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Vol. 16, Issue 4, 1823-1838, April 2005
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* Laboratory of Nucleopore Biology, Institute of Molecular and Cell Biology, Singapore 138673, Republic of Singapore;
Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076, India
Submitted July 13, 2004;
Accepted January 11, 2005
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The full-length transcript of the Tf1 retroelement consisting of Gag, PR, RT, and IN proteins assemble along with copies of RNA to form virus-like particles (VLP) or preintegration complexes (PIC) that contain a 26-fold molar excess of Gag relative to IN (Atwood et al., 1996). The presence of Gag is essential for the formation of VLPs and reverse transcription of the RNA into double-stranded cDNA (Teysset et al., 2003). To integrate its cDNA into the host genome, the Tf1 VLP/PIC containing the IN and cDNA must be imported into the nucleus (Dang and Levin, 2000).
HIV-1 Vpr is a 14-kDa, 96 amino acid virion-associated protein that is highly conserved among primate lentiviruses HIV-1, HIV-2, and the simian immunodeficiency virus (SIV; Bukrinsky and Haffar, 1997; Sherman et al., 2002a; Tungaturthi et al., 2003). Unlike the accessory proteins Vif and Nef, incorporation of Vpr has been shown to be specific, involving a distinct domain, the p6 region in HIV-1 Gag and thus, its relative amount within the virion may be closely linked with that of Gag (Lu et al., 1993, 1995; Selig et al., 1999; Tungaturthi et al., 2003). Vpr has been implicated as one of the significant agents in AIDS pathogenesis (Sherman et al., 2002b; Tungaturthi et al., 2003) although the molecular mechanisms underlying such claims are far from understood.
In a manner analogous to HIV-1 Vpr requirement for nuclear import of its PIC (Lu et al., 1993; Mahalingam et al., 1995b, 1997a, 1997b; Fouchier et al., 1998; Popov et al., 1998a, 1998b; Fouchier and Malim, 1999; Le Rouzic et al., 2002), the transposition efficiency of the Tf1-retrotransposon correlated with its ability to import TF1-Gag into the nucleus (Balasundaram et al., 1999; Dang and Levin, 2000). Though the Tf1 retrotransposon lacks the accessory protein Vpr, its Gag protein plays a critical role in the packaging and nuclear import of its VLP resulting in elevated transposition levels (Levin et al., 1993; Atwood et al., 1996; Dang and Levin, 2000; Teysset et al., 2003). HIV-1 Vpr and Tf1-Gag proteins (a) display evident karyophilic properties and localize to the nucleus although they do not contain any canonical NLS (b) are required for the nuclear import of the genome of retrotransposon Tf1 (Gag) and HIV-1 (Vpr), (c) form an integral part of VLP in the case of Tf1 Gag and HIV-1 preintegration complex in the case of Vpr, and (d) are essential for optimal HIV-1 replication in macrophages (Vpr) and transposition of Tf1 (Gag; Fouchier et al., 1998; Fouchier and Malim, 1999; Dang and Levin, 2000; Teysset et al., 2003).
Given the importance of nuclear transport and the NPC in the etiology of human disease (Hutchison, 2002; Cronshaw and Matunis, 2004; Kau et al., 2004), the question of how retroviral elements transcend this physical barrier of the nuclear membrane is both an interesting and an important one to consider as a possible means of blocking viral replication. We had previously reported that a nonessential nucleoporin Nup124p of S. pombe, was essential for Tf1 transposition (Balasundaram et al., 1999). Here, we have used retrotransposition in the fission yeast as a genetic tool and the involvement of Nup124p to understand the mechanism underlying nuclear transport of Tf1-Gag and the HIV-1 accessory protein Vpr.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Strain YNB19, h–ura4-294 leu 1-32 
his3 nup124::HIS3+ ade6-M216 and YNB16, h–leu1-32 ura4-294 were the principal yeast strains used in this study and referred to in the text as null mutant or nup124 and wild-type or WT, respectively. For some experiments YNB38 was used, ade6-M216, leu1-32, his3-D1, ura4-D18 h–. Although the construction of strain YNB19 (YHL7143) was described earlier (Balasundaram et al., 1999), experiments using the same were not elaborated till this report. YNB19 and YNB16 were transformed with various plasmids (Tables 1 and 2). Transformants were propagated in EMM –ura–leu + 15 µM thiamine routinely. When expression of the desired sequence was required, strains were grown in EMM –ura/+/–leu without thiamine to induce the nmt1 or nmt81 promoter.
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Plasmid Constructions
DNA fragments used to create plasmids for this study (Table 2) were generated by PCR using high-fidelity enzyme Turbo Pfu (Stratagene, La Jolla, CA). Oligonucleotide primers are listed in Table 3. All constructs were confirmed by DNA sequencing. huNup153 in pcDNA1 (BNB405, Table 2) was a generous gift from Michael J. Matunis, Johns Hopkins University. pCDL280 and pCDL28 (Table 2) were a generous gift from Mohan Balasubramanian, Temasek Life Sciences Laboratory.
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Molecular and Genetic Techniques
Tf1 transposition and Tf1 cDNA recombination were assayed as previously described (Balasundaram et al., 1999). Briefly, Tf1 transposition was monitored by placing a neo-marked Tf1 element under the control of an inducible nmt1 promoter. The bacterial neo gene allowed cells to grow in the presence of 500 µg of G418/ml. Tf1 cDNA in the nucleus was examined by cDNA recombination assays and is correlated with the ability of cells to grow on G418-containing medium. Tf1 transposition activity was assayed in strains with the neoAI-marked Tf1 plasmid that were induced for the expression of Tf1 on EMM –Ura plates without thiamine for 4 d at 32°C. The plates were then replica printed to EMM plates containing 1 mg of 5-fluoroorotic acid (FOA)/ml. Recombination between cDNA and cellular transposon sequences was scored on FOA-G418 plates after 38 h of growth at 32°C.
Immunofluorescence Microscopy
The nuclear envelope was stained using MAb414 antibodies (Covance, Berkley, CA) that recognize FXFG-repeats in nucleoporins (Davis and Blobel, 1986). HA-tagged proteins were visualized with antihemagglutinin antibody (clone 12CA5, Roche Biochemicals, Basel, Switzerland). Cells were harvested at 0.4–0.8 OD600 and treated for indirect immunofluorescence as previously described (Balasundaram et al., 1999) with the primary antibody anti-HA at a dilution of 1:1000 or MAb414 at 1:200 dilution. Either Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR) was used as the secondary antibody at a 1:1000 dilution and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). All epi-fluorescence microscopy were performed at 1000x magnification. A Leica (Deerfield, IL) DMLB microscope equipped with an Optronics DEI-750T coded CCD camera with Leica Qwin proprietary software was used to capture images that were processed for figure presentation using Adobe Photoshop 5.0 software. Laser-scanning confocal microscopy was performed using a Zeiss LSM 510 confocal microscope equipped with a krypton/argon laser as the light source. Images were captured for CFP (excitation at 458 nm at 11% and emission at 475–500 nm), GFP (excitation at 488 nm at 11% and collection of the signal from 505 nm), YFP (excitation at 488 nm at 11% and emission at 610–640 nm) and DAPI (excitation at 780 nm at 100% and collection of the signal from 435–485 nm). Images were processed using Metamorph software (Universal Imaging, West Chester, PA).
Coimmunoprecipitation and In Vitro Binding Assay
Nup124, TF1-Gag:FLAG, HIV-1 Vpr:FLAG, SIV Vpr:FLAG, SIV Gag:FLAG, Nup153, and GST:6His:S.tag:Kap95 from BNB401, BNB349, BNB422, BNB430, BNB429, BNB405, and BNB400, respectively (Table 2), were transcribed and translated in the presence of [35S]methionine using a TNT-coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's instructions. Equimolar amounts of in vitro–translated proteins were mixed and incubated for 60 min in a binding buffer containing 25 mM HEPES (pH 7.9), 150 mM KCl, 0.1%, Nonidet P-40, 5% glycerol, 0.5 mM dithiothreitol, and 0.4 mM phenylmethylsulfonyl fluoride. Respective antibodies were added to each tube with 400 µl of binding buffer and incubated for 90 min. Protein G-Sepharose (5 mg per tube) was added to all the tubes, which were then incubated for 90 min on a nutator. The beads were washed three times with the binding buffer. The immunoprecipitated protein complexes were eluted from the Sepharose beads and subjected to SDS/PAGE in 8–16% gels. The gels were processed for fluorography. All incubations were carried out at 4°C unless otherwise noted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To differentiate whether this behavior resulted from lack of recruitment of the Tf1-Gag to the nuclear envelope or translocation of Tf1-Gag into the nucleus in a nup124 mutant, we expressed an inducible YFP-tagged Tf1-Gag construct in Wild-type (WT) and nup124 host strains. The nuclear envelope was stained with MAb414 that specifically reacts with FXFG-containing nucleoporins at the nuclear envelope (Davis and Blobel, 1986) and nuclear DNA was stained with DAPI. As depicted in Figure 1, both strains exhibited aggregates in the cytoplasm, a feature that is suggestive of the presence of VLPs (Teysset et al., 2003). The WT strain exhibited YFP-Gag fluorescence within the nucleus, colocalizing with the DAPI stain. However, in contrast, the nup124 strain exhibited a perinuclear staining of YFP-Gag at the nuclear envelope (NE) that colocalized with the MAb414 staining corresponding to FXFG-nucleoporins. No YFP-Gag fluorescence was visible within the nucleus. These observations were confirmed by confocal-laser scanning Z-series imaging (unpublished data). We may thus conclude that Tf1 is recruited to the nuclear envelope even in the absence of Nup124p but is unable to be translocated into the nucleus as is depicted for the wild-type.
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Domains of Nup124p Required for Its Function
As deduced from its amino acid sequence, Nup124p contains two major domains. An N-terminal domain containing five clusters of basic amino acids and a C-terminal domain containing eleven FXFG repeats (Figure 2A). To determine the functionality of these domains with respect to activity of Tf1-Gag as well as nuclear localization, we deleted the abovementioned domains. Mutant nup124 constructs were expressed in S. pombe along with a Tf1 reporter plasmid, pHL449–1 (Balasundaram et al., 1999) (Figure 2A) to determine the levels of Tf1 cDNA recombination and transposition (Figure 2B) by in vivo complementation of the defect in a null mutant using previously described genetic assays (Balasundaram et al., 1999). In addition, immunofluorescent approaches were applied to visualize these mutant HA-tagged Nup124 proteins inside the cell (Figure 2C). As seen in Figure 2B, deleting either the entire eleven FXFG-repeat domain nup124AA571–1150 or nup124AA111–333 containing all five N-terminal basic amino acid clusters exhibited levels of Tf1 cDNA recombination and transposition comparable to the null mutant suggesting that both domains were independently required for Tf1 activity. Next, we asked whether loss of Tf1 transposition activity observed in these mutants was caused by altering their ability to localize to the NPC. Strains with plasmids expressing 3HA:Nup124p and mutants 3HA: nup124pAA571–1150 or 3HA:nup124pAA111–333 (Figure 2C) were examined for localization by indirect immunofluorescence using a monoclonal antibody (mAb) to the HA epitope. Both mutant strains exhibited similar pattern of perinuclear staining when compared with the wild-type. It is therefore possible that in the absence of the either domain, Tf1 VLPs are recruited to the NPC but unable to execute the subsequent steps of the translocation mechanism.
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Activity of HIV-1 Vpr in S. pombe Is Nup124p-dependent
HIV-1 Vpr is known to localize to the nuclear envelope in mammalian cells (Mahalingam et al., 1995a; Vodicka et al., 1998), S. cerevisiae and S. pombe (Zhao and Elder, 2000; Chang et al., 2004). Because Nup124p localizes to the nuclear envelope in S. pombe and is required for the nuclear import of Tf1-Gag (Balasundaram et al., 1999), we asked if Nup124p was one of the targets of Vpr at the NPC. We therefore constructed a plasmid expressing HIV-1 CFP:Vpr (BNB198) whose expression in WT and nup124 strains of S. pombe was under the control of the thiamine-repressible promoter, nmt1. The HIV-1 Vpr was amplified from the pathogenic dual tropic clone 89.6 of HIV-1 (Mahalingam et al., 1997a). In the presence of thiamine, cells showed no inhibition in growth (Figure 3A, left panel). When HIV-1 Vpr expression was induced, wild-type cells were growth-arrested and died subsequently, whereas the nup124 cells survive (Figure 3A, right panel). To test whether a certain population of cells lacking Nup124p had a distinct growth advantage when Vpr was expressed, we grew up cultures in thiamine and spread 1 x 103 and 1 x 106 cells on plates containing thiamine. After 2 d, these plates were printed to ones without thiamine to induce Vpr expression. When plates incubated for 4–6 d at 32°C were examined for growth, we observed that wild-type cells did not form colonies, whereas the null mutant formed a titratable number of colonies (Figure 3B). Our results demonstrate that Nup124p may be one of the targets of Vpr at the NPC because absence of the nucleoporin supports survival of the host strain. Another possibility is that Nup124p allows Vpr access to critical nuclear components required for cell growth and viability. In the absence of Nup124p, Vpr may be mislocalized, so its interactions or effects on nuclear components may be altered or lost.
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HIV-1 Vpr Is Mislocalized in the Absence of Nup124p
Because absence of Nup124 abrogated Vpr-mediated growth inhibition and cell death, we asked whether the localization of HIV-1 Vpr was altered in WT and nup124 strains. WT and nup124 strains expressing the CFP:Vpr fusion protein were examined by confocal microscopy. Vpr was localized at the nuclear envelope (NE) with the FXFG containing nucleoporins (stained with MAb414) both in WT as well as the nup124 strains (Figure 4). However, significant differences in the localization patterns were observed. Accumulation of CFP:Vpr signal appeared usually in the form of a "bleb" or blister in the wild strain exclusively. Z-stack and projection images showed that this bleb characteristically appeared on the inner side of the nuclear envelope and was embedded within the MAb414 staining (Figure 4, top panel, and online Supplementary Video YNB560Z-Stack.MOV). In contrast, no blebs were visible within the nucleus of the mutant. Instead, the NE formed projections into the cytoplasm (Figure 4, bottom panel, and online Supplemmentary Video YNB562Z-Stack.MOV). Indeed, Z-series images confirmed those observations (see Supporting Online Material). Like Tf1-Gag, the HIV-1 Vpr is recruited to the NE, but may be mislocalized in the absence of Nup124p and thus, may not have access to the inner face of the nucleus. Such a result may be consistent with our observation in Figure 3 that absence of Nup124p abrogates loss of cell viability and growth.
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Nup124p Interacts with Tf1-Gag and HIV-1 Vpr In Vitro
Our genetic and localization data imply a relationship between Nup124p and Tf1-Gag or HIV-1 Vpr. To determine whether Vpr and Tf1-Gag were capable of physically interacting with Nup124p, we performed coimmunoprecipitation experiments using individually translated products from rabbit reticulocyte lysates in vitro. DNAs encoding Nup124p, HIV-1 Vpr, SIV-Vpr, Tf1-Gag, and SIV-Gag (Table 2) were individually transcribed and translated in the presence of [35S]methionine. Figure 5A shows the yield and size of the products when 1.0-µl aliquots of the synthesized protein were separated on an 8–16% SDS-PAGE gel. We noted that in each case the synthesized products corresponded to the expected molecular size as indicated. However, we do see some additional species that were possibly incomplete translational products. Approximately equimolar amounts of each translation product shown in Figure 5A were mixed in various combinations and immunoprecipitated with antibodies as indicated in Figure 5B. Immunoprecipitates were analyzed on an 8–16% gradient SDS-PAGE gel and subjected to fluorography. Figure 5B shows that a mixture of Nup124p and Tf1-Gag:FLAG was immunoprecipitated by either anti-FLAG (lane 1) or the MAb414 (lane 2). Similarly, a mixture of Nup124p and HIV-1 Vpr were immunoprecipitated by either the MAb414 (lane 3) or anti-Vpr (lane 4). To exclude the possibility of nonspecific interactions, we used Gag and Vpr or Vpx from the SIV belonging to the same family of lentiviral proteins. To ascertain whether the nuclear import of SIV-Gag, SIV-Vpr, or SIV-Vpx was not dependent on Nup124p, they were expressed from a thiamine-repressible reporter, nmt1 as C-terminal fusions (Table 2) with GFP and transformed into WT and nup124 strains (Table 1). Microscopic examination revealed that localization of SIV-Gag:GFP was cytoplasmic whereas SIV-Vpr:GFP and SIV-Vpx:GFP were nuclear irrespective of the absence or presence of Nup124p (unpublished data). We reasoned therefore that those viral proteins would be appropriate negative controls. Plasmids expressing SIV-Gag: FLAG, SIV-Vpr:FLAG, and SIV-Vpx:FLAG were individually transcribed and translated in the presence of [35S]methionine (Figure 5A). Figure 5B, lanes 5 and 6, show Nup124p does not coimmunoprecipitate with SIV-Gag: FLAG. However, when a mixture of Nup124p and SIV-Vpr: FLAG is treated with MAb414, a 14-kDa band corresponding to SIV-Vpr is observed with MAb414 and a very faint band with anti-FLAG (lanes 7 and 8, respectively), suggesting there might be a weak interaction between Nup124p and SIV-Vpr. No interaction was observed between Nup124p and SIV-Vpx (unpublished data).
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Search for a Mammalian Homolog of Nup124p
Because Nup124p interacted with Tf1-Gag and HIV-1 Vpr in vitro and because of the latter's possible role in AIDS pathogenesis (Sherman et al., 2002b; Tungaturthi et al., 2003), we asked if a human homolog of the nucleoporin might do the same. A BLASTp search revealed three FXFG-containing proteins that share a low similarity with the S. pombe Nup124p (Figure 6A). They were the S. cerevisiae (Sc) nucleoporins ScNup1p, ScNsp1p, and the vertebrate (human) nucleoporin huNup153p. Proteomic analysis of mammalian NPC proteins and comparison with those from S. cerevisiae previously identified Nup1p as a homolog of Nup153 (Stoffler et al., 1999; Cronshaw et al., 2002). Interestingly, all three proteins contain very large numbers of FG-repeats scattered over the entire length of the protein but differ in the position of FXFG-repeat domains. Nup153 resembles Nup124p closely in that the FXFG-repeat domain is at the C-terminus, whereas for Nup1p it is in the middle and Nsp1p, at the N-terminus (Figure 6A). Comparison of Nup124p and Nup153 sequences revealed a significant number of short conserved stretches of residues at the N and C-termini (Figure 6, B and C). We asked therefore, if Nup153 was able to interact with both Tf1-Gag and HIV-1 Vpr in vitro just as Nup124p did. Equal amounts of in vitro translated Nup153, HIV-1 Vpr, and Tf1-Gag proteins were subjected to a coimmunoprecipitation assay using MAb414, anti-Vpr, or anti-FLAG antibodies as described for Figure 5. Figure 7 shows that a mixture of Nup153 and Tf1-Gag:FLAG was immunoprecipitated by either anti-FLAG (lane 1) or the MAb414 (lane 2). Similarly, a mixture of Nup153 and HIV-1 Vpr were immunoprecipitated by either the MAb414, (lane 3) or anti-Vpr (lane 4). As negative controls, [35S]methionine SIV-Gag: FLAG and SIV-Vpr:FLAG, or SIV-Vpx:FLAG were individually incubated with Nup153. As observed for Nup124p, no immunoprecipitate is recovered with either MAb414 or anti-FLAG when treated with SIV-Gag:FLAG (lanes 5 and 6, respectively), whereas faint bands were observed with SIV-Vpr:FLAG (lanes 7 and 8). No interaction was observed between Nup153 and SIV-Vpx (unpublished data). Our data therefore show that Nup153p interacts with HIV1-Vpr and Tf1-Gag in a manner identical to that of Nup124p. Lane 9 depicts the positive control interaction between SIV-Gag and SIV-Vpr. Because both nucleoporins Nup124p and Nup153 localize to the NPC we wanted to eliminate the possibility that any non-nucleoporin that also localizes to the NPC would interact with Tf1-Gag or HIV-1 Vpr. The S. pombe Kap95 is an importin 
that concentrates at the nuclear rim (Chen et al., 2004). We cloned Kap95 into an expression vector pET41b so as to generate (BNB400, Table 2) an N-terminally tagged GST-6His-S.Tag-Kap95 fusion product. [35S]methionine-GST-6His-S.Tag-Kap95 treated with [35S]HIV-1 Vpr or [35S]Tf1-Gag was subjected to a coimmunoprecipitation assay using anti-FLAG (Figure 7B, lane 1) or anti-Vpr (Figure 7B, lane 2) exactly as described for Figure 5. No immunoprecipitate was recovered with anti-FLAG, suggesting that Kap95 did not interact with Tf1-Gag. Interestingly, Kap95 did interact with HIV1-Vpr (lane 2). The above reactions were also conducted with anti-His, confirming the above observation (unpublished data).
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A Unique Domain of Nup153 Is Absolutely Essential for Tf1 Activity in a nup124 Mutant
Because we had previously demonstrated an N-terminal Nup124p-Tf1-Gag interaction in a yeast two-hybrid analysis (Balasundaram et al., 1999) and in this report both Nup124p and Nup153 independently interacted with Tf1-Gag in vitro, we asked if the Tf1-Gag-binding activity was generated by a homologous domain within these two nucleoporins. The human Nup153 is a large nucleoporin comprising three domains (McMorrow et al., 1994). The N-terminal domain (AA1–339) that has NE-binding activity (Enarson et al., 1998) and an M 9-like domain (Nakielny et al., 1999). The middle domain contains four C2-C2 type zinc-finger motifs (AA657–880) comprising the Ran-GDP–binding domain. Nup124p on the other hand, lacks at least the M 9-like domain and C2-C2 zinc-finger motifs domains (Figure 6A). Nup153 has a C-terminal domain (572 residues) comprising all 15 FXFG-repeats (AA903–1475). Similarly, all 11 FXFG repeats of Nup124p are in the 588-residue C-terminal domain (AA571–1159) (Figure 6D). The number of identical residues within the FXFG region was significantly high, although no significant alignment of FG-/FXFG repeats was evident (Figure 6D). Interestingly, Nup124p shares an unrecognizable domain that lies between the defined M9-like (AA235–300) and Zn-finger (AA657–880) regions of Nup153 (Figure 6, B and C). Using consensus amino acid residues from each of boxes 1–4 (Figure 6C) to scan protein sequences shown in Figure 6A, we asked whether similar subdomain structures were present. We noted that boxes 1–4 were absent in Nsp1p, Nup1p, and CAN/Nup214 and that box 2 was found only in Nup124p and the human Nup153 among the known nucleoporins (including Nup153 from nonhuman species) to date (unpublished data). Because Nup124pAA272–454 was part of the Tf1-Gag-binding domain (Balasundaram et al., 1999) and we have shown in this report that Nup124pAA111–331 was required for Tf1-transposition (Figure 2), we reasoned that this domain (Figure 6, B and C, boxes 1–4) could be a critical determinant in the functioning of these two nucleoporins as mediators of Tf1 transposition. We therefore asked if amino acids 272–454 of Nup124p or 448–634 of Nup153 were critical for Tf1 activity. nmt81-Nup124 (BNB439) expressing the full-length (WT) Nup124 gene was used as the parent plasmid into which all further manipulations were made. In the first instance amino acids 272–454 were deleted in Nup124p (nup124AA272–454). Nucleotides encoding the Nup124AA272–454 deletion were cloned into BNB439 to form BNB469. We next replaced Nup124AA272–454 with amino acids 448–634 from Nup153 (nup124AA272–454::Nup153AA448–634). Nucleotides encoding the Nup124AA272–454::Nup153AA448–634 fusion were cloned into BNB439 to form BNB470. The nmt81-nup124AA1–1159, nmt81-nup124AA272–454, and nmt81-nup124AA272–454::Nup153AA448–634 constructs were expressed in the nup124 mutant along with a Tf1 reporter plasmid, pHL449–1 (Table 1) to determine the levels of Tf1 cDNA recombination and transposition by genetic assays described earlier under Figure 2, A and B. As depicted in Figure 8A, absence of amino acids 272–454 in Nup124p caused a dramatic fall in Tf1 activity (patches 3–4 comparable to that of the null mutant as negative control, patch 1) when compared with the full-length Nup124p (patch 2), indicating that those amino acids were critical for Tf1 function. However, replacing those deleted amino acids with AA448–634 from the human Nup153 completely restored Tf1 activity (patches 5 and 6) to WT levels. These results clearly indicate that the unique domain(s) described above are transferable and therefore, functionally conserved between Nup124p and Nup153. To know if heterologous expression of full-length Nup153 in a nup124 mutant would reinstate wild-type levels of Tf1 cDNA recombination and transposition, the full-length human Nup153 gene was cloned from BNB405 into an S. pombe vector to form nmt81:Nup153 AA1–1475 (BNB492A/B, Table 2). nup124 null mutant and WT strains bearing these constructs as indicated in Figure 8B were subjected to Tf1 cDNA recombination and transposition assays as previously described. Tf1 cDNA recombination and transposition from strains expressing nmt81:Nup153 AA1–1475 did not complement the Tf1 activity in a nup124 mutant when compared with an equivalent strain expressing nmt81:Nup124 AA1–1159 (Figure 8B). Taken together, our results demonstrate that expression of Nup153AA448–634 within the context of an nup124AA272–454 deletion mutation facilitated Tf1 transposition, whereas the full-length Nup153 is unable to do so in a nup124 null mutant.
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Abrogation of Tf1 Activity when AA 264–454 of Nup124p or AA447–634 from Human Nup153 Is Overexpressed
Overexpression studies may also be used to ascertain the functionality of a gene or its product if it results in a loss-/gain-of-function or a dominant-negative phenotype. By overexpressing the unique domains of Nup124p and Nup153 in a WT background we reasoned that should these domains be interacting with either a viral component or some other factor, then the ratio of interacting components may not be favorable and would result in lowering transposition. To test whether the AA 272–454 of Nup124p or AA448–634 from human Nup153 was mechanistically required for the Tf1 transposition process, we asked if overexpression of the aforementioned fragments would cause an inhibition of Tf1 transposition in a WT background without inhibiting growth of the host. Assays for dominant negative phenotypes have been successfully used to identify and dissect nuclear transport processes involving two or more interacting species (Iovine et al., 1995; Bastos et al., 1996; Fornerod et al., 1997; Shah and Forbes, 1998) and in this particular context, would be consistent with the titration of a Nup124p-interacting protein by an excess of the Nup124p or Nup153-fragment. Furthermore, such a result would also imply a specific (Tf1-related) function rather than a global effect. Nucleotides encoding Nup124AA264–454 and Nup153AA447–634 were cloned (AA264–454 and AA447–634 were used instead of AA272–634 and AA448–634, respectively, for purely cloning reasons) into vectors so that their transcription was under the control of either the thiamine repressible nmt81 (lowest strength) or an nmt1 promoter (highest strength) (Figure 9B) and expressed in the WT along with the Tf1 reporter plasmid, pHL449–1 (Table 1) to determine the levels of Tf1 cDNA recombination and transposition by genetic assays described earlier under Figure 2, A and B. As depicted in Figure 9A, overexpression of both nup124 and nup153 fragments at low levels (nmt81, patches 7–8 and 9–10, respectively) did not inhibit Tf1 activity. However, when overexpressed from the highest strength promoter, nmt1 both fragments caused an equal and almost complete knock-down of Tf1 activity (nmt1, patches 11–12 and 13–14, respectively) when compared with the positive (patches 2–6) or negative (patch 1) controls for the Tf1 assay. A 10–20% decrease in colony size was observed between nmt81- and nmt1-induced cultures when compared with noninduced cultures (Figure 9C), suggesting that full-strength induction of the fragments had a slight to moderate effect on growth. Two conclusions may be drawn from the experiment illustrated in Figure 9. First, overexpression of the domain knocks down Tf1 transposition specifically in a dose-dependent manner without significantly affecting growth and second, the Nup153pAA447–634 domain acts in a similar way to the Nup124pAA264–454 fragment. Taken together, our data from Figures 8 and 9 demonstrate that a unique domain found in these two nucleoporins Nup124p and Nup153 is essential for Tf1 retrotransposon activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Balasundaram et al., 1999). The ability of Tf1-Gag to be imported into the nucleus in these cells correlated well with wild-type levels of Tf1 cDNA recombination and transposition activity. Interestingly, Tf1-Gag appeared exclusively at the NE colocalizing with MAb414-stained FG-nucleoporins even in the absence of Nup124p. It is noteworthy that mere recruitment of Tf1-Gag to the nuclear envelope in the null mutant is not sufficient to promote Tf1 activity implying that Nup124p was not required for recruitment and docking of Tf1-Gag to the NE but rather for translocation of Tf1-Gag into the nucleus. Consistent with the genetic (in vivo) evidence presented here on the involvement of Nup124p in Tf1 transposition is the observation that Nup124p appears to interact with TF1-Gag rather than with SIV-Gag in vitro in our immunoprecipitation assay. On the basis of yeast two-hybrid and GST-pull down assays, we had previously shown that the N-terminal half of Nup124p binds to Tf1-Gag (Balasundaram et al., 1999). Furthermore, the NLS activity of Gag when expressed in a heterologous protein was specifically dependent on the presence of Nup124p (Dang and Levin, 2000). It is not clear, however, whether Nup124p facilitates Tf1-Gag independently or within context of the entire virus-like particle. Tf1-Gag is required for particle assembly in the cytoplasm and is also found associated with 50-nm particles inside the nucleus, implying that Tf1 particles were able to pass through nuclear pores without first being disassembled before nuclear import (Teysset et al., 2003).
Cellular Effects of HIV-1 Vpr Are Mediated via Nup124p
In vivo, Vpr shows multiple activities both in mammalian and S. pombe cells, which include nuclear transport, induction of cell cycle G2 arrest, morphological changes, and cell death (Di Marzio et al., 1995; Rogel et al., 1995; Elder et al., 2000, 2002; de Noronha et al., 2001; Chang et al., 2004). Several lines of evidence presented here suggest that nuclear import of HIV-1 Vpr in the fission yeast involves Nup124p. Our investigations show that absence of Nup124p gives cells expressing Vpr a distinct growth advantage in contrast to wild-type cells. Indeed, such an observation is strong evidence in favor of a critical role for Nup124p in Vpr-mediated cell toxicity. Second, presence or absence of Nup124p affects the nuclear localization of HIV-1 Vpr. Finally, Nup124p appears to interact weakly with Tf1-Gag or HIV-1 Vpr in an in vitro coimmunoprecipitation assay. To eliminate the possibility of artifacts in immunoprecipitation arising from the use of a single antibody, we tested the ability of both proteins to be recovered from immunoprecipitates in any given set of reactions independently by two different antibodies. Despite numerous attempts, we were unable to secure stoichiometric amounts of both sets of proteins in our immunoprecipitates. Three possibilities may be entertained. One is that the interaction between Nup124p and Tf1-Gag or Vpr is weak or transient. The other is because our interaction data were derived from in vitro reactions, it is possible that some putative cofactors in the reticulocyte lysate could be different from those found in the fission yeast. Finally, that our observed interactions may not be direct. It is possible that Nup124p is part of a subcomplex at the NPC and that we may be missing additional elements or yeast specific factors not present in the reticulocyte lysate that may in fact, contribute to binding at the pore.
Because a significant fraction of Vpr could interact with components of NPC (Fouchier et al., 1998; Popov et al., 1998a, 1998b; Fouchier and Malim, 1999; Le Rouzic et al., 2002), it is possible that Vpr needs to localize to the interior of the nuclear rim or to associate with certain proteins at the nuclear face of the nuclear envelope to be effective in its cell killing activity. We had previously observed that Nup124p concentrated within the region between the nuclear and cytoplasmic faces of the NPC (Balasundaram et al., 1999). Thus, in the absence of Nup124p from the NPC, Vpr may be denied access to nuclear components it would otherwise interact with. The ability of Vpr to disrupt normal nuclear morphology was noted in previous studies in S. pombe (Zhao et al., 1998; Chang et al., 2004). Recently, Chang et al. (2004) have observed defects in the assembly and function of the mitotic spindle in S. pombe expressing HIV-1 Vpr. Their results suggested that perturbations in nuclear architecture, in fact, might lead to delocalization of two proteins sad1p and the polo kinase plo1p from the spindle pole body (SPB), resulting in defects in the assembly and function of the mitotic spindle. de Noronha et al. (2001) observed that Vpr expression induced transient herniations or blebbing in the nuclear lamina, leading to local bursting and mixing of nuclear and cytoplasmic components, especially some key cell cycle regulators like Cdc25, cyclin B1, Wee 1, and perhaps many other soluble components. Although yeast nuclei do not possess a nuclear lamina, these NE herniations or bleb-like structures are clearly visible in wild-type cells of S. pombe when Vpr is expressed (depicted in Figure 4 and Y. Zhao, personal communication), suggesting a common mechanism that underlies Vpr activity in both mammalian systems and yeast.
Is the Vertebrate (human) Nup153p a Functional Homolog of the Fission Yeast Nup124p?
Proteomic comparisons of nucleoporins between yeast (S. cerevisiae) and mammals reveal a moderate level of conservation despite anatomical differences and a very low sequence homology (Rout et al., 2000; Cronshaw et al., 2002). However, the extent of functional homology has yet to be fully realized. In general, sequence comparisons between nucleoporins from mammalian and yeast (S. cerevisiae) yield very poor alignments and in many cases direct sequence homology is not apparent or even feasible. The mammalian Nup153 and its possible homologues in yeast is a case in point with some reports implying that the vertebrate Nup153 has no identifiable yeast homolog (Vasu et al., 2001; Walther et al., 2001), whereas others recording Nup1p of S. cerevisiae as its homolog (Stoffler et al., 1999; Cronshaw et al., 2002). Similarly, Nup60p of S. cerevisiae was considered a yeast "version" of Nup153 based on common binding partners rather than comprehensive sequence homology (Hase and Cordes, 2003). Our own analysis summarized in Figure 6 shows that Nup124p may be a possible homolog of Nup153 based on sequence comparison. We therefore reasoned that in a manner analogous to that of Nup124p, the mammalian Nucleoporin Nup153p might be the responsible for translocating Vpr into the nucleus. Nup153, like Nup124p, binds weakly to both Vpr and Tf1-Gag. Furthermore, both nucleoporins did not bind SIV-Gag or SIV-Vpx, indicating that their interaction with Tf1-Gag was selective, whereas they exhibited a very weak binding to SIV-Vpr. The latter result is possible given the high sequence conservation between Vpr from HIV-1 and SIV isolates (see Figure 1 in Zhou and Ratner, 2000). However, expression of full-length Nup153 failed to complement the Tf1 transposition defect in a nup124 null mutant. An immediate admission to our findings reported here therefore is that the vertebrate Nup153 is not a homolog of Nup124p. Yet, swapping the fission yeast nup124 gene fragment (encoding AA272–454) with the nup153 gene fragment (encoding AA448–634) was indeed able to restore wild-type levels of Tf1 cDNA recombination and transposition in a nup124pAA272–454 mutant. Furthermore, the ability of Nup124pAA264–454 and Nup153AA447–634 to similarly cause a dose-dependent inhibition of the Tf1 cDNA recombination and transposition processes (specifically inhibiting viral replication by overexpression of critical nucleoporin domains is an attractive antiviral strategy) may be seen as additional evidence in support of that domain in Nup153 as being the functional homolog of the respective domain in Nup124p. AA448–634 of huNup153 is devoid of any recognizable motif (Sukegawa and Blobel, 1993; McMorrow et al., 1994), whereas Nup124pAA272–454 fell within the domain required for Tf1-Gag binding (Balasundaram et al., 1999) and the nup124pAA111–333 mutant did not support Tf1 activity (see Figure 2B). Thus, in the context of our results, Nup153 may have evolutionarily retained one Tf1-transposition competent domain and lost one (or more) other required domain(s) or conversely, acquired other specialized domains that may, within the context of the full-length protein, inhibit Tf1 transposition activity. In the latter scenario, multiple domains unique to Nup153 do not appear to have apparent equivalents in Nup124p (or in Nup1p for that matter). These domains are the TRN1-binding domain or M9 NLS, AA235–300 (Nakielny et al., 1999), the RNA-binding domain, AA250–400 (Dimaano et al., 2001), and the Zn-finger domain, AA657–880 (Sukegawa and Blobel, 1993; McMorrow et al., 1994).
The presence of short stretches of conserved amino acids between Nup124p and huNup153 might indicate that both proteins derive from a common ancestor. This is perhaps equally true of Nup60 (Hase and Cordes, 2003) or Nup1p (Stoffler et al., 1999; Cronshaw et al., 2002). Nup153 having acquired additional domains concomitant with its involvement in specialized tasks like in nuclear envelope breakdown and reformation during cell division (Bodoor et al., 1999; Daigle et al., 2001) through its interaction with lamin B3 (Smythe et al., 2000), the COP1 complex (Liu et al., 2003), and ability to anchor other proteins at the NPC (Walther et al., 2001). In the fission yeast, such a role for Nup124p is expected to be redundant since there is no "open" mitosis. Furthermore, in vertebrate cells, Nup153 is believed to play key roles in import of proteins into the nucleus (Radu et al., 1995; Shah and Forbes, 1998; Shah et al., 1998), export of proteins and RNA from the nucleus (Bastos et al., 1996; Ullman et al., 1999; Ball et al., 2004), and in the architecture, assembly, and functioning of the NPC (Nakielny et al., 1999; Smythe et al., 2000; Walther et al., 2001; Fahrenkrog et al., 2002; Hutchison, 2002; Griffis et al., 2004). In the fission yeast, such essential tasks may be distributed among other nucleoporins because Nup124p may be deleted without loss of nuclear transport function or viability and to the best of our knowledge has no other phenotype other than the Tf1 transposition defect (Balasundaram et al., 1999).
Future Perspectives
Nup153p is described to have close structural and functional associations with the nuclear lamina (Smythe et al., 2000), the site of Vpr-induced NE herniations or blebs and showing significant changes in patterns of lamins A, B and C expression (de Noronha et al., 2001). A direct interaction between Vpr and lamins was sought but not detected (de Noronha et al., 2001). Though the mechanism behind the formation and bursting of Vpr-induced blebs remains unknown (de Noronha et al., 2001), it is tempting to speculate that Nup153 may be one of the key components affected, given its location on the distal nucleoplasmic ring of the NPC (Pante et al., 1994) and its association with lamin B3 (Smythe et al., 2000). Recent work in S. cerevisiae and S. pombe has identified and described the presence of nucleoplasmic rings, basket or fishtrap, and other NPC structures similar to those found in higher eukaryotic species (Kiseleva et al., 2004). Although yeasts do not possess a lamin structure undergirding the nuclear envelope, it is proposed that there may be equivalent structures and components including the yeast equivalent of Nup153 (Hutchison, 2002). Because in our studies, Nup153, like Nup124p was able to bind HIV-1 Vpr in vitro and the overexpression of the Nup153AA447–634 domain was able to inhibit replication of the Tf1 retrotransposon, our observation of the Vpr-induced blebs and death in wild-type cells of S. pombe may be consistent with a similar pathology found in mammalian cells. It still, however, remains to be determined if overexpression of Nup153AA448–634 in HeLa cells will prevent the Vpr-induced nuclear envelope blebbing or inhibit HIV-1 infection of macrophages. Another exciting prospect is to find binding partners of Nup124p or Nup153 or Nup124p/Nup153 chimeras required for Tf1 transposition or HIV-1 Vpr activity in the fission yeast. It is likely that these binding partners in the fission yeast will lead to identification of proteins with corresponding functions in mammalian systems that affect nuclear import of viral components. The recent availability of mutants for nucleoporins and nuclear transport factors as those described for budding yeast (Strawn et al., 2004) or fission yeast (Chen et al., 2004) will serve as powerful tools to understand the mechanism of nuclear import and action of viruses or viral components that do require an obligatory nuclear passage.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: David Balasundaram (davidb{at}imcb.astar.edu.sg).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Balasundaram, D., Benedik, M. J., Morphew, M., Dang, V. D., and Levin, H. L. ((1999). ). Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1. Mol. Cell. Biol. 19, , 5768–5784.
Ball, J. R., Dimaano, C., and Ullman, K .S. ((2004). ). The RNA binding domain within the nucleoporin Nup153 associates preferentially with single-stranded RNA. RNA 10, , 19–27.
Bastos, R., Lin, A., Enarson, M., and Burke, B. ((1996). ). Targeting and function in mRNA export of nuclear pore complex protein Nup153. J. Cell Biol. 134, , 1141–1156.
Bodoor, K., Shaikh, S., Enarson, P., Chowdhury, S., Salina, D., Raharjo, W. H., and Burke, B. ((1999). ). Function and assembly of nuclear pore complex proteins. Biochem. Cell Biol. 77, , 321–329.[CrossRef][Medline]
Bukrinsky, M. I., and Haffar, O. K. ((1997). ). HIV-1 nuclear import: in search of a leader. Front. Biosci. 2, , d578–587.[Medline]
Chang, F., Re, F., Sebastian, S., Sazer, S., and Luban, J. ((2004). ). HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes. Mol. Biol. Cell. 15, , 1793–1801.
Chen, X. Q., Du, X., Liu, J., Balasubramanian, M. K., and Balasundaram, D. ((2004). ). Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis. Yeast 21, , 495–509.[CrossRef][Medline]
Cronshaw, J. M., Krutchinsky, A. N., Zhang, W., Chait, B. T., and Matunis, M. J. ((2002). ). Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, , 915–927.
Cronshaw, J. M., and Matunis, M. J. ((2004). ). The nuclear pore complex: disease associations and functional correlations. Trends Endocrinol. Metab. 15, , 34–39.[CrossRef][Medline]
Daigle, N., Beaudouin, J., Hartnell, L., Imreh, G., Hallberg, E., Lippincott-Schwartz, J., and Ellenberg, J. ((2001). ). Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J. Cell Biol. 154, , 71–84.
Dang, V. D., and Levin, H. L. ((2000). ). Nuclear import of the retrotransposon Tf1 is governed by a nuclear localization signal that possesses a unique requirement for the FXFG nuclear pore factor Nup124p. Mol. Cell. Biol. 20, , 7798–7812.
Davis, L. I., and Blobel, G. ((1986). ). Identification and characterization of a nuclear pore complex protein. Cell 45, , 699–709.[CrossRef][Medline]
de Noronha, C. M., Sherman, M. P., Lin, H. W., Cavrois, M. V., Moir, R. D., Goldman, R. D., and Greene, W. C. ((2001). ). Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr. Science 294, , 1105–1108.
Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R., and Landau, N. R. ((1995). ). Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69, , 7909–7916.[Abstract]
Dimaano, C., Ball, J. R., Prunuske, A. J., and Ullman, K. S. ((2001). ). RNA association defines a functionally conserved domain in the nuclear pore protein Nup153. J. Biol. Chem. 276, , 45349–45357.
Elder, R. T., Benko, Z., and Zhao, Y. ((2002). ). HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints. Front. Biosci. 7, , d349–357.[Medline]
Elder, R. T., Yu, M., Chen, M., Edelson, S., and Zhao, Y. ((2000). ). Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint. Virus Res. 68, , 161–173.[CrossRef][Medline]
Enarson, P., Enarson, M., Bastos, R., and Burke, B. ((1998). ). Amino-terminal sequences that direct nucleoporin nup153 to the inner surface of the nuclear envelope. Chromosoma 107, , 228–236.[CrossRef][Medline]
Fahrenkrog, B., Maco, B., Fager, A. M., Koser, J., Sauder, U., Ullman, K. S., and Aebi, U. ((2002). ). Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. J. Struct. Biol. 140, , 254–267.[CrossRef][Medline]
Fornerod, M., van Deursen, J., van Baal, S., Reynolds, A., Davis, D., Murti, K. G., Fransen, J., and Grosveld, G. ((1997). ). The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 16, , 807–816.[CrossRef][Medline]
Fouchier, R. A., and Malim, M. H. ((1999). ). Nuclear import of human immunodeficiency virus type-1 preintegration complexes. Adv. Virus Res. 52, , 275–299.[Medline]
Fouchier, R. A., Meyer, B. E., Simon, J. H., Fischer, U., Albright, A. V., Gonzalez-Scarano, F., and Malim, M. H. ((1998). ). Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J. Virol. 72, , 6004–6013.
Griffis, E. R., Craige, B., Dimaano, C., Ullman, K. S., and Powers, M. A. ((2004). ). Distinct functional domains within nucleoporins Nup153 and Nup98 mediate transcription dependent mobility. Mol. Biol. Cell Mol. Biol. Cell 15, , 1991–2002.
Hase, M. E., and Cordes, V. C. ((2003). ). Direct interaction with nup153 mediates binding of tpr to the periphery of the nuclear pore complex. Mol. Biol. Cell 14, , 1923–1940.
Hutchison, C. J. ((2002). ). Lamins: building blocks or regulators of gene expression? Nat. Rev. Mol. Cell. Biol. 3, , 848–858.[CrossRef][Medline]
Iovine, M. K., Watkins, J. L., and Wente, S. R. ((1995). ). The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor. J. Cell Biol. 131, , 1699–1713.
Kau, T. R., Way, J. C., and Silver, P. A. ((2004). ). Nuclear transport and cancer: from mechanism to intervention. Nat. Rev. Cancer 4, , 106–117.[Medline]
Kiseleva, E., Allen, T. D., Rutherford, S., Bucci, M., Wente, S. R., and Goldberg, M. W. ((2004). ). Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments. J. Struct. Biol. 145, , 272–288.[CrossRef][Medline]
Le Rouzic, E., Mousnier, A., Rustum, C., Stutz, F., Hallberg, E., Dargemont, C., and Benichou, S. ((2002). ). Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J. Biol. Chem. 277, , 45091–45098.
Levin, H. L., Weaver, D. C., and Boeke, J. D. ((1993). ). Novel gene expression mechanism in a fission yeast retroelement: Tf1 proteins are derived from a single primary translation product. EMBO J. 12, , 4885–4895.[Medline]
Liu, J., Prunuske, A. J., Fager, A. M., and Ullman, K. S. ((2003). ). The COPI complex functions in nuclear envelope breakdown and is recruited by the nucleoporin Nup153. Dev. Cell 5, , 487–498.[CrossRef][Medline]
Lu, Y. L., Bennett, R. P., Wills, J. W., Gorelick, R., and Ratner, L. ((1995). ). A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles. J. Virol. 69, , 6873–6879.[Abstract]
Lu, Y. L., Spearman, P., and Ratner, L. ((1993). ). Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J. Virol. 67, , 6542–6550.
Mahalingam, S., Ayyavoo, V., Patel, M., Kieber-Emmons, T., and Weiner, D. B. ((1997a). ). Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J. Virol. 71, , 6339–6347.[Abstract]
Mahalingam, S., Collman, R. G., Patel, M., Monken, C. E., and Srinivasan, A. ((1995a). ). Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization. Virology 212, , 331–339.[CrossRef][Medline]
Mahalingam, S., Khan, S. A., Jabbar, M. A., Monken, C. E., Collman, R. G., and Srinivasan, A. ((1995b). ). Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for virion incorporation. Virology 207, , 297–302.[CrossRef][Medline]
Mahalingam, S., MacDonald, B., Ugen, K. E., Ayyavoo, V., Agadjanyan, M. G., Williams, W. V., and Weiner, D. B. ((1997b). ). In vitro and in vivo tumor growth suppression by HIV-1 Vpr. DNA Cell Biol. 16, , 137–143.[Medline]
McMorrow, I., Bastos, R., Horton, H., and Burke, B. ((1994). ). Sequence analysis of a cDNA encoding a human nuclear pore complex protein, hnup153. Biochim. Biophys. Acta 1217, , 219–223.[Medline]
Nakielny, S., Shaikh, S., Burke, B., and Dreyfuss, G. ((1999). ). Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain. EMBO J. 18, , 1982–1995.[CrossRef][Medline]
Pante, N., Bastos, R., McMorrow, I., Burke, B., and Aebi, U. ((1994). ). Interactions and three-dimensional localization of a group of nuclear pore complex proteins. J. Cell Biol. 126, , 603–617.
Popov, S., Rexach, M., Ratner, L., Blobel, G., and Bukrinsky, M. ((1998a). ). Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex. J. Biol. Chem. 273, , 13347–13352.
Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M. A., Ratner, L., Lane, C. M., Moore, M. S., Blobel, G., and Bukrinsky, M. ((1998b). ). Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J. 17, , 909–917.[CrossRef][Medline]
Radu, A., Blobel, G., and Moore, M. S. ((1995). ). Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc. Natl. Acad. Sci. USA 92, , 1769–1773.
Rogel, M. E., Wu, L. I., and Emerman, M. ((1995). ). The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. J. Virol. 69, , 882–888.[Abstract]
Rout, M. P., Aitchison, J. D., Suprapto, A., Hjertaas, K., Zhao, Y., and Chait, B. T. ((2000). ). The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, , 635–651.
Selig, L., Pages, J. C., Tanchou, V., Preveral, S., Berlioz-Torrent, C., Liu, L. X., Erdtmann, L., Darlix, J., Benarous, R., and Benichou, S. ((1999). ). Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses. J. Virol. 73, , 592–600.
Shah, S., and Forbes, D. J. ((1998). ). Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors. Curr. Biol. 8, , 1376–1386.[CrossRef][Medline]
Shah, S., Tugendreich, S., and Forbes, D. ((1998). ). Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr. J. Cell Biol. 141, , 31–49.
Sherman, M. P., De Noronha, C. M., Williams, S. A., and Greene, W. C. ((2002a). ). Insights into the biology of HIV-1 viral protein R. DNA Cell Biol. 21, , 679–688.[CrossRef][Medline]
Sherman, M. P., Schubert, U., Williams, S. A., de Noronha, C. M., Kreisberg, J. F., Henklein, P., and Greene, W. C. ((2002b). ). HIV-1 Vpr displays natural protein-transducing properties: implications for viral pathogenesis. Virology 302, , 95–105.[CrossRef][Medline]
Smythe, C., Jenkins, H. E., and Hutchison, C. J. ((2000). ). Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs. EMBO J. 19, , 3918–3931.[CrossRef][Medline]
Stoffler, D., Fahrenkrog, B., and Aebi, U. ((1999). ). The nuclear pore complex: from molecular architecture to functional dynamics. Curr. Opin. Cell Biol. 11, , 391–401.[CrossRef][Medline]
Strawn, L. A., Shen, T., Shulga, N., Goldfarb, D. S., and Wente, S. R. ((2004). ). Minimal nuclear pore complexes define FG repeat domains essential for transport. Nat. Cell Biol. 6, , 197–206.[Medline]
Sukegawa, J., and Blobel, G. ((1993). ). A nuclear pore complex protein that contains zinc finger motifs, binds DNA, and faces the nucleoplasm. Cell 72, , 29–38.[CrossRef][Medline]
Teysset, L., Dang, V. D., Kim, M. K., and Levin, H. L. ((2003). ). A long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe expresses a Gag-like protein that assembles into virus-like particles which mediate reverse transcription. J. Virol. 77, , 5451–5463.
Tungaturthi, P. K., Sawaya, B. E., Singh, S. P., Tomkowicz, B., Ayyavoo, V., Khalili, K., Collman, R. G., Amini, S., and Srinivasan, A. ((2003). ). Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr. Biomed. Pharmacother. 57, , 20–24.[CrossRef][Medline]
Ullman, K. S., Shah, S., Powers, M. A., and Forbes, D. J. ((1999). ). The nucleoporin nup153 plays a critical role in multiple types of nuclear export. Mol. Biol. Cell 10, , 649–664.
Vasu, S., Shah, S., Orjalo, A., Park, M., Fischer, W. H., and Forbes, D. J. ((2001). ). Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export. J. Cell Biol. 155, , 339–354.
Vodicka, M. A., Koepp, D. M., Silver, P. A., and Emerman, M. ((1998). ). HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 12, , 175–185.
Walther, T. C., Fornerod, M., Pickersgill, H., Goldberg, M., Allen, T. D., and Mattaj, I. W. ((2001). ). The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J. 20, , 5703–5714.[CrossRef][Medline]
Zhao, Y., and Elder, R. T. ((2000). ). Yeast perspectives on HIV-1 VPR. Front. Biosci. 5, , D905–916.[Medline]
Zhao, Y., Yu, M., Chen, M., Elder, R. T., Yamamoto, A., and Cao, J. ((1998). ). Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline. Virology 246, , 266–276.[CrossRef][Medline]
Zhou, Y., and Ratner, L. ((2000). ). Phosphorylation of human immunodeficiency virus type 1 Vpr regulates cell cycle arrest. J. Virol. 74, , 6520–6527.
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 T. Kobori, M. Kodama, K. Hizume, S. H. Yoshimura, T. Ohtani, and K. Takeyasu Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit J. Electron Microsc. (Tokyo), January 1, 2006; 55(1): 31 - 40. [Abstract] [Full Text] [PDF]
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X. Du, M. R.K. S. Rao, X. Q. Chen, W. Wu, S. Mahalingam, and D. Balasundaram The Homologous Putative GTPases Grn1p from Fission Yeast and the Human GNL3L Are Required for Growth and Play a Role in Processing of Nucleolar Pre-rRNA Mol. Biol. Cell, January 1, 2006; 17(1): 460 - 474. [Abstract] [Full Text] [PDF]
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nti-GFP antibody, respectively. Nuclear DNA was stained with DAPI. Arrows indicate nuclear blebs or cytoplasmic projections. Experimental and image-capture conditions were identical to those detailed in 
