Importin- Family Members Mediate Alpharetrovirus G
http://www.100md.com
病菌学杂志 2006年第4期
Departments of Biochemistry and Molecular Biology Medicine
Microbiology and Immunology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033
ABSTRACT
The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin- family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-/ (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.
INTRODUCTION
Retroviruses, as obligate intracellular parasites, exist in a dynamic relationship with the host cell. These significant pathogens coopt host cell machinery to facilitate viral entry, gene expression, protein synthesis, and virion release. Identification of cellular factors involved in the retroviral life cycle could be facilitated by the use of a well-characterized genetic system. Saccharomyces cerevisiae serves as the ideal candidate, as each gene has been systematically deleted or mutated, enabling a search for gene products that participate in viral processes. Although the complete retrovirus replication cycle cannot be recapitulated in yeast, discrete steps of the life cycle can be studied in detail to identify host factors that are required.
The gag gene, one of the three major genes shared by all retroviruses, encodes the Gag polyprotein, which drives the formation and release of virus particles from the host cell membrane. Gag proteins are synthesized on cytosolic ribosomes, and previously it was thought that Gag was transported directly to the plasma membrane to drive virus assembly. However, we discovered that the Gag protein of the alpharetrovirus Rous sarcoma virus (RSV), a tumor-producing virus that infects domestic fowl, undergoes nucleocytoplasmic shuttling prior to its transport to the plasma membrane (36). The role of Gag nuclear trafficking in the viral life cycle is not well understood; however, both virion release and infectivity are inhibited when Gag nuclear export is prevented (36; L. Scheifele and L. Parent, unpublished data, 2004).
Following virion release, the RSV Gag polyprotein undergoes proteolytic cleavage into the MA, p2, p10, CA, NC, and PR proteins. Extracellular virus particles bind to receptors and undergo fusion with the cell membrane, and the viral core enters the cytosol. Reverse transcription proceeds, and the viral ribonuclear complex (known as the preintegration complex [PIC]) enters the nucleus, where proviral DNA integrates into the cellular genome. For human immunodeficiency virus type 1 (HIV-1), which infects nondividing cells, the PIC traverses through the nuclear pore complex (NPC) by use of redundant nuclear localization signals (NLSs) in the integrase, Vpr, and MA proteins (40, 49). In contrast, RSV primarily infects dividing cells, and therefore it was believed that the import of the PIC depended on the breakdown of the nuclear envelope during mitosis. However, RSV does replicate at low levels in quiescent cells (15, 18); thus, there is some level of active transport of the PIC, and nuclear entry likely depends on the karyophilic properties of viral proteins.
Nuclear transport occurs through the NPC, which is composed of 30 nucleoporins (35), via interactions between NLSs on protein cargoes and a family of soluble nuclear receptors known as importins or karyopherins (reviewed in reference 45). The classical import pathway involves direct recognition of a basic NLS by importin- (Kap95p) or the use of importin- as an adaptor for binding to importin-. The diversity of nonclassical NLSs is still being elaborated, and transport of proteins bearing nonclassical import signals is incompletely understood; however, karyopherin family members other than importin- appear to transport these specialized cargoes.
Using S. cerevisiae strains bearing deletions or conditional mutations in genes encoding essential or nonessential importin- family members, we determined that the route of nuclear entry for the RSV MA protein, which contains a noncanonical NLS, depends on Mtr10p and Kap120p. We also identified a novel nuclear targeting activity within the Gag NC domain that depends on the recycling of importin-. Importantly, in avian cells (the natural host for RSV), the MA NLS had demonstrable but weak activity both when expressed alone as a green fluorescent protein (GFP) fusion protein and in the context of the Gag polyprotein. However, the NC protein exhibited stronger nuclear localization and appeared to be the major karyophilic signal for the nuclear import of Gag. Thus, the use of the powerful yeast genetic system led to our discovery that independent NLSs in MA and NC utilize distinct nuclear import receptors that may play critical roles in the virus life cycle.
MATERIALS AND METHODS
Yeast strains and methods. For initial studies, yeast strain W303 (MATa ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100) was used. For analysis of nonessential importin- family members, strains from the yeast deletion collection (Open Biosystems) were used (13). Strains used to assess essential importin- family members were as follows: Y1706 (MAT ura3-52 ade2-101 his3-11 trp1-901 CSE1) and Y1709 (MATa ura3-52 ade2-101 his3-11 trp1-901 cse1-1) (52), both from M. Fitzgerald-Hayes; PSY589 (MATa PSE1 ura3-52 trp163 leu2) and PSY1201 (MATa pse1-1 ura3-52 trp163 leu21); PSY 1213 (MATa pse1-7 ura3-52 trp163 leu21) (37); PSY 870 (MATa RSL1/KAP95 rna1-1 ade2 ade3 leu2 ura3 and carrying pPS714 with a wild-type [wt] RNA1) and PSY871 (MATa rsl1-1/kap95 rna1-1 ade2 ade3 leu2 ura3 and carrying pPS714 with a wild-type RNA1) (19), all from P. Silver; NOY 388 (MATa SRP1/KAP60 ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100) and NOY 612 (MATa srp1-31/kap60-310 ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100) (53), both from M. Nomura; DF5 (MAT KAP104 lys2-801 leu2-3,2-112 ura3-52 his3200 trp1-1) and kap104-16 (MAT kap104::ura3::HIS3 lys2-801 leu2-3,2-112 ura3-52 his3200 trp1-1 and containing pRS314kap104-16) from L. Pemberton (1); the W303 XPO1 (MATa xpo1::LEU2 ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100 ura3-1 and containing plasmid pKW440 encoding XPO1) and W303 xpo1-1 (MATa xpo1::LEU2 ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100 ura3-1 and containing plasmid pKW457 encoding xpo1-1) strains (43) from C. Guthrie; and LLY1044 (MAT crm1::KANr leu2– his3– trp1– ura3– and containing a functional CRM1-HA on pRS315) and MNY8 (MAT crm1::KANr leu2– his3– trp1– ura3– and containing pDC-CRM1T539C encoding a leptomycin B [LMB]-sensitive Crm1p on pRS315), both from M. Rosbash. The MTR10 gene is unessential in the genetic background used to construct the mtr10::Kanr strain and was constructed by gene replacement as described previously (3).
Yeast growth and transformations followed standard procedures (39). For the induction of protein expression using pEMBLyex4 and pIGinA/outA vectors, cells were transferred to media with 2% galactose as the carbon source. Induction generally proceeded for 6 h, although induction of some constructs proceeded more slowly. For analysis of protein localization in temperature-sensitive strains, cells were grown at a permissive temperature (23°C) with 2% glucose, induced for 6 to 8 h in media with 2% galactose, and moved from permissive to nonpermissive temperatures (37°C for heat-sensitive mutations and 16°C for cold-sensitive mutations), and images were captured after 30 to 60 min at the restrictive temperature.
Plasmids. Plasmid constructions were performed in Escherichia coli strain DH5 using standard methods. The pIGin/out vectors (Fig. 1A) were created using pRS426 (New England Biolabs) (41) as a backbone. pIGinA contained the GAL1 promoter to allow inducible gene expression only, while pIGinB utilized the hybrid GAL10-CYC1 promoter to permit both constitutive and inducible expression. Histone 2B (H2B) sequences either with or without the nuclear localization signal were obtained using PCR amplification. Two tandem GFP sequences were amplified by PCR, with the insertion of a stop codon after the second copy of the gene. Each PCR product was ligated into the pGEM-T (Promega) or Zero Blunt TOPO (Invitrogen) vectors, digested with the appropriate restriction enzyme, and ligated into pRS426. Control sequences encoding NLSs from SV40 large T antigen (22) and Mod5p-II (46) or nuclear export signals (NESs) from Gle1p and Rna1p (8) were inserted between BamHI-EcoRI sites in pIGinA/B or pIGoutA/B. All gag-derived coding sequences were obtained from the wild-type RSV Prague C genome of pATV-8 by use of PCR amplification, and fragments were subcloned into the pIGinA/B, pIGoutA/B, pEGFP.N2, or pECFP.C1 (Promega) vector. Plasmids pGag-GFP (4), pMA-GFP (11), and pGag.NC.GFP (5) have been previously described, and pYFP.NC was a kind gift from E. Callahan and J. Wills. All plasmid sequences were confirmed by automated DNA sequencing.
Microscopic imaging. Yeast cells grown on solid medium were suspended in phosphate-buffered saline on glass slides and photographed using a Nikon Microphot-FX microscope with a 62x objective and a SenSys charge-coupled-device camera (Photometrics Ltd). Images were processed using QED software, with identical manual exposure settings used for experimental and control cell imaging. Avian cells (transformed quail fibroblasts [QT6 cells]) were maintained in culture and transfected as described previously (11), fixed with 2% paraformaldehyde, and imaged using a Leica TCS SP2 AOBS confocal microscope at an excitation wavelength of 488 nm. Images were captured at appropriate microscope settings so that no pixels were overexposed, and image processing was performed using Corel software to make minor adjustments in the overall intensity of the gray scale images.
RESULTS
Creation of inducible vectors for subcellular localization studies. The objective of these studies was to identify the nuclear transport machinery that interacts with the RSV Gag protein to facilitate nucleocytoplasmic trafficking. To this end, a Gag-GFP fusion protein (4) was transferred into the pEMBLyex4 (47) and pIGinB vectors, which allow constitutive protein expression in addition to inducible expression via galactose regulation. However, expression levels of the fusion protein were very low, with distribution of the Gag-GFP protein either throughout the entire cell or with a slightly enhanced nuclear pool, and some cells appeared enlarged or misshapen (data not shown). Although unfavorable codon utilization could explain the low level of protein expression, attempts to induce higher expression levels had a paradoxical effect, with fewer cells expressing the Gag-GFP fusion protein, suggesting instead cellular toxicity. To limit the putative toxic effects of Gag-GFP in yeast, we utilized vector pGP54a (a kind gift from J.E. Hopper), which carries only the GAL1 promoter to allow for cell growth in the absence of the Gag protein until its induction with galactose. Even under these conditions, expression levels were very low, with <10% of cells expressing Gag-GFP.
Because the difficulty in expressing the full-length Gag protein limited a comprehensive analysis of protein localization, we reasoned that smaller portions of Gag (MA, p10, and NC) might be used to identify domains of Gag that govern nucleus/cytosol dynamics. For these experiments, we developed a new inducible expression system for the assessment of protein location in live cells (Fig. 1A). The pIGinA/outA vectors contain the 2μ origin of replication, allowing for multicopy plasmid replication (5 to 20 copies per cell), with expression of the target gene driven by the inducible GAL1 promoter. The target protein was inserted as a fusion with two tandem, in-frame copies of GFP to amplify the fluorescent signal and to prevent the fusion protein from undergoing passive diffusion through the NPC. The inserted protein sequence was not initiated from its own translation start site but was cloned as a fusion with the yeast histone 2B protein (23) because of the favorable translation initiation context. The pIGinA vector contained the N-terminal 14 amino acids of the histone 2B protein, which possesses no specific targeting activity, to ascertain whether the inserted test sequence has karyophilic properties. The pIGoutA vector contains the first 67 amino acids of histone 2B, which includes its NLS, to permit identification of test sequences that possess nuclear export activity that counteracts the NLS and redirects the fusion protein into the cytoplasm.
Control NLS or NES sequences were inserted into the appropriate pIGinA/outA parental vector to test the utility of the system for studying nuclear trafficking in live cells with fluorescence microscopy (Fig. 1B). Following induction, expression of the pIGinA vector, which encodes tandem GFP proteins without a specific targeting signal, revealed fluorescence confined to the cytoplasm. Insertion of the SV40 large T antigen NLS or the cellular Mod5p NLS (46) into the pIGinA vector resulted in the localization of the majority of the GFP fusion protein pool within the yeast cell nucleus (Fig. 1B, data shown for SV40 T antigen NLS). The nucleus in budding yeast is located near the interface between the mother cell and the daughter cell, and if cell division is not complete, nuclear contents can be seen streaming between the cells (Fig. 1, pIGinA NLS induced and pIGoutA induced). The location of the nucleus was confirmed by staining live cells with DAPI (4',6'-diamidino-2-phenylindole) and by immunofluorescence of fixed cells costained with DAPI and an antibody against GFP (data not shown).
Induced expression of the pIGoutA vector, which encodes tandem GFP proteins with the histone 2B NLS, revealed fluorescence confined to the nucleus (Fig. 1B). Addition of the Gle1p NES (8) to pIGoutA led to a partial redirection of the GFP signal from the nucleus into the cytoplasm, although a minor nuclear pool was apparent (data not shown). In contrast, the dual NESs of Rna1p (8) led to redistribution of the majority of the nuclear pool into the cytoplasm (Fig. 1B, pIGoutANES). Western blot analysis revealed that all of the proteins were stably expressed as predominantly full-length proteins, with negligible unconjugated GFP detected (data not shown). Thus, the pIGinA/outA vectors are effective reporters of NES and NLS activities in living cells. Additional versions of the pIGin/out plasmids were generated using other selectable markers with and without centromeric sequences and were found to function similarly.
Crm1p dependence of the p10 NES. To test whether these inducible expression vectors would be useful for our studies of retroviral Gag targeting sequences, we examined whether the nuclear export activity identified previously in the p10 domain of RSV Gag (36) was a transferable signal recognized by the transport machinery in S. cerevisiae. Initially, the entire p10 sequence was expressed using pIGoutA, which contains the histone 2B NLS fused to two copies of GFP. Wild-type yeast cells induced to express pIGoutAp10 displayed cytoplasmic localization of the GFP reporter, suggesting that a sequence in p10 redirected the protein out of the nucleus (Fig. 2A, left panel). To identify the amino acids in p10 that are sufficient for nuclear export, residues 216GPALTDWARVREELAST232 were substituted for the entire p10 sequence and expressed in the context of pIGoutA. This 17-amino-acid motif comprising the p10 NES conferred cytoplasmic localization to the nuclear reporter protein, suggesting that residues 216 to 232 of p10 constitute a transferable NES that is active in wild-type yeast cells (pIGoutAp10NES) (Fig. 2A, left panel).
Although these results suggested that p10 residues 216 to 232 exhibited nuclear export activity, it was possible that the lack of nuclear localization of pIGoutAp10 and pIGoutAp10NES was due to a failure of the protein to undergo nuclear entry, despite the presence of the histone 2B NLS. Because the nuclear export of Gag in avian cells is disrupted by LMB, a specific inhibitor of CRM1 activity (36), interfering with the Crm1p pathway in yeast would be predicted to cause an accumulation of the p10 or p10NES fusion proteins within the nucleus. In S. cerevisiae, Crm1p (also called Xpo1p or Exportin1) is essential, so a conditional, temperature-sensitive mutant strain was utilized (the xpo1-1 strain [43]). Fusion protein expression in transformed XPO1 (wild-type) and xpo1-1 cells was induced at 23°C, and cells were shifted to 37°C for 30 to 60 min and imaged with fluorescence microscopy (Fig. 2A). As expected, there was no difference in the appearances of wild-type and xpo1-1 mutant cells expressing the pIGinA or pIGoutA control proteins. However, in the xpo1-1 strain, the localizations of pIGoutAp10 and pIGoutAp10NES were dramatically altered from the cytoplasm to the nucleus (Fig. 2A, compare left and right panels).
To quantitate the pattern of nuclear/cytosolic expression in the population of transformed cells, 100 individual cells were scored according to subcellular localization as predominantly cytoplasmic (C>N) or predominantly nuclear (NC; this fraction included a small percentage of cells that expressed GFP equally in the nucleus and cytoplasm). As shown in Fig. 2B, the control protein expressed by pIGoutA was nuclear in the wt (NC, 98%) and xpo1-1 (NC, 96%) strains. In contrast, pIGoutAp10 and pIGoutAp10NES fusion proteins were located in the cytoplasm in the majority of wt cells (C>N, 78.6% and 81.7%, respectively), but each protein was nuclear in xpo1-1 cells (NC, 80.5% and 70%, respectively).
Although the p10 NES operated through Crm1p when nuclear import was directed by the NLS of histone 2B, we asked whether the NES would function properly in yeast when nuclear entry was also governed by the NLS in the Gag MA domain. To address this question, the N-terminal portion of Gag containing the MA, p2, and p10 sequences was fused to tandem GFPs in the context of the pIGoutA vector. For pIGoutAMAp2p10 in wild-type XPO1 cells, 36% of transformed cells demonstrated primarily nuclear localization, in contrast to the 98% of cells expressing pIGoutA, suggesting that the p10 NES functioned in yeast in the context of the native Gag sequence (Fig. 2B). In xpo1-1 cells expressing pIGoutAMAp2p10, there was an increase in the fraction of cells with nuclear localization (54.9%), confirming the Crm1p dependence of export. It is not clear why the percentage of xpo1-1 cells demonstrating fluorescence within the nucleus was less for pIGoutAMAp2p10 than for pIGoutAp10, although it is possible that the addition of the MA and p2 sequences influences the optimal exposure of the Gag NLS or NES in this context. Nonetheless, these data confirm that the p10 NES provides nuclear export capabilities when in proximity to its normal cis-acting sequences in Gag.
To verify the results found with the xpo1-1 mutant, we also inhibited Crm1p with LMB using strain MNY8 (27), which bears a mutant of Crm1p that is sensitive to LMB due to a C539T substitution, and a similar outcome was observed (data not shown). Taken together, these data indicate that the p10 NES functions in a Crm1p-dependent fashion that is recapitulated in S. cerevisiae, signifying that this is a relevant and informative genetic system for the study of host factors that govern subcellular localization of RSV Gag proteins.
Nuclear localization of MA and NC proteins using the pIGinA/outA vector system. To ascertain whether the nuclear targeting activity of MA that was previously identified in avian cells would be amenable to study with S. cerevisiae, the MA protein was fused in frame to the tandem GFP protein. Expression of pIGinAMA revealed an accumulation of the fusion protein in the nucleus with residual cytoplasmic localization (Fig. 3). This distribution of the MA-GFP protein is the same as that in avian cells (36), suggesting a similar targeting mechanism might be shared in lower and higher eukaryotic cells. In a minor subpopulation of cells, a small cytoplasmic dot distinct from the nucleus was observed in cells induced for longer periods of time ranging from 6 to 24 h. These cytosolic dots might represent misfolded MA-GFP fusion proteins that accumulate in the cytoplasm.
Despite evidence that MA possesses nuclear targeting activity, the sequence does not contain a classical motif of basic residues that is characteristic of either monopartite or bipartite NLSs. Unlike MA, the NC domain of Gag contains several clusters of basic residues, with 11 of 52 amino acids being Lys or Arg. Therefore, we postulated that NC might also possess karyophilic properties. Consistent with this idea, expression of pIGinANC resulted in nearly complete nuclear localization of NC-GFP with very little cytoplasmic signaling, indicating robust nuclear targeting activity (Fig. 3). Thus, we identified a novel NLS in NC, suggesting that the RSV Gag polyprotein might contain two independent signals for nuclear entry.
Utilization of MA and NC NLSs for nuclear import of the Gag polyprotein in avian cells. Finding a new karyophilic signal in NC that is recognized by the yeast nuclear transport machinery raised the possibility that this sequence might play a role in the admittance of Gag proteins into the nucleus during RSV infection. Although we previously observed (36) nuclear targeting activity of the RSV MA protein in avian cells, as shown in Fig. 4, the localization of the NC protein in these cells had not been examined. To ascertain whether the NLS in NC that we discovered in yeast was also active in avian cells, the sequence encoding NC was linked to yellow fluorescent protein (YFP) as a C-terminal fusion, and QT6 cells expressing YFP-NC were examined using confocal microscopy. Although the distribution of YFP was throughout the entire cell, the YFP-NC protein was strongly localized to the nucleus and was further concentrated within subnuclear structures identified as nucleoli (Fig. 4B and data not shown).
Because MA and NC each possess nuclear import activity, we asked whether both NLSs contribute equally to the nuclear entry of the Gag polyprotein or whether one might be the predominant signal. To test this idea, constructs with deletions in the MA or NC domains of Gag-GFP were expressed in avian cells in the presence or absence of LMB, and the degree of nuclear localization was assessed by confocal microscopy (Fig. 4C). GagNC-GFP contains a C-terminal deletion of NC and removes all but two basic amino acids as well as the zinc finger-binding domain. Like the full-length Gag-GFP protein, GagNC-GFP was located in the cytoplasm of untreated QT6 cells, but upon exposure to LMB, a portion of the protein pool became located within the nucleus (Fig. 4C) (5). However, there was more fluorescence present in the cytoplasm of LMB-treated GagNC-GFP-expressing cells than in the cytoplasm of those expressing Gag-GFP. The nuclear import of GagNC-GFP presumably depends upon the NLS in MA, and nuclear localization is incomplete. The data are consistent with NC contributing to the ability of Gag-GFP to localize to the nucleus.
To determine whether deletion of the MA NLS would similarly reduce the nuclear accumulation of Gag-GFP, two constructs with deletions either of the N-terminal portion of MA (MA5-86 Gag-GFP) or of nearly the entire MA sequence (MA5-148 Gag-GFP) were examined in QT6 cells (Fig. 4A). For both MA deletions, localization was cytoplasmic, with nuclear exclusion in untreated cells, although the larger deletion (MA5-148 Gag-GFP) resulted in a more punctate appearance (Fig. 4C). Treatment with LMB led to the nuclear localization of the MA-deleted GFP fusion protein to a degree similar to that for full-length Gag-GFP, indicating that the remaining NLS within NC was sufficient for the nuclear entry of Gag. Therefore, in the context of Gag, it appears that the NC domain contains a stronger NLS than MA, implying that NC might be the dominant signal for nuclear translocation of the Gag polyprotein.
Identification of importin- family members utilized for import of MA and NC. We undertook these studies to identify both cis and trans elements involved in the transport of RSV Gag proteins into the nucleus. Based on the absence of a canonical NLS motif within the MA sequence, we predicted that its nuclear import might depend on a trans-acting factor other than the classical importin- (Kap95p) transport pathway. To examine this question with an unbiased approach, we screened a deletion collection of nonessential nuclear transport genes in S. cerevisiae to identify strains that were deficient in nuclear localization of pIGinAMA (Fig. 5 and Table 1). The MA fusion protein displayed enriched nuclear localization in all of the deletion strains except those strains lacking mtr10 or kap120, indicating that Mtr10p and Kap120p are important for MA nuclear entry. Because both Mtr10p and Kap120p appear to facilitate the import of MA, we asked whether these importins might be involved in redundant pathways. Double mutants generated by mating, sporulation, and dissection had growth much poorer than that of either single mutant (data not shown). The "synthetically sick" phenotype of the mtr10 kap120 segregants suggests overlapping functions of these import factors. Of the essential importin- family members tested, xpo1-1 had no effect on localization. However, no conclusions could be drawn about the roles of Cse1p, the transporter involved in the cytoplasmic recycling of importin- (21, 42, 52) or Kap104p in pIGinAMA localization, because at the nonpermissive temperature there were no nuclear pools in either the wt or the conditional cse1-1 and kap104-16 mutant strains.
Analysis of pIGinANC localization in the importin- mutant strains revealed that none of the nonessential genes was involved (Fig. 4 and Table 1). However, at restrictive temperatures, the cse1-1 mutant strain displayed reduced nuclear signaling and enhanced cytoplasmic fluorescence, implicating a role for the classical importin-/ pathway in the nuclear transport of NC. The fact that pIGinASV40 (SV40 large T antigen NLS), which utilizes importin-/ for nuclear entry, also had increased cytoplasmic accumulation in the cse1-1 strain at the nonpermissive temperature verified that the mutant strain behaved appropriately. The involvement of importin- (Srp1p), importin- (Rsl1p), or RanBP6 (Pse1p) could not be directly tested because of an inability to induce expression of NC or MA fusion proteins in the corresponding conditional mutant strains, most likely due to the dependence of the GAL induction system on the classical nuclear transport system (30).
DISCUSSION
The nuclear trafficking of RSV Gag was only recently discovered, and the possible role(s) Gag proteins play in the nucleus during virus assembly remains uncertain (36). However, nuclear entry of retroviral DNA during early infection is a well-described feature of all retroviruses and is vital to the establishment of infection. The viral signals that mediate nuclear import at this critical early stage of infection are poorly characterized for RSV, yet it is likely that at least some of the mature Gag proteins are involved. In fact, we found that both the mature MA and NC proteins possess nuclear targeting signals. In an effort to shed light on the nuclear transport pathways used by these Gag proteins, we utilized genetic tools developed in studies with S. cerevisiae to identify importin- family members that mediate the import of MA and NC. Understanding the cellular factors usurped by Gag proteins for nuclear entry may shed light on the function(s) of Gag in the nucleus and may begin to elucidate the molecular mechanisms involved in import of the PIC.
The initial aim of studying the nuclear transport pathway of the Gag polyprotein was thwarted by its apparent cellular toxicity in yeast. We can imagine several possible mechanisms of Gag cytotoxicity. Gag proteins interface with the machinery associated with vacuolar sorting through multivesicular bodies during the latter stages of virus assembly (reviewed in reference 24). Because the rigid yeast cell wall precludes viral budding, it is possible that Gag proteins accumulate in vacuolar bodies and impair normal functions of the vacuolar sorting machinery. As well, Gag proteins targeted to the yeast cell membrane might interfere with cell growth and division. Both of these ideas are consistent with the elongated appearance of yeast cells expressing Gag proteins. Alterations in cellular morphology and toxicity are also observed in avian cells that express high levels of Gag-GFP, and the degree of cytotoxicity is more evident with Gag mutants that are strongly plasma membrane associated than in those mutants having a cytosolic distribution (5, 11, 36). Furthermore, the nuclear trafficking of Gag might itself be toxic to yeast, particularly if the import factors used by Gag are needed for other cellular functions. Efforts to isolate spontaneous mutants of yeast that are able to tolerate high levels of Gag expression may prove to be informative for identifying additional host factors that interact with the virus assembly pathway.
S. cerevisiae serves as a model system for nucleus/cytosol exchange because the nuclear transport pathways are very highly conserved among lower and higher eukaryotes (7). We constructed a set of inducible expression vectors to identify cis- and trans-acting factors involved in Gag nucleus/cytoplasm dynamics in live yeast cells. Using these new pIGin/pIGout vectors, our data demonstrated that residues 216 to 232 in the p10 domain of Gag are sufficient to mediate nuclear export through the Crm1p pathway. The NLS in the MA protein we previously described (36) was also confirmed, and we discovered a novel NLS within the NC domain. Verification that the NC NLS was functional in avian cells validated the use of the yeast expression system to identify new targeting elements in heterologous viral proteins.
There are examples of proteins that possess multiple NLSs, some of which are served by different importin- family members (25), but in general, little is known about how each individual signal is utilized. Accordingly, we wonder why there are two independent NLSs in the RSV Gag polyprotein. One possibility is that both signals are used during the nuclear trafficking of Gag during virus assembly, and the presence of redundant targeting signals in Gag would imply that nuclear trafficking is an important step in replication. However, our experiments suggest that the novel NLS in NC is the predominant sequence used for nuclear import of Gag in avian cells. Another possible explanation for the dual import signals is that the MA and NC NLSs might function either at separate steps or within distinct import complexes to facilitate Gag nuclear entry. Finally, perhaps either the MA or NC NLS has no function in nuclear entry, since the basic residues in NC are also important for viral RNA binding and the basic residues in MA mediate plasma membrane targeting.
It is also conceivable that the NLSs in MA and/or NC are active during the early steps of infection, when mature Gag proteins enter the cell with the incoming virion. It is uncertain whether MA is a component of the RSV PIC, although genetic evidence supports this idea (12). Most certainly, NC is present within the PIC because it is bound to the RNA genome in the virion and is involved in reverse transcription and integration (2, 6, 16, 34, 48). An NLS in RSV integrase was previously identified (20), and thus there are three candidate karyophilic proteins that might promote nuclear transport of the viral genome early in infection. The nuclear import of the HIV-1 PIC has been intensively studied, and it appears that there are multiple signals for nuclear targeting, including MA, integrase, Vpr, and an intermediate DNA product of reverse transcription (40). As well, HIV-1 NC enters the nucleus early after infection and enhances the expression of spliced viral mRNAs after integration (54).
Interestingly, our screen of deletion strains and conditional mutants involving importin- family members indicated that the MA and NC proteins enter the nucleus through interactions with different soluble nuclear transport receptors. Import of the MA protein was dependent on Mtr10p and Kap120p. Mtr10p (mRNA transport regulator) is involved in nuclear translocation of RNA-binding proteins that contain serine/arginine (SR)-rich domains, including Np13p, Gbp2p, and Hrb1p, which are nucleocytoplasmic shuttling proteins implicated in the nuclear export of mRNA in yeast (14, 26, 29, 38, 50). These shuttling SR proteins are imported by Mtr10p and recruited to mRNA during transcription and then accompany the ribonuclear complex into the cytoplasm, where they may regulate translation (51). The dual involvement of Mtr10p in the import of MA and in the import of shuttling mRNA binding proteins is intriguing because we have postulated that RSV Gag enters the nucleus to bind newly transcribed viral RNA and transport it into the cytoplasm for encapsidation into assembling virus particles (36). However, there is no identifiable sequence homology between MA and SR proteins, including the NLS in Npl3p (10, 38; also, data not shown). Because little is known about the substrate specificity or molecular interaction between cargoes for the human (transportin-SR or importin 12 [17]) or avian homologues of Mtr10, RSV MA may prove to be an important future tool for studying this import pathway in higher eukaryotes.
We also found that the deletion of Kap120p, proposed to be a bidirectional transporter through the NPC (9, 44), impaired MA nuclear import. Kap120p functions in the export of the 60S ribosomal subunit (44) and its human homologue, importin 11, has been implicated in the import of ribosomal protein L12 (31, 32). Importin 11 also serves as the import receptor for the ubiquitin-conjugating enzyme UbcM2, and covalent attachment of ubiquitin to UbcM2 is a prerequisite for nuclear transport (33). This is an intriguing observation, given the dependence of RSV particle assembly on ubiquitination (28), prompting us to speculate that ubiquitin modification of Gag might be involved in its nuclear translocation.
Because deletions of both mtr10 and kap120 interfered with the nuclear accumulation of MA, we proposed that these importins might be involved in redundant pathways. Indeed, double mutants (mtr10 kap120) grew very poorly, suggesting overlapping functions for these import receptors. Future studies of MA-Kap120p interactions may reveal new insights into the substrate specificity and structural determinants of this poorly understood import receptor pathway.
In contrast to MA, the NC protein has a basic motif that resembles the canonical NLSs, and accordingly, its nuclear entry appeared to be mediated by the classical importin-/ pathway. It will be of interest to identify the specific residues in NC that are sufficient to mediate nuclear entry. Furthermore, we do not yet know whether there is a direct or indirect interaction between NC and the avian homologue of importin-. Finally, the relative reliance of Gag nuclear entry on the avian Kap120p, Mtr10p, or importin-/ homologues will be worthwhile to pursue.
The results of these studies raise many intriguing questions. Why does the Gag polyprotein contain two independent NLSs that utilize distinct import receptors Are the NLSs in MA and NC that mediate Gag nuclear entry also involved in the nuclear targeting of the PIC and the entry of the incoming viral genome during early infection, or do they have other intranuclear roles Are there cellular proteins that compete with RSV MA for utilization of Mtr10p and Kap120p in avian cells The use of the S. cerevisiae genetic system has provided the initial key insights needed to address these compelling questions, which are critical for understanding the molecular interactions of RSV with its intracellular environment.
ACKNOWLEDGMENTS
We thank M. L. Whitney for construction of mtr10::Kanr; J. E. Hopper, M. Fitzgerald-Hayes, P. Silver, M. Nomura, L. Pemberton, C. Guthrie, M. Robash, E. Callahan, and J. Wills for reagents; Scott Kenney for critical review of the manuscript; and the Penn State College of Medicine Core Facility for assistance with DNA sequencing and confocal microscopy.
We greatly appreciate support from the NIH (grants R01CA76534 to L.J.P. and R01GM27930 to A.K.H.), the NSF (predoctoral fellowship to L.Z.S.), the Penn State College of Medicine Dean's Feasibility Award (L.J.P. and A.K.H.), and the M.D. Research Facilitation Award (L.J.P.). This project was funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds, and the Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.
These authors contributed equally to this work.
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Microbiology and Immunology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033
ABSTRACT
The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin- family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-/ (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.
INTRODUCTION
Retroviruses, as obligate intracellular parasites, exist in a dynamic relationship with the host cell. These significant pathogens coopt host cell machinery to facilitate viral entry, gene expression, protein synthesis, and virion release. Identification of cellular factors involved in the retroviral life cycle could be facilitated by the use of a well-characterized genetic system. Saccharomyces cerevisiae serves as the ideal candidate, as each gene has been systematically deleted or mutated, enabling a search for gene products that participate in viral processes. Although the complete retrovirus replication cycle cannot be recapitulated in yeast, discrete steps of the life cycle can be studied in detail to identify host factors that are required.
The gag gene, one of the three major genes shared by all retroviruses, encodes the Gag polyprotein, which drives the formation and release of virus particles from the host cell membrane. Gag proteins are synthesized on cytosolic ribosomes, and previously it was thought that Gag was transported directly to the plasma membrane to drive virus assembly. However, we discovered that the Gag protein of the alpharetrovirus Rous sarcoma virus (RSV), a tumor-producing virus that infects domestic fowl, undergoes nucleocytoplasmic shuttling prior to its transport to the plasma membrane (36). The role of Gag nuclear trafficking in the viral life cycle is not well understood; however, both virion release and infectivity are inhibited when Gag nuclear export is prevented (36; L. Scheifele and L. Parent, unpublished data, 2004).
Following virion release, the RSV Gag polyprotein undergoes proteolytic cleavage into the MA, p2, p10, CA, NC, and PR proteins. Extracellular virus particles bind to receptors and undergo fusion with the cell membrane, and the viral core enters the cytosol. Reverse transcription proceeds, and the viral ribonuclear complex (known as the preintegration complex [PIC]) enters the nucleus, where proviral DNA integrates into the cellular genome. For human immunodeficiency virus type 1 (HIV-1), which infects nondividing cells, the PIC traverses through the nuclear pore complex (NPC) by use of redundant nuclear localization signals (NLSs) in the integrase, Vpr, and MA proteins (40, 49). In contrast, RSV primarily infects dividing cells, and therefore it was believed that the import of the PIC depended on the breakdown of the nuclear envelope during mitosis. However, RSV does replicate at low levels in quiescent cells (15, 18); thus, there is some level of active transport of the PIC, and nuclear entry likely depends on the karyophilic properties of viral proteins.
Nuclear transport occurs through the NPC, which is composed of 30 nucleoporins (35), via interactions between NLSs on protein cargoes and a family of soluble nuclear receptors known as importins or karyopherins (reviewed in reference 45). The classical import pathway involves direct recognition of a basic NLS by importin- (Kap95p) or the use of importin- as an adaptor for binding to importin-. The diversity of nonclassical NLSs is still being elaborated, and transport of proteins bearing nonclassical import signals is incompletely understood; however, karyopherin family members other than importin- appear to transport these specialized cargoes.
Using S. cerevisiae strains bearing deletions or conditional mutations in genes encoding essential or nonessential importin- family members, we determined that the route of nuclear entry for the RSV MA protein, which contains a noncanonical NLS, depends on Mtr10p and Kap120p. We also identified a novel nuclear targeting activity within the Gag NC domain that depends on the recycling of importin-. Importantly, in avian cells (the natural host for RSV), the MA NLS had demonstrable but weak activity both when expressed alone as a green fluorescent protein (GFP) fusion protein and in the context of the Gag polyprotein. However, the NC protein exhibited stronger nuclear localization and appeared to be the major karyophilic signal for the nuclear import of Gag. Thus, the use of the powerful yeast genetic system led to our discovery that independent NLSs in MA and NC utilize distinct nuclear import receptors that may play critical roles in the virus life cycle.
MATERIALS AND METHODS
Yeast strains and methods. For initial studies, yeast strain W303 (MATa ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100) was used. For analysis of nonessential importin- family members, strains from the yeast deletion collection (Open Biosystems) were used (13). Strains used to assess essential importin- family members were as follows: Y1706 (MAT ura3-52 ade2-101 his3-11 trp1-901 CSE1) and Y1709 (MATa ura3-52 ade2-101 his3-11 trp1-901 cse1-1) (52), both from M. Fitzgerald-Hayes; PSY589 (MATa PSE1 ura3-52 trp163 leu2) and PSY1201 (MATa pse1-1 ura3-52 trp163 leu21); PSY 1213 (MATa pse1-7 ura3-52 trp163 leu21) (37); PSY 870 (MATa RSL1/KAP95 rna1-1 ade2 ade3 leu2 ura3 and carrying pPS714 with a wild-type [wt] RNA1) and PSY871 (MATa rsl1-1/kap95 rna1-1 ade2 ade3 leu2 ura3 and carrying pPS714 with a wild-type RNA1) (19), all from P. Silver; NOY 388 (MATa SRP1/KAP60 ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100) and NOY 612 (MATa srp1-31/kap60-310 ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100) (53), both from M. Nomura; DF5 (MAT KAP104 lys2-801 leu2-3,2-112 ura3-52 his3200 trp1-1) and kap104-16 (MAT kap104::ura3::HIS3 lys2-801 leu2-3,2-112 ura3-52 his3200 trp1-1 and containing pRS314kap104-16) from L. Pemberton (1); the W303 XPO1 (MATa xpo1::LEU2 ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100 ura3-1 and containing plasmid pKW440 encoding XPO1) and W303 xpo1-1 (MATa xpo1::LEU2 ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100 ura3-1 and containing plasmid pKW457 encoding xpo1-1) strains (43) from C. Guthrie; and LLY1044 (MAT crm1::KANr leu2– his3– trp1– ura3– and containing a functional CRM1-HA on pRS315) and MNY8 (MAT crm1::KANr leu2– his3– trp1– ura3– and containing pDC-CRM1T539C encoding a leptomycin B [LMB]-sensitive Crm1p on pRS315), both from M. Rosbash. The MTR10 gene is unessential in the genetic background used to construct the mtr10::Kanr strain and was constructed by gene replacement as described previously (3).
Yeast growth and transformations followed standard procedures (39). For the induction of protein expression using pEMBLyex4 and pIGinA/outA vectors, cells were transferred to media with 2% galactose as the carbon source. Induction generally proceeded for 6 h, although induction of some constructs proceeded more slowly. For analysis of protein localization in temperature-sensitive strains, cells were grown at a permissive temperature (23°C) with 2% glucose, induced for 6 to 8 h in media with 2% galactose, and moved from permissive to nonpermissive temperatures (37°C for heat-sensitive mutations and 16°C for cold-sensitive mutations), and images were captured after 30 to 60 min at the restrictive temperature.
Plasmids. Plasmid constructions were performed in Escherichia coli strain DH5 using standard methods. The pIGin/out vectors (Fig. 1A) were created using pRS426 (New England Biolabs) (41) as a backbone. pIGinA contained the GAL1 promoter to allow inducible gene expression only, while pIGinB utilized the hybrid GAL10-CYC1 promoter to permit both constitutive and inducible expression. Histone 2B (H2B) sequences either with or without the nuclear localization signal were obtained using PCR amplification. Two tandem GFP sequences were amplified by PCR, with the insertion of a stop codon after the second copy of the gene. Each PCR product was ligated into the pGEM-T (Promega) or Zero Blunt TOPO (Invitrogen) vectors, digested with the appropriate restriction enzyme, and ligated into pRS426. Control sequences encoding NLSs from SV40 large T antigen (22) and Mod5p-II (46) or nuclear export signals (NESs) from Gle1p and Rna1p (8) were inserted between BamHI-EcoRI sites in pIGinA/B or pIGoutA/B. All gag-derived coding sequences were obtained from the wild-type RSV Prague C genome of pATV-8 by use of PCR amplification, and fragments were subcloned into the pIGinA/B, pIGoutA/B, pEGFP.N2, or pECFP.C1 (Promega) vector. Plasmids pGag-GFP (4), pMA-GFP (11), and pGag.NC.GFP (5) have been previously described, and pYFP.NC was a kind gift from E. Callahan and J. Wills. All plasmid sequences were confirmed by automated DNA sequencing.
Microscopic imaging. Yeast cells grown on solid medium were suspended in phosphate-buffered saline on glass slides and photographed using a Nikon Microphot-FX microscope with a 62x objective and a SenSys charge-coupled-device camera (Photometrics Ltd). Images were processed using QED software, with identical manual exposure settings used for experimental and control cell imaging. Avian cells (transformed quail fibroblasts [QT6 cells]) were maintained in culture and transfected as described previously (11), fixed with 2% paraformaldehyde, and imaged using a Leica TCS SP2 AOBS confocal microscope at an excitation wavelength of 488 nm. Images were captured at appropriate microscope settings so that no pixels were overexposed, and image processing was performed using Corel software to make minor adjustments in the overall intensity of the gray scale images.
RESULTS
Creation of inducible vectors for subcellular localization studies. The objective of these studies was to identify the nuclear transport machinery that interacts with the RSV Gag protein to facilitate nucleocytoplasmic trafficking. To this end, a Gag-GFP fusion protein (4) was transferred into the pEMBLyex4 (47) and pIGinB vectors, which allow constitutive protein expression in addition to inducible expression via galactose regulation. However, expression levels of the fusion protein were very low, with distribution of the Gag-GFP protein either throughout the entire cell or with a slightly enhanced nuclear pool, and some cells appeared enlarged or misshapen (data not shown). Although unfavorable codon utilization could explain the low level of protein expression, attempts to induce higher expression levels had a paradoxical effect, with fewer cells expressing the Gag-GFP fusion protein, suggesting instead cellular toxicity. To limit the putative toxic effects of Gag-GFP in yeast, we utilized vector pGP54a (a kind gift from J.E. Hopper), which carries only the GAL1 promoter to allow for cell growth in the absence of the Gag protein until its induction with galactose. Even under these conditions, expression levels were very low, with <10% of cells expressing Gag-GFP.
Because the difficulty in expressing the full-length Gag protein limited a comprehensive analysis of protein localization, we reasoned that smaller portions of Gag (MA, p10, and NC) might be used to identify domains of Gag that govern nucleus/cytosol dynamics. For these experiments, we developed a new inducible expression system for the assessment of protein location in live cells (Fig. 1A). The pIGinA/outA vectors contain the 2μ origin of replication, allowing for multicopy plasmid replication (5 to 20 copies per cell), with expression of the target gene driven by the inducible GAL1 promoter. The target protein was inserted as a fusion with two tandem, in-frame copies of GFP to amplify the fluorescent signal and to prevent the fusion protein from undergoing passive diffusion through the NPC. The inserted protein sequence was not initiated from its own translation start site but was cloned as a fusion with the yeast histone 2B protein (23) because of the favorable translation initiation context. The pIGinA vector contained the N-terminal 14 amino acids of the histone 2B protein, which possesses no specific targeting activity, to ascertain whether the inserted test sequence has karyophilic properties. The pIGoutA vector contains the first 67 amino acids of histone 2B, which includes its NLS, to permit identification of test sequences that possess nuclear export activity that counteracts the NLS and redirects the fusion protein into the cytoplasm.
Control NLS or NES sequences were inserted into the appropriate pIGinA/outA parental vector to test the utility of the system for studying nuclear trafficking in live cells with fluorescence microscopy (Fig. 1B). Following induction, expression of the pIGinA vector, which encodes tandem GFP proteins without a specific targeting signal, revealed fluorescence confined to the cytoplasm. Insertion of the SV40 large T antigen NLS or the cellular Mod5p NLS (46) into the pIGinA vector resulted in the localization of the majority of the GFP fusion protein pool within the yeast cell nucleus (Fig. 1B, data shown for SV40 T antigen NLS). The nucleus in budding yeast is located near the interface between the mother cell and the daughter cell, and if cell division is not complete, nuclear contents can be seen streaming between the cells (Fig. 1, pIGinA NLS induced and pIGoutA induced). The location of the nucleus was confirmed by staining live cells with DAPI (4',6'-diamidino-2-phenylindole) and by immunofluorescence of fixed cells costained with DAPI and an antibody against GFP (data not shown).
Induced expression of the pIGoutA vector, which encodes tandem GFP proteins with the histone 2B NLS, revealed fluorescence confined to the nucleus (Fig. 1B). Addition of the Gle1p NES (8) to pIGoutA led to a partial redirection of the GFP signal from the nucleus into the cytoplasm, although a minor nuclear pool was apparent (data not shown). In contrast, the dual NESs of Rna1p (8) led to redistribution of the majority of the nuclear pool into the cytoplasm (Fig. 1B, pIGoutANES). Western blot analysis revealed that all of the proteins were stably expressed as predominantly full-length proteins, with negligible unconjugated GFP detected (data not shown). Thus, the pIGinA/outA vectors are effective reporters of NES and NLS activities in living cells. Additional versions of the pIGin/out plasmids were generated using other selectable markers with and without centromeric sequences and were found to function similarly.
Crm1p dependence of the p10 NES. To test whether these inducible expression vectors would be useful for our studies of retroviral Gag targeting sequences, we examined whether the nuclear export activity identified previously in the p10 domain of RSV Gag (36) was a transferable signal recognized by the transport machinery in S. cerevisiae. Initially, the entire p10 sequence was expressed using pIGoutA, which contains the histone 2B NLS fused to two copies of GFP. Wild-type yeast cells induced to express pIGoutAp10 displayed cytoplasmic localization of the GFP reporter, suggesting that a sequence in p10 redirected the protein out of the nucleus (Fig. 2A, left panel). To identify the amino acids in p10 that are sufficient for nuclear export, residues 216GPALTDWARVREELAST232 were substituted for the entire p10 sequence and expressed in the context of pIGoutA. This 17-amino-acid motif comprising the p10 NES conferred cytoplasmic localization to the nuclear reporter protein, suggesting that residues 216 to 232 of p10 constitute a transferable NES that is active in wild-type yeast cells (pIGoutAp10NES) (Fig. 2A, left panel).
Although these results suggested that p10 residues 216 to 232 exhibited nuclear export activity, it was possible that the lack of nuclear localization of pIGoutAp10 and pIGoutAp10NES was due to a failure of the protein to undergo nuclear entry, despite the presence of the histone 2B NLS. Because the nuclear export of Gag in avian cells is disrupted by LMB, a specific inhibitor of CRM1 activity (36), interfering with the Crm1p pathway in yeast would be predicted to cause an accumulation of the p10 or p10NES fusion proteins within the nucleus. In S. cerevisiae, Crm1p (also called Xpo1p or Exportin1) is essential, so a conditional, temperature-sensitive mutant strain was utilized (the xpo1-1 strain [43]). Fusion protein expression in transformed XPO1 (wild-type) and xpo1-1 cells was induced at 23°C, and cells were shifted to 37°C for 30 to 60 min and imaged with fluorescence microscopy (Fig. 2A). As expected, there was no difference in the appearances of wild-type and xpo1-1 mutant cells expressing the pIGinA or pIGoutA control proteins. However, in the xpo1-1 strain, the localizations of pIGoutAp10 and pIGoutAp10NES were dramatically altered from the cytoplasm to the nucleus (Fig. 2A, compare left and right panels).
To quantitate the pattern of nuclear/cytosolic expression in the population of transformed cells, 100 individual cells were scored according to subcellular localization as predominantly cytoplasmic (C>N) or predominantly nuclear (NC; this fraction included a small percentage of cells that expressed GFP equally in the nucleus and cytoplasm). As shown in Fig. 2B, the control protein expressed by pIGoutA was nuclear in the wt (NC, 98%) and xpo1-1 (NC, 96%) strains. In contrast, pIGoutAp10 and pIGoutAp10NES fusion proteins were located in the cytoplasm in the majority of wt cells (C>N, 78.6% and 81.7%, respectively), but each protein was nuclear in xpo1-1 cells (NC, 80.5% and 70%, respectively).
Although the p10 NES operated through Crm1p when nuclear import was directed by the NLS of histone 2B, we asked whether the NES would function properly in yeast when nuclear entry was also governed by the NLS in the Gag MA domain. To address this question, the N-terminal portion of Gag containing the MA, p2, and p10 sequences was fused to tandem GFPs in the context of the pIGoutA vector. For pIGoutAMAp2p10 in wild-type XPO1 cells, 36% of transformed cells demonstrated primarily nuclear localization, in contrast to the 98% of cells expressing pIGoutA, suggesting that the p10 NES functioned in yeast in the context of the native Gag sequence (Fig. 2B). In xpo1-1 cells expressing pIGoutAMAp2p10, there was an increase in the fraction of cells with nuclear localization (54.9%), confirming the Crm1p dependence of export. It is not clear why the percentage of xpo1-1 cells demonstrating fluorescence within the nucleus was less for pIGoutAMAp2p10 than for pIGoutAp10, although it is possible that the addition of the MA and p2 sequences influences the optimal exposure of the Gag NLS or NES in this context. Nonetheless, these data confirm that the p10 NES provides nuclear export capabilities when in proximity to its normal cis-acting sequences in Gag.
To verify the results found with the xpo1-1 mutant, we also inhibited Crm1p with LMB using strain MNY8 (27), which bears a mutant of Crm1p that is sensitive to LMB due to a C539T substitution, and a similar outcome was observed (data not shown). Taken together, these data indicate that the p10 NES functions in a Crm1p-dependent fashion that is recapitulated in S. cerevisiae, signifying that this is a relevant and informative genetic system for the study of host factors that govern subcellular localization of RSV Gag proteins.
Nuclear localization of MA and NC proteins using the pIGinA/outA vector system. To ascertain whether the nuclear targeting activity of MA that was previously identified in avian cells would be amenable to study with S. cerevisiae, the MA protein was fused in frame to the tandem GFP protein. Expression of pIGinAMA revealed an accumulation of the fusion protein in the nucleus with residual cytoplasmic localization (Fig. 3). This distribution of the MA-GFP protein is the same as that in avian cells (36), suggesting a similar targeting mechanism might be shared in lower and higher eukaryotic cells. In a minor subpopulation of cells, a small cytoplasmic dot distinct from the nucleus was observed in cells induced for longer periods of time ranging from 6 to 24 h. These cytosolic dots might represent misfolded MA-GFP fusion proteins that accumulate in the cytoplasm.
Despite evidence that MA possesses nuclear targeting activity, the sequence does not contain a classical motif of basic residues that is characteristic of either monopartite or bipartite NLSs. Unlike MA, the NC domain of Gag contains several clusters of basic residues, with 11 of 52 amino acids being Lys or Arg. Therefore, we postulated that NC might also possess karyophilic properties. Consistent with this idea, expression of pIGinANC resulted in nearly complete nuclear localization of NC-GFP with very little cytoplasmic signaling, indicating robust nuclear targeting activity (Fig. 3). Thus, we identified a novel NLS in NC, suggesting that the RSV Gag polyprotein might contain two independent signals for nuclear entry.
Utilization of MA and NC NLSs for nuclear import of the Gag polyprotein in avian cells. Finding a new karyophilic signal in NC that is recognized by the yeast nuclear transport machinery raised the possibility that this sequence might play a role in the admittance of Gag proteins into the nucleus during RSV infection. Although we previously observed (36) nuclear targeting activity of the RSV MA protein in avian cells, as shown in Fig. 4, the localization of the NC protein in these cells had not been examined. To ascertain whether the NLS in NC that we discovered in yeast was also active in avian cells, the sequence encoding NC was linked to yellow fluorescent protein (YFP) as a C-terminal fusion, and QT6 cells expressing YFP-NC were examined using confocal microscopy. Although the distribution of YFP was throughout the entire cell, the YFP-NC protein was strongly localized to the nucleus and was further concentrated within subnuclear structures identified as nucleoli (Fig. 4B and data not shown).
Because MA and NC each possess nuclear import activity, we asked whether both NLSs contribute equally to the nuclear entry of the Gag polyprotein or whether one might be the predominant signal. To test this idea, constructs with deletions in the MA or NC domains of Gag-GFP were expressed in avian cells in the presence or absence of LMB, and the degree of nuclear localization was assessed by confocal microscopy (Fig. 4C). GagNC-GFP contains a C-terminal deletion of NC and removes all but two basic amino acids as well as the zinc finger-binding domain. Like the full-length Gag-GFP protein, GagNC-GFP was located in the cytoplasm of untreated QT6 cells, but upon exposure to LMB, a portion of the protein pool became located within the nucleus (Fig. 4C) (5). However, there was more fluorescence present in the cytoplasm of LMB-treated GagNC-GFP-expressing cells than in the cytoplasm of those expressing Gag-GFP. The nuclear import of GagNC-GFP presumably depends upon the NLS in MA, and nuclear localization is incomplete. The data are consistent with NC contributing to the ability of Gag-GFP to localize to the nucleus.
To determine whether deletion of the MA NLS would similarly reduce the nuclear accumulation of Gag-GFP, two constructs with deletions either of the N-terminal portion of MA (MA5-86 Gag-GFP) or of nearly the entire MA sequence (MA5-148 Gag-GFP) were examined in QT6 cells (Fig. 4A). For both MA deletions, localization was cytoplasmic, with nuclear exclusion in untreated cells, although the larger deletion (MA5-148 Gag-GFP) resulted in a more punctate appearance (Fig. 4C). Treatment with LMB led to the nuclear localization of the MA-deleted GFP fusion protein to a degree similar to that for full-length Gag-GFP, indicating that the remaining NLS within NC was sufficient for the nuclear entry of Gag. Therefore, in the context of Gag, it appears that the NC domain contains a stronger NLS than MA, implying that NC might be the dominant signal for nuclear translocation of the Gag polyprotein.
Identification of importin- family members utilized for import of MA and NC. We undertook these studies to identify both cis and trans elements involved in the transport of RSV Gag proteins into the nucleus. Based on the absence of a canonical NLS motif within the MA sequence, we predicted that its nuclear import might depend on a trans-acting factor other than the classical importin- (Kap95p) transport pathway. To examine this question with an unbiased approach, we screened a deletion collection of nonessential nuclear transport genes in S. cerevisiae to identify strains that were deficient in nuclear localization of pIGinAMA (Fig. 5 and Table 1). The MA fusion protein displayed enriched nuclear localization in all of the deletion strains except those strains lacking mtr10 or kap120, indicating that Mtr10p and Kap120p are important for MA nuclear entry. Because both Mtr10p and Kap120p appear to facilitate the import of MA, we asked whether these importins might be involved in redundant pathways. Double mutants generated by mating, sporulation, and dissection had growth much poorer than that of either single mutant (data not shown). The "synthetically sick" phenotype of the mtr10 kap120 segregants suggests overlapping functions of these import factors. Of the essential importin- family members tested, xpo1-1 had no effect on localization. However, no conclusions could be drawn about the roles of Cse1p, the transporter involved in the cytoplasmic recycling of importin- (21, 42, 52) or Kap104p in pIGinAMA localization, because at the nonpermissive temperature there were no nuclear pools in either the wt or the conditional cse1-1 and kap104-16 mutant strains.
Analysis of pIGinANC localization in the importin- mutant strains revealed that none of the nonessential genes was involved (Fig. 4 and Table 1). However, at restrictive temperatures, the cse1-1 mutant strain displayed reduced nuclear signaling and enhanced cytoplasmic fluorescence, implicating a role for the classical importin-/ pathway in the nuclear transport of NC. The fact that pIGinASV40 (SV40 large T antigen NLS), which utilizes importin-/ for nuclear entry, also had increased cytoplasmic accumulation in the cse1-1 strain at the nonpermissive temperature verified that the mutant strain behaved appropriately. The involvement of importin- (Srp1p), importin- (Rsl1p), or RanBP6 (Pse1p) could not be directly tested because of an inability to induce expression of NC or MA fusion proteins in the corresponding conditional mutant strains, most likely due to the dependence of the GAL induction system on the classical nuclear transport system (30).
DISCUSSION
The nuclear trafficking of RSV Gag was only recently discovered, and the possible role(s) Gag proteins play in the nucleus during virus assembly remains uncertain (36). However, nuclear entry of retroviral DNA during early infection is a well-described feature of all retroviruses and is vital to the establishment of infection. The viral signals that mediate nuclear import at this critical early stage of infection are poorly characterized for RSV, yet it is likely that at least some of the mature Gag proteins are involved. In fact, we found that both the mature MA and NC proteins possess nuclear targeting signals. In an effort to shed light on the nuclear transport pathways used by these Gag proteins, we utilized genetic tools developed in studies with S. cerevisiae to identify importin- family members that mediate the import of MA and NC. Understanding the cellular factors usurped by Gag proteins for nuclear entry may shed light on the function(s) of Gag in the nucleus and may begin to elucidate the molecular mechanisms involved in import of the PIC.
The initial aim of studying the nuclear transport pathway of the Gag polyprotein was thwarted by its apparent cellular toxicity in yeast. We can imagine several possible mechanisms of Gag cytotoxicity. Gag proteins interface with the machinery associated with vacuolar sorting through multivesicular bodies during the latter stages of virus assembly (reviewed in reference 24). Because the rigid yeast cell wall precludes viral budding, it is possible that Gag proteins accumulate in vacuolar bodies and impair normal functions of the vacuolar sorting machinery. As well, Gag proteins targeted to the yeast cell membrane might interfere with cell growth and division. Both of these ideas are consistent with the elongated appearance of yeast cells expressing Gag proteins. Alterations in cellular morphology and toxicity are also observed in avian cells that express high levels of Gag-GFP, and the degree of cytotoxicity is more evident with Gag mutants that are strongly plasma membrane associated than in those mutants having a cytosolic distribution (5, 11, 36). Furthermore, the nuclear trafficking of Gag might itself be toxic to yeast, particularly if the import factors used by Gag are needed for other cellular functions. Efforts to isolate spontaneous mutants of yeast that are able to tolerate high levels of Gag expression may prove to be informative for identifying additional host factors that interact with the virus assembly pathway.
S. cerevisiae serves as a model system for nucleus/cytosol exchange because the nuclear transport pathways are very highly conserved among lower and higher eukaryotes (7). We constructed a set of inducible expression vectors to identify cis- and trans-acting factors involved in Gag nucleus/cytoplasm dynamics in live yeast cells. Using these new pIGin/pIGout vectors, our data demonstrated that residues 216 to 232 in the p10 domain of Gag are sufficient to mediate nuclear export through the Crm1p pathway. The NLS in the MA protein we previously described (36) was also confirmed, and we discovered a novel NLS within the NC domain. Verification that the NC NLS was functional in avian cells validated the use of the yeast expression system to identify new targeting elements in heterologous viral proteins.
There are examples of proteins that possess multiple NLSs, some of which are served by different importin- family members (25), but in general, little is known about how each individual signal is utilized. Accordingly, we wonder why there are two independent NLSs in the RSV Gag polyprotein. One possibility is that both signals are used during the nuclear trafficking of Gag during virus assembly, and the presence of redundant targeting signals in Gag would imply that nuclear trafficking is an important step in replication. However, our experiments suggest that the novel NLS in NC is the predominant sequence used for nuclear import of Gag in avian cells. Another possible explanation for the dual import signals is that the MA and NC NLSs might function either at separate steps or within distinct import complexes to facilitate Gag nuclear entry. Finally, perhaps either the MA or NC NLS has no function in nuclear entry, since the basic residues in NC are also important for viral RNA binding and the basic residues in MA mediate plasma membrane targeting.
It is also conceivable that the NLSs in MA and/or NC are active during the early steps of infection, when mature Gag proteins enter the cell with the incoming virion. It is uncertain whether MA is a component of the RSV PIC, although genetic evidence supports this idea (12). Most certainly, NC is present within the PIC because it is bound to the RNA genome in the virion and is involved in reverse transcription and integration (2, 6, 16, 34, 48). An NLS in RSV integrase was previously identified (20), and thus there are three candidate karyophilic proteins that might promote nuclear transport of the viral genome early in infection. The nuclear import of the HIV-1 PIC has been intensively studied, and it appears that there are multiple signals for nuclear targeting, including MA, integrase, Vpr, and an intermediate DNA product of reverse transcription (40). As well, HIV-1 NC enters the nucleus early after infection and enhances the expression of spliced viral mRNAs after integration (54).
Interestingly, our screen of deletion strains and conditional mutants involving importin- family members indicated that the MA and NC proteins enter the nucleus through interactions with different soluble nuclear transport receptors. Import of the MA protein was dependent on Mtr10p and Kap120p. Mtr10p (mRNA transport regulator) is involved in nuclear translocation of RNA-binding proteins that contain serine/arginine (SR)-rich domains, including Np13p, Gbp2p, and Hrb1p, which are nucleocytoplasmic shuttling proteins implicated in the nuclear export of mRNA in yeast (14, 26, 29, 38, 50). These shuttling SR proteins are imported by Mtr10p and recruited to mRNA during transcription and then accompany the ribonuclear complex into the cytoplasm, where they may regulate translation (51). The dual involvement of Mtr10p in the import of MA and in the import of shuttling mRNA binding proteins is intriguing because we have postulated that RSV Gag enters the nucleus to bind newly transcribed viral RNA and transport it into the cytoplasm for encapsidation into assembling virus particles (36). However, there is no identifiable sequence homology between MA and SR proteins, including the NLS in Npl3p (10, 38; also, data not shown). Because little is known about the substrate specificity or molecular interaction between cargoes for the human (transportin-SR or importin 12 [17]) or avian homologues of Mtr10, RSV MA may prove to be an important future tool for studying this import pathway in higher eukaryotes.
We also found that the deletion of Kap120p, proposed to be a bidirectional transporter through the NPC (9, 44), impaired MA nuclear import. Kap120p functions in the export of the 60S ribosomal subunit (44) and its human homologue, importin 11, has been implicated in the import of ribosomal protein L12 (31, 32). Importin 11 also serves as the import receptor for the ubiquitin-conjugating enzyme UbcM2, and covalent attachment of ubiquitin to UbcM2 is a prerequisite for nuclear transport (33). This is an intriguing observation, given the dependence of RSV particle assembly on ubiquitination (28), prompting us to speculate that ubiquitin modification of Gag might be involved in its nuclear translocation.
Because deletions of both mtr10 and kap120 interfered with the nuclear accumulation of MA, we proposed that these importins might be involved in redundant pathways. Indeed, double mutants (mtr10 kap120) grew very poorly, suggesting overlapping functions for these import receptors. Future studies of MA-Kap120p interactions may reveal new insights into the substrate specificity and structural determinants of this poorly understood import receptor pathway.
In contrast to MA, the NC protein has a basic motif that resembles the canonical NLSs, and accordingly, its nuclear entry appeared to be mediated by the classical importin-/ pathway. It will be of interest to identify the specific residues in NC that are sufficient to mediate nuclear entry. Furthermore, we do not yet know whether there is a direct or indirect interaction between NC and the avian homologue of importin-. Finally, the relative reliance of Gag nuclear entry on the avian Kap120p, Mtr10p, or importin-/ homologues will be worthwhile to pursue.
The results of these studies raise many intriguing questions. Why does the Gag polyprotein contain two independent NLSs that utilize distinct import receptors Are the NLSs in MA and NC that mediate Gag nuclear entry also involved in the nuclear targeting of the PIC and the entry of the incoming viral genome during early infection, or do they have other intranuclear roles Are there cellular proteins that compete with RSV MA for utilization of Mtr10p and Kap120p in avian cells The use of the S. cerevisiae genetic system has provided the initial key insights needed to address these compelling questions, which are critical for understanding the molecular interactions of RSV with its intracellular environment.
ACKNOWLEDGMENTS
We thank M. L. Whitney for construction of mtr10::Kanr; J. E. Hopper, M. Fitzgerald-Hayes, P. Silver, M. Nomura, L. Pemberton, C. Guthrie, M. Robash, E. Callahan, and J. Wills for reagents; Scott Kenney for critical review of the manuscript; and the Penn State College of Medicine Core Facility for assistance with DNA sequencing and confocal microscopy.
We greatly appreciate support from the NIH (grants R01CA76534 to L.J.P. and R01GM27930 to A.K.H.), the NSF (predoctoral fellowship to L.Z.S.), the Penn State College of Medicine Dean's Feasibility Award (L.J.P. and A.K.H.), and the M.D. Research Facilitation Award (L.J.P.). This project was funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds, and the Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.
These authors contributed equally to this work.
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