Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

Expression vectors, plasmids, and cells.RSV Gag expression plasmid pGagΔPR was described previously (46). The Gag p10 NES mutations encoding L219A, LWVL-A, and NES-A in pGag-GFP (6, 45) were transferred into pGag.CFP and pGag.YFP expression vectors (a gift from V. Vogt, Cornell University) (27) using SstI-SdaI fragment exchange. pΔNC.CFP and pΔNC.YFP were created using SacI-ApaI fragment exchange between pΔNC.GFP (a gift from J. Wills, Penn State College of Medicine) (7) and pGag.CFP or pGag.YFP. pGag.mCherry was made by PCR amplification of the fluorophore sequence from pRSET₈-mCherry (a gift from R. Tsien, University of California at San Diego) (49) and transfer into pGag-GFP using ApaI-NotI. pGFP.NLS.PK was created using QuikChange mutagenesis (Stratagene) to insert the simian virus 40 T-antigen nuclear localization signal (NLS) into pGFP-PK (a gift from Warner Greene, University of California at San Francisco) (51). pRSVGag.VN173 and pRSVGag.VC155 were constructed by using PCR amplification of bJunVN173 or bFosVC155 (a gift from C. Hu, Purdue University) (53) and insertion into the pRSVGag.GFP vector in place of green fluorescent protein (GFP) using ApaI-NotI. pMLV.Gag.VN173 and pMLV.Gag.VC155 were made by PCR and NheI-SmaI fragment exchange into pMLVGag.YFP (50) (a gift from W. Mothes, New Haven, CT). Mutants were screened using restriction endonuclease digestion and confirmed with automated DNA sequencing. All experiments were performed using either the chemically transformed QT6 quail fibroblast cell line or immortalized chicken embryo fibroblast DF-1 cells, maintained as previously described (19, 38). Transfections were performed by using the calcium phosphate method or Fugene 6 transfection reagent (Roche Applied Science).

Radioimmunoprecipitation assays.Budding assays were performed as previously described (39, 59). Immunoprecipitated RSV Gag proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed using a PhosphorImager (Molecular Dynamics). Budding efficiency was calculated as the ratio of Gag proteins in the media divided by the sum of the Gag proteins expressed in the cell lysates and media.

Confocal imaging and subcellular quantification.Live cells were plated onto 35-mm glass-bottomed dishes (MatTek Corporation) and imaged using a Leica AOBS SP2 confocal microscope with sequential scan settings at 17 to 24 h posttransfection. Quantification of the total fluorescent intensity of the fluorescently tagged Gag proteins was performed using a single optical slice through the nuclear plane, and image analysis was performed using Leica Microsystems software. The percent nuclear fluorescence was calculated as the fluorescence intensity of the nucleus divided by the fluorescence intensity of the entire cell.

FRET measurements.Acceptor photobleaching FRET was performed on transfected cells fixed with 4% paraformaldehyde. Prebleach images of both cyan fluorescent protein (CFP) (excitation at 458 nm, emission at 460 to 500 nm, and 20% laser power) and yellow fluorescent protein (YFP) (excitation at 514 nm, emission at 550 to 600 nm, and 10% laser power) channels were acquired. YFP was specifically photobleached using the 514-nm laser at 100% laser power until the fluorescence intensity was decreased to 10% of the prebleach level. Postbleach images were acquired at the prebleach settings. FRET efficiency was calculated using the formula $$mathtex$$\(\mathrm{FRET}_{\mathrm{Eff}}{=}\frac{\mathrm{donor}_{\mathrm{Post}}{-}\mathrm{donor}_{\mathrm{Pre}}}{\mathrm{donor}_{\mathrm{Pre}}}\)$$mathtex$$, when donor_Post is greater than donor_Pre. FRET analysis was performed on at least two separate days using a minimum of 10 different cells per day. Nuclear FRET was performed by bleaching the entire nucleus through a single optical section of the nuclear plane.

BiFC analysis.QT6 cells were transfected in duplicate with 100 ng of each plasmid DNA using Fugene 6. At 4.5 h posttransfection, cells were fixed in 4% paraformaldehyde, stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Calbiochem) at a 1:10,000 dilution, washed in phosphate-buffered saline, mounted using a Slowfade antifade kit (Invitrogen), and imaged using a Leica SP2 confocal microscope. Overall intensity was increased equally for each bimolecular fluorescence complementation (BiFC) image after acquisition using CorelDRAW X3 (Corel Corporation). Duplicate plates were lysed in Laemmli sample buffer for analysis of intracellular protein expression levels using Western blotting with polyclonal anti-GFP (ab290) antibody (Abcam).

Subcellular localization of p10 NES mutant Gag proteins coexpressed with wild-type Gag.To determine whether the expression of NES mutant Gag proteins would alter the subcellular localization of the wild-type Gag protein, each Gag variant was fused to either YFP or CFP at the C terminus (Fig. 1). Because the wild-type Gag protein shuttles through the nucleus, we tested the possibility that it might dimerize with the nucleus-restricted NES mutant. Upon coexpression of the differentially tagged proteins, we envisioned three possible outcomes. First, if the NES mutant and wild-type Gag proteins did not associate either prior to nuclear entry or within the nucleus, the intracellular distribution of each protein population would be unchanged. Second, if the NES mutant exerted a trans-dominant negative effect on trafficking of the wild-type protein, there would be an increase in the amount of wild-type Gag in the nucleus. Third, the wild-type Gag protein might associate with the NES mutant within the nucleus, restoring cytoplasmic relocalization to the mutant that is normally “trapped” within the nucleus.

Using sequential scanning to eliminate spectral overlap between the YFP and CFP channels, optical slices through the nuclear plane of each cell were obtained using confocal microscopy. Coexpression of wild-type Gag-YFP and wild-type Gag-CFP revealed primarily cytoplasmic fluorescence with punctate foci within the cytoplasm and along the plasma membrane (Fig. 2a and b, where pseudocolored images show YFP as red and CFP as green). Localization appeared to be similar to data from previous reports of singly and coexpressed RSV Gag proteins in avian cells (6, 27, 44). Of note, the nuclei of these cells did not display substantial fluorescence, reflecting the transient nature of Gag nuclear trafficking and the greater efficiency of nuclear export compared to import. In contrast, when a mutant NES Gag-CFP protein was expressed with the wild-type Gag-YFP protein, a distinct change in the localization of the wild-type Gag protein was observed (Fig. 2c to h). In cells marked with white arrows, the wild-type Gag protein was sequestered in the nucleus, as indicated by an increase in the nuclear fluorescence. The accumulation of wild-type Gag within the nucleus was most striking in those cells expressing higher levels of the NES Gag mutant, as shown clearly in the case of the LWVL-A.Gag-CFP protein (Fig. 2d). These results support the hypothesis that the NES mutant Gag proteins act in a trans-dominant negative fashion to alter the nuclear export of wild-type Gag through protein-protein interactions within the nucleus.

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FIG. 2.

Confocal microscopic images of cells transfected with Gag-YFP and NES mutant Gag-CFP. Live cells expressing wild-type Gag-YFP (left) (pseudocolored red) and NES mutants LWVL-A.Gag-CFP, NES-A.Gag-CFP, and L219A.Gag-CFP (right) (pseudocolored green) were analyzed using sequential scanning laser confocal microscopy at wavelengths of 458 nm (CFP) and 514 nm (YFP). A single optical section through the nuclear plane is shown. Arrows indicate the nuclear accumulation of wild-type Gag-YFP with the coexpression of NES mutant Gag-CFP proteins.

To quantify the amount of nuclear accumulation of the wild-type Gag protein, confocal images of living cells were analyzed for nuclear fluorescence compared to total cellular fluorescence (Fig. 3). This method was preferred over subcellular fractionation because we used a transient transfection system, and therefore, it was not possible to ensure that every cell in the population expressed both the NES mutant and wild-type Gag proteins. The confocal imaging allowed us to choose to analyze only those cells coexpressing the mutant and wild-type proteins. To avoid complications resulting from potential weak interactions between the YFP and CFP domains (65), we substituted a monomeric far-red fluorescent tag (mCherry) for the fluorophore on the wild-type Gag protein, and CFP was used to tag the NES mutant Gag proteins (49). Western blot analysis of each fusion protein used for analysis revealed little or no free mCherry or CFP (data not shown).

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FIG. 3.

Quantification of the nuclear localization of wild-type and NES mutant Gag proteins. QT6 cells transfected with the indicated plasmid DNA were imaged with laser confocal microscopy using an excitation wavelength of 543 nm for mCherry. Total cellular and nuclear fluorescence intensities were measured, and Gag-mCherry nuclear fluorescence was calculated as the nuclear intensity divided by the whole-cell intensity. Each bar represents the average ratio of the nuclear to total cellular fluorescence. At least 100 cells were measured from three separate transfections, except for GFP.NLS.PK, for which 46 cells from two transfections were analyzed.

The wild-type Gag-mCherry fusion protein produced a subcellular distribution that was indistinguishable from that of Gag-GFP, Gag-YFP, or Gag-CFP (data not shown), and 7.3% of the wild-type protein was detected in the nucleus under steady-state conditions (Fig. 3). The coexpression of Gag-mCherry with LWVL-A.Gag-CFP, L219A.Gag-CFP, or NES-A.Gag-CFP resulted in an increase in the nuclear fluorescence of Gag-mCherry to 14.7%, 15.5%, or 16.4%, respectively, of the total cellular fluorescence. As a control, we expressed Gag-CFP with GFP.NLS.PK, which contains the simian virus 40 large-T-antigen NLS fused to chicken pyruvate kinase (51). The coexpression of the nucleus-localized GFP.NLS.PK protein with wild-type Gag-mCherry led to a slight increase in the amount of wild-type Gag in the nucleus (9.8%). However, the nuclear accumulation of wild-type Gag was significantly increased in the presence of each of the NES mutants compared to intranuclear levels of wild-type Gag expressed alone or with GFP.NLS.PK (P value of <0.0001). This finding indicated that the interaction between the wild-type and NES mutant Gag proteins was specific. Together, these results suggest that the NES mutant forms dimers or oligomers with wild-type Gag in the nucleus, retarding the nuclear egress of the wild-type protein.

Because Gag nuclear sequestration was not complete under conditions of coexpression with the NES mutants, we tested the possibility that the intranuclear interactions between Gag and the NES mutant facilitated the trafficking of the mutant into the cytoplasm. The cytoplasmic localization of each NES mutant was measured using confocal microscopy in cells coexpressing wild-type and NES mutant Gag proteins (data not shown). There was very little change in the cytoplasmic relocalization of the NES mutant proteins. However, we considered it likely that interactions between the wild-type protein and the NES Gag mutant would result in a loss of the mutant protein from the cytoplasm due to its release into the medium during budding. If the coexpression of wild-type Gag partially reversed the budding defect of the NES mutants, the cytoplasmic fluorescence of the NES mutants might be reduced.

Rescue of budding by complementation between wild-type and mutant Gag proteins.To determine whether the wild-type Gag protein could enhance the incorporation of NES mutant Gag proteins into VLPs, we performed budding assays on cells coexpressing wild-type Gag-GFP and NES mutants LWVL-A.Gag-CFP, NES-A.Gag-CFP, and L219A.Gag-CFP (Fig. 4). Cells were metabolically labeled for 2.5 h, cell lysates and medium samples were immunoprecipitated with anti-RSV serum, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by PhosphorImager analysis. Budding efficiency was calculated as the amount of Gag protein released into the medium compared to the total amount of Gag protein detected in the cell lysate and medium. The expression of each p10 NES mutant Gag-CFP protein with wild-type Gag-GFP resulted in an increase in budding for each mutant, although LWVL-A.Gag-CFP (11.4% increased to 50.0%) and L219A.Gag-CFP (10.5% increased to 34.3%) demonstrated the greatest degree of rescue (Fig. 4A and B). Of note, the magnitude of the increase in budding might be underestimated in these experiments because particle release for wild-type Gag-CFP was reduced in comparison to the budding efficiency of wild-type Gag-GFP.

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FIG. 4.

Rescue of budding for NES mutant Gag proteins coexpressed with wild-type Gag. (A) Autoradiogram of a typical budding rescue assay (see Materials and Methods). Gag-GFP can be differentiated from Gag-CFP by its faster migration during electrophoresis. Note the increase in the amount of Gag in the medium for L219A.Gag-CFP when coexpressed with wild-type Gag.GFP. (B) PhosphorImager quantification of budding efficiency. Particle release for wild-type Gag-GFP was arbitrarily set to 100%, and each coexpressed CFP protein was compared to Gag-GFP. Error bars represent standard errors of the means. (C) Analysis of budding efficiency for untagged NES GagΔPR mutants expressed with wild-type Gag-GFP resulted in an increase in NES mutant budding.

To eliminate any potential influence of the C-terminal CFP/GFP tags with Gag-Gag interactions, we performed budding complementation experiments using untagged NES mutants in which the PR domains were deleted (Fig. 4C). For LWVL-A.GagΔPR and NES-A.Gag ΔPR, budding efficiency was rescued from 9.3% to 41.6% and 45.5% of wild-type levels, respectively. The restoration of budding for the NES mutants suggests that the wild-type Gag protein interacts with the NES mutants in the nucleus. While it is possible that the Gag-mutant interaction occurs prior to nuclear entry, this conclusion is less likely given the confocal microscopy results shown in Fig. 2. However, to test rigorously whether true intermolecular Gag-Gag interactions were established in the nucleus, a biophysical method was utilized to detect protein-protein associations.

Analysis of intranuclear Gag-Gag interactions.Although Gag-Gag interactions in the cytoplasm and at the plasma membrane have been reported using FRET analysis (27), there are no studies of intranuclear Gag-Gag binding. For FRET to occur, two proteins must be between 10 and 100 Å from one another, a distance consistent with a biologically relevant, direct, and physical interaction (21, 54, 57). The magnitude of the energy transfer between the CFP and YFP fluorophores depends on proximity, so more closely associated proteins demonstrate higher FRET efficiencies (48).

For the FRET experiments, cells were transfected with Gag-CFP/Gag-YFP pairs, and representative images obtained for the YFP channel are shown in Fig. 5. To assess intranuclear interactions between wild-type Gag proteins, cells were treated with LMB to concentrate Gag within the nucleus. Two patterns of intranuclear distribution were observed: either Gag proteins were diffuse throughout the nucleoplasm, excluding nucleoli (Fig. 5a), or there were distinct punctate foci within the nucleoplasm, again excluding nucleoli (Fig. 5a′). The discrete puncta likely represent large aggregates of Gag proteins that form more frequently with higher levels of intracellular Gag expression.

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FIG. 5.

Intranuclear distribution patterns of Gag proteins and FRET analysis. Fixed QT6 cells were imaged through the nuclear plane using confocal microscopy. Diffuse intranuclear patterns are shown on the left, punctate distributions are on the right, and cells having different subnuclear distribution patterns were derived from a single transfection. Cells treated with LMB are indicated as +LMB. Punctate foci were not obtained for ΔNCGag-YFP/CFP or Gag.Zip-YFP/CFP.

To determine whether LMB was affecting the pattern of intranuclear localization, the NES-A.Gag-YFP/CFP proteins were examined without LMB treatment; however, the same diffuse and punctate distributions were observed (Fig. 5b and b′). Interestingly, the deletion of the Gag NC domain, which mediates Gag-RNA and Gag-Gag interactions, abrogated the formation of punctate foci (Fig. 5c), indicating that the puncta were NC dependent. To test whether the punctate pattern was mediated through protein-protein or protein-RNA interactions, we examined LMB-treated cells expressing the Gag.Zip-YFP/CFP proteins, which have the human CREB binding domain leucine zipper substituted for NC (22). The Gag.Zip proteins interact via a nucleic acid-independent mechanism and failed to form punctate foci, suggesting that the Gag aggregates are mediated through NC-RNA interactions in the nucleus.

Acceptor photobleaching FRET analysis (23) was performed using single confocal z sections in which the maximal nuclear radius was evident in cells expressing wild-type or mutant Gag-CFP/YFP pairs. Representative images from a FRET experiment for NES-A.Gag-CFP coexpressed with NES-A.Gag-YFP are shown in Fig. 6A. A region of interest encompassing the entire nucleus was specifically bleached using the 514-nm laser (excitation wavelength for YFP) at 100% laser intensity. A marked decrease in YFP fluorescence was evident in the postbleach YFP panel compared to the prebleach image, as expected (Fig. 6A, right). A concomitant increase in the intensity of the postbleach CFP fluorescence compared to the prebleach level was observed, indicating that FRET had occurred (Fig. 6A, left).

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FIG. 6.

Intranuclear Gag-Gag interactions assessed by acceptor photobleaching FRET analysis. (A) FRET analysis was performed on cells coexpressing NES-A.Gag-CFP and NES-A.Gag-YFP. Confocal images through the nuclear plane were obtained prior to photobleaching (top). The nuclear region of interest, indicated by the white circle, was subjected to bleaching of the YFP (acceptor) fluorophore using a high-intensity laser at a wavelength of 514 nm, resulting in a significant loss of the signal in the postbleach NES-A.Gag-YFP image (lower right). The increased intensity of the NES-A.Gag-CFP fluorescence in the postbleach image (lower left) resulted from the decreased transfer of energy from the CFP fluorophore (donor) to YFP. (B) QT6 cells expressing the indicated proteins demonstrating punctate (P) or diffuse (D) phenotypes (as shown in Fig. 5) were analyzed using FRET. The FRET efficiency (percent) for each condition was calculated according to the equation shown in Materials and Methods. FRET experiments were performed a minimum of 10 times from at least two separate transfections. The mean FRET efficiency was calculated and graphed, with error bars representing standard errors of the means.

Average FRET efficiency values were obtained from at least 10 cells derived from two separate transfections in which Gag was distributed in diffuse or punctate patterns (Fig. 6B). As a negative control, cells expressing free CFP and YFP produced very low FRET efficiencies (0.6%) in the nucleus. Cells coexpressing wild-type Gag-CFP and Gag-YFP fusion proteins were treated with LMB, causing the relocalization of Gag to the nucleus, and FRET efficiencies of 2.0% were observed in nuclei with a diffuse pattern and 9.4% in cells containing punctate foci. For the NES mutants, the range of nuclear FRET values was 1.4% to 4.9% for nuclei with a diffuse distribution and 8.5% to 12.8% for nuclei with punctate fluorescence. Deletion of the NC domain did not eliminate Gag-Gag associations, as the FRET efficiency was 4.9%, although there were no large multimeric intranuclear complexes of Gag seen in these cells. For the Gag.Zip-CFP/YFP pair, the FRET efficiency was 11.1%, indicating that the proteins were interacting in the nucleus. However, even though the Gag.Zip proteins were in close proximity based on the FRET results, they were unable to form punctate fluorescent complexes in the nucleus.

Detection of Gag-Gag interactions using BiFC.Accurate results with FRET required that Gag proteins be expressed at high levels. Although both FRET and BiFC detect protein-protein interactions within distinct subcellular compartments, BiFC offers an advantage because it identifies transitory protein complexes (20). Furthermore, the specificity of interactions detected using BiFC requires low expression levels of the test proteins (53), avoiding potential problems arising from protein overexpression.

To determine whether Gag-Gag interactions could be detected at lower intracellular levels of Gag, we utilized BiFC analyses. For these experiments, wild-type and mutant Gag proteins were fused to either the N-terminal 173 residues of the YFP variant Venus (VN173) or the C-terminal 155 amino acids (VC155) (Fig. 1 and 7). A low level of protein expression was needed to preserve the specificity of the interactions, so a small amount of plasmid DNA (100 ng) was used for transfection, and cells were examined by confocal microscopy a short time (4.5 h) after transfection (20, 53). If Gag proteins were closely juxtaposed, the N- and C-terminal halves of Venus would be brought together and fold into a functional fluorophore (63). The expression of each fusion protein containing VN or VC in the absence of the complementing fluorophores revealed no fluorescence (data not shown). However, the coexpression of RSV Gag-VN with RSV Gag-VC led to perinuclear, cytoplasmic, and plasma membrane epifluorescence (Fig. 7a and a′). The NES-A.Gag-VN/VC proteins appeared predominantly within the nucleus and faintly along the plasma membrane, indicating that these sites were the major sites of interaction. Removal of the NC domain resulted in cytoplasmic interactions without plasma membrane epifluorescence. As a negative control, RSV Gag-VN was expressed with MLV Gag-VC, and no fluorescence was detected, as expected, since these heterologous Gag proteins are not copackaged into VLPs (1, 4). The lack of fluorescence was not due to defective MLV Gag proteins, as complementation between MLV Gag-VN/VC resulted in Venus expression within discrete cytoplasmic foci. Thus, interactions between intranuclear RSV Gag proteins occurred under conditions that allowed the discrimination of specific intermolecular interactions.

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FIG. 7.

BiFC analysis of Gag-Gag complex formation. Cells were transfected with 100 ng of each plasmid construct bearing N- or C-terminal halves of the Venus fluorophore fused to the indicated Gag protein. Cells were fixed 4.5 h posttransfection and imaged using confocal microscopy. Excitation with the 514-nm laser produced a fluorescent signal when Gag dimers formed. The YFP (Venus) images are shown on the left, and Venus overlaid with DAPI shows the location of nuclei on the right.