATCCCGGCGGCCGCCCTCTGGACC30. The ZAP/398–776-NLS2 mutant was generated by replacing the KpnI–BamHI fragment with a PCR derived fragment using primers ZAP-NLS2m-FP and ZAP-NLS2m-RP. The KpnI site and the codon for alanine are built in the forward primer. The primer sequences are listed below. ZAP-NLS2m-FP 5 0 -CGGGGTACCTCGGTCTCAGGAATTCCAGCGGAGGATTCA CA-3 0 ; ZAP-NLS2m-RP 5 0 CGCGGATCCCGGCCGCCCTGTGACC-3 0 .
L. Liu et al. / Biochemical and Biophysical Research Communications 321 (2004) 517–523 ZAP-NLS2m was generated by replacing the 33 bp EcoRI fragment of ZAP/398-776-NLS2m with a 1.2 kb EcoRI fragment from pEGFPN1-ZAP. Replacement of the 33 bp fragment with the 1.2 kb fragment from ZAP-NLS1m created ZAP-NLSdm. ZAP-NESdm was generated by replacing the 405 bp NheI fragment of ZAP-NESm with the corresponding fragment from ZAP-pNESm. Cell culture and transfection. HeLa cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37 C with 5% CO2. Transient transfection was performed using Lipofectamine 2000 (Boehringer–Mannheim) following manufacturerÕs instruction. Eight hours posttransfection, the cells were re-plated onto coverslips. 24 h posttransfection, the cells were fixed for further processing. For LMB treatment, the cells were cultured in fresh medium containing 5 or 10 ng/ml LMB for 6 h before fixation. Immunofluorescence staining and PI staining. The HeLa cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS three times for 5 min each, permeabilized with 0.4% Triton X-100 in PBS for 10 min, and then washed with PBS three times for 10 min each. Subsequently, the samples were blocked in staining buffer for 5 min, washed once with PBS, and then incubated with 9E10 anti-myc antibody (Santa Cruz) at room temperature for 1 h. The coverslips were washed three times with PBS and incubated with FITC-conjugated anti-mouse IgG (Sigma) at room temperature for 45 min. The samples were then washed three times for 10 min each and mounted with Vectashield (Vector Laboratories). The subcellular localizations of the stained proteins were photographed using a Laser Confocal Microscope (Leica PCS SP2) with the 100· Plan-NeoFluor oil objective. In some cases, the cells were also stained with propidium iodide (PI) to mark the nuclei. The cells were fixed and permeabilized as described above. The samples were treated with 10 U/ml RNase A at 37 C in a humidified chamber for 30 min to remove the cellular RNA. The samples were then washed three times with PBS for a total of 5 min and incubated with PI (Sigma) at a final concentration of 2.5 lg/ml for 1 h. The samples were washed three times with PBS for 10 min each time, mounted, and photographed as described above.
Results ZAP is localized in the cytoplasm at steady state and shuttles between the nucleus and the cytoplasm To analyze the subcellular localization of ZAP, GFP was fused to the C-terminus of ZAP for convenient visualization. The subcellular localization of ZAP in HeLa cells at steady state was analyzed by confocal microscopy. The results are shown in Fig. 1A. GFP alone was expressed in both nucleus and cytoplasm (Fig. 1A, I), whereas ZAP-GFP was localized predominantly in the cytoplasm (Fig. 1A, II). When the cells were treated with leptomycin B (LMB), a specific inhibitor of the nuclear export receptor CRM1 [13–15], a large fraction of ZAP shifted to the nucleus (Fig. 1A, III). These data indicate that ZAP shuttles between the nucleus and the cytoplasm and this shuttling activity is CRM1-dependent. To test whether fusion of GFP to ZAP affected the localization of ZAP, myc-tagged ZAP was expressed in HeLa cells and analyzed for subcellular localization. ZAP-myc displayed the same localization pattern as ZAP-GFP (Fig. 1A, IV), indicating that fusion with GFP does not change the localization of
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ZAP. In the following experiments, GFP was used as a tag for convenient visualization of the subcellular localization. The subcellular localization of NZAP-Zeo-myc and NZAP-myc In an attempt to understand the difference between NZAP-Zeo and NZAP-myc in the activity to inhibit the expression of retroviral vectors, we analyzed the subcellular localization of the two proteins in HeLa cells. For side-by-side comparison with NZAP-myc and for immunofluorescence detection, NZAP-Zeo was tagged with the myc epitope. As shown in Fig. 1B, NZAPZeo-myc is predominantly cytoplasmic. In contrast, NZAP-myc is predominantly nuclear. The different subcellular localization seems to explain the different activities between NZAP-myc and NZAP-Zeo. Mapping the NLS and NES All the shuttling proteins contain specific sequences for translocation. Typically, a short positively charged nuclear localization sequence (NLS) is responsible for importing the protein into the nucleus and a nuclear export sequence (NES) is responsible for exporting the protein out of the nucleus [16]. The well-characterized classical NLS either contains a cluster of basic amino acids or is bipartite, with two basic amino acids in one cluster and three or more basic amino acids in the other cluster [17–19]. The NLS-containing proteins are imported to the nucleus by the heterodimeric carrier importin a/b [8,20,21]. Importin a recognizes NLS and importin b mediates the interaction with the nuclear pore [22]. The classical NES is composed of a short stretch of leucine-rich sequence [18,23]. This NES element directly interacts with the nuclear pore complex, which comprises the nuclear export receptor CRM1 and the FG-repeats of nucleoporin Can/Nup 214 [24– 27]. This export activity can be blocked by LMB, an inhibitor of CRM1 [13–15]. To map the NLS and NES motifs in ZAP, a series of C-terminal deletion mutants were constructed, tagged with GFP at the C-terminus, and analyzed for subcellular localization in HeLa cells. The results are shown in Fig. 2. Deletion up to amino acid 325 (ZAP/1–325) did not change the cytoplasmic localization of ZAP (Fig. 2, I–V). However, ZAP/1–275 is predominantly nuclear (Fig. 2, VI), suggesting that there is a NES domain between amino acids 276 and 325. The nuclear localization of ZAP/1–275 also suggested that there is a NLS domain in this fragment. The truncation mutants ZAP/1–175 and ZAP/1–75 displayed the same nuclear localization as ZAP/1–275 (Fig. 2, VII and VIII), indicating that the NLS domain is localized between amino acids 1 and 75.
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Mapping the second NLS
Fig. 1. The subcellular localization of ZAP. HeLa cells were transfected with plasmids expressing the indicated proteins. Twelve hours posttransfection, the cells were replated on 24-well chamber multiwell slides. The cells expressing GFP or ZAP-GFP were grown for an additional 12 h and then fixed with 4% paraformaldehyde and permeabilized with Triton X-100; the cells expressing myc-tagged proteins were grown for an additional 24 h and then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and probed with 9E10 anti-myc antibody and FITC-conjugated anti-mouse IgG. For LMB treatment, and 10 ng/ml LMB was added to the cells 6 h before fixation. The cells were visualized by confocal microscopy.
In the course of mapping the NLS and NES motifs, we found that LMB treatment of ZAP/179–776, which does not include the NLS identified above (referred to as NLS1), caused change of the localization from the cytoplasm to the nucleus (Fig. 3A). This result suggested that there is another NLS (referred to as NLS2) domain between amino acids 179 and 776. To map NLS2, a series of N-terminal deletion mutants were constructed. In these mutants, GFP was fused to the N-terminus of ZAP. The subcellular localizations of the deletion mutants are shown in Fig. 3B. The nuclear localization of ZAP/379–776 (Fig. 3B, III) is consistent with the above result that there is a NES domain between amino acids 276 and 325. The cytoplasmic localization of ZAP/479– 776 indicated that NLS2 is localized between amino acids 379 and 479 (Fig. 3B, IV). That ZAP/429–776 (Fig. 3B, V) is cytoplasmic whereas ZAP/398–776 is nuclear (Fig. 3B, VI) further mapped NLS2 to between amino acids 398 and 429.
Fig. 2. C-terminal deletion mutagenesis to map NLS1 and NES. Top panel: confocal microscopy of the subcellular localization in HeLa cells of ZAP deletion mutants with GFP fused to the C-terminus. Bottom panel: schematic representation of the mutants, showing the amino acid numbers and GFP (shaded ovals).
Fig. 3. Mapping NLS2. (A) ZAP/179–776 contains a NLS domain. HeLa cells transfected with the plasmid expressing ZAP/179–776 with GFP fused to the N-terminus were treated with 5 ng/ml LMB or 75% methanol. The cells were visualized by confocal microscopy. (B) Deletion mutagenesis to map NLS2. Top panel: confocal microscopy of the subcellular localization in HeLa cells of ZAP deletion mutants with GFP fused to the N-terminus. Bottom panel: schematic representation of the mutants, showing the amino acid numbers and GFP (shaded ovals).
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Fig. 4. Identification of NLS1 and NLS2 by site-directed mutagenesis. HeLa cells were transfected with plasmids expressing the indicated proteins. Twenty-four hours posttransfection the cells were fixed, permeabilized, treated with 10 U/ml RNase A to remove the RNA, and stained with propidium iodide (PI) to mark the nucleus. The cells were visualized by confocal microscopy. (A) The subcellular localization of ZAP/68–179 and the mutant ZAP/68–179-R68A/R70A/R73A/R74A (ZAP/68–179-NLS1m). GFP was fused to the N-terminus of ZAP/68–179. (B) The subcellular localization of ZAP/398–776 and the mutant ZAP/398–776-K405A/K406A (ZAP/398–776-NLS2m). GFP was fused to the N-terminus of ZAP/398776. (C) The subcellular localization of full-length ZAP harboring mutations in NLS1 (ZAP-NLS1m), in NLS2 (ZAP-NLS2m) or in both NLS1 and NLS2 (ZAP-NLSdm) in the absence (top two panels) or presence (bottom panels) of LMB (10 ng/ml). GFP was fused to the C-terminus of ZAP and the mutants.
Fig. 5. Identification of NES by site-directed mutagenesis. HeLa cells were transfected with plasmids expressing the indicated proteins. Twenty-four hours posttransfection the cells were fixed, permeabilized, treated with 10 U/ml RNase A to remove the RNA, and stained with propidium iodide (PI) to mark the nucleus. The cells were visualized by confocal microscopy. (A) The subcellular localization of ZAP/179–776 harboring the L284A, V287A, V289A, V291A or L284A/V287A/V289A/V291A mutations. GFP was fused to the N-terminus of ZAP/179–776. (B) The subcellular localization of full-length ZAP harboring mutations V287A (ZAP-NESm), L29A/L31A (ZAP-pNESm) or both (ZAP-NESdm).
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Identification of NLS1 and NLS2 As mentioned above, the traditional NLS motifs are characterized by a cluster of basic residues. Sequence analysis of ZAP/1–75, in which NLS1 was mapped, suggested that 68-RARVCRRK-75 might meet this criterion. To validate this notion, the basic residues R68, R70, R73, and R74 were substituted with alanines. As predicted, ZAP/68–179 is predominantly nuclear, whereas the mutant ZAP/68–179-R68A/R70A/R73A/ R74A (referred to as ZAP/68–179-NLS1m) is localized in both the nucleus and the cytoplasm (Fig. 4A). The disperse distribution of the mutant could be due to diffusion of the relatively small protein, which is about 35 kDa. In the region between amino acids 398 and 429, where NLS2 was mapped, the only clustered basic residues are K405 and K406. Substitution of these two residues with alanines changed the subcellular localization of ZAP/398–776 from both nuclear and cytoplasmic to predominantly cytoplasmic (Fig. 4B). To further analyze the nuclear localization function of NLS1 and NLS2, the mutations of NLS1 and NLS2 were introduced into full-length ZAP. The mutants were analyzed for the subcellular localization in the absence or presence of LMB. The results are shown in Fig. 4C. Without LMB treatment, all the mutants were localized predominantly in the cytoplasm (Fig. 4C, upper panels). When treated with LMB, the NLS1 mutant (ZAP-NLS1m) and NLS2 mutant (ZAPNLS2m) displayed the same localization pattern as ZAP, both cytoplasmic and nuclear (Fig. 4C, lower panels). In contrast, the double mutant (ZAP-NLSdm) remained predominantly cytoplasmic (Fig. 4C, lower panels). These results indicated that both NLS1 and NLS2 are functional in the full-length ZAP. Identification of the NES The typical CRM1-dependent NES motifs are characterized by a cluster of bulky hydrophobic residues with conserved spacing. Sequence analysis of the fragment between amino acids 276 and 325, where the NES was mapped, identified a potential NES motif: 284-LEDVSVDV-291. To test whether this motif is required to direct the nuclear export, L284, V287, V289, and V291 of ZAP/179–776 were substituted with alanines. To analyze the functions of these residues, they were also individually substituted with alanine. The mutants were analyzed for the subcellular localization and the results are shown in Fig. 5A. Substitution of the leucine with alanine did not change the cytoplasmic localization of ZAP/179–776 (Fig. 5A, L284A). However, substitution of any one of the valines with alanine resulted in the localization in both nucleus and cytoplasm (Fig. 5A, V287A, V289A, and V291A). These results in-
dicated that amino acids V287, V289, and V291 are critical for the nuclear export of ZAP. Sequence analysis revealed that in the N-terminus of ZAP there is a stretch of amino acids (22-LEELLGEIRL-31) that has the typical NES consensus sequence. To test whether this motif (referred to as pNES for pseudo-NES) is a functional NES, I29 and L31 were substituted with alanines in full-length ZAP. For comparison, the above NES mutation V287A was also introduced into full-length ZAP. The subcellular localizations of the mutants are shown in Fig. 5B. Consistent with the above result, the NES mutation rendered ZAP both cytoplasmic and nuclear (Fig. 5B, ZAPNESm). However, the pNES mutant remained predominantly cytoplasmic (Fig. 5B, ZAP-pNESm), suggesting that pNES is not a functional NES. Consistently, the NES and pNES double mutant displayed the same localization pattern as the NES single mutant (Fig. 5B, ZAP-NESdm).
Discussion In this report, we demonstrated that ZAP is a nucleocytoplasmic shuttling protein and is localized predominantly in the cytoplasm at steady state. Two NLS and one functional NES motifs were identified. NLS1 was mapped to 68-RARVCRRK and NLS2 was mapped to K405 and K406. The functional CRM1-dependent NES was mapped to 284-LEDVSVDV, which is similar to but not so characteristic of the classical NES consensus sequences. Mutation of this NES motif or treatment with LMB caused the shift of ZAP from the cytoplasm to the nucleus. Consistent with the location of the NLS and NES motifs, NZAP-myc and NZAP-GFP are localized predominantly in the nucleus. But surprisingly, NZAP-Zeo is localized predominantly in the cytoplasm. A possible explanation is that the NLS motif is not well exposed in NZAP-Zeo. Our previous work demonstrated that ZAP specifically prevents the accumulation of MMLV RNA in the cytoplasm but not in the nucleus [1]. Since the viral mRNA is transcribed in the nucleus and exported into the cytoplasm, the reduced level of viral mRNA in the cytoplasm could be accounted for either by blocked nuclear export of the RNA or by degradation of the RNA in the cytoplasm. The predominant cytoplasmic localization at steady state and the shuttling capability of ZAP could explain either possibility. Many shuttling proteins have been demonstrated to be involved in RNA export or in modulating RNA stability [28–32]. However, the fact that ZAP also specifically inhibits the replication of SIN, which replicates entirely in an RNA state in the cytoplasm, favors the possibility that the RNA is degraded in the cytoplasm. If this were true, one would predict that disruption of the NES motif should abolish
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the activity of ZAP. But this is technically difficult to prove because even substitution of all the leucine and valine residues in the NES motif with alanines caused only partial nuclear localization of ZAP (Fig. 5). A large fraction of the protein remained in the cytoplasm, which is sufficient for ZAPÕs activity. The results presented in this report are consistent with our previous observations that ZAP inhibits the replication of SIN, which replicates in the cytoplasm, and that ZAP specifically eliminates MMLV RNA in the cytoplasm. The nucleocytoplasmic shuttling capability suggests that besides modulating the mRNA level in the cytoplasm ZAP may also function in the nucleus.
Acknowledgments This work is supported by grants to Guangxia Gao from the National Science Foundation (30225002) and the Ministry of Science and Technology (2002CB513001) of China. We thank Drs. Hong Tang and Quan Chen for helpful discussion, and Guangjun Dong and Shiwen Li for technical support during the course of this work.
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