The Cellular RNA Export Receptor TAP/NXF1 Is Required for ICP27-Mediated Export of Herpes Simplex Virus 1 RNA, but the TREX Complex Adaptor Protein Aly/REF Appears To Be Dispensable

Abstract

Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)⁺ RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)⁺ RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

In eukaryotes, the export of mRNA from the nucleus to the cytoplasm is a complex and carefully orchestrated process. In mammalian cells, the major mRNA export receptor is termed TAP/NXF1 (1, 4, 11, 16, 20, 49), and the homologue in Saccharomyces cerevisiae is Mex67p (14, 18, 40, 42, 45). TAP/NXF1 possesses a C-terminal domain that interacts with FG-repeats present on nucleoporins, which comprise the nuclear pore complex (NPC), and these interactions move TAP/NXF1 and its bound cargo RNA through the NPC to the cytoplasm (1, 2, 11, 16, 21). TAP/NXF1 does not bind directly to most RNA transcripts; instead, it interacts with export adaptor proteins that bind RNA and interact directly with TAP/NXF1 (47, 51). Aly/REF and its yeast homologue Yra1 have been shown to bind poly(A)⁺ RNA and to interact directly with TAP/NXF1 and Mex67p, respectively (18, 37, 40, 42, 47). Aly/REF was originally reported to be part of a complex of proteins termed the exon junction complex, which is deposited just upstream of exon junctions during a late stage of pre-mRNA splicing to mark the mRNA for export, mRNA surveillance, and localization in the cytoplasm (9, 24-26, 35, 50). Aly/REF requires UAP56, a splicing factor, for its recruitment to mRNA (13, 30, 31). More recent studies have provided evidence that Aly/REF has a only transient association with the exon junction complex and instead forms part of the TREX complex, which binds the 5′ end of the mRNA (8, 30, 33, 34, 46) through the cap binding protein CBP80 and which is essential for the export of both spliced and intronless mRNAs (8, 33, 46). UAP56 recruits Aly/REF and forms part of the TREX complex (3).

The herpesvirus family of multifunctional regulatory proteins, typified by the herpes simplex virus type 1 (HSV-1) protein ICP27, have been reported to interact with Aly/REF (3, 6, 7, 17, 32) and/or UAP56 (28, 48) and to use the TAP/NXF1 pathway for viral mRNA export (3, 6, 7, 19, 22). We have reported that ICP27 interacts directly with both Aly/REF and TAP/NXF1 (6, 7) and that ICP27 recruits Aly/REF but not TAP/NXF1 to viral replication compartments (6). Overexpression of Aly/REF stimulated export of several viral transcripts (7, 22). We have also shown that bulk poly(A)⁺ RNA and the majority of HSV-1 transcripts were retained in the nuclei of cells infected with viral ICP27 mutants that either cannot bind RNA or that do not interact with TAP/NXF1 (19), demonstrating that ICP27 accesses the TAP/NXF1 pathway to mediate viral RNA export. These studies did not address whether Aly/REF is also required for viral RNA export. Another complication is that both the N and C termini of ICP27 must be intact for interaction with TAP/NXF1 (6), and these regions have also been found to be required for ICP27's interaction with RNA polymerase II (10) and Hsc70 (27) so that deletion mutations in the N and C termini may have pleiotropic effects on HSV-1 replication. To directly monitor the dynamics of ICP27 localization and its interactions with Aly/REF and TAP/NXF1 in living infected cells, we constructed viral recombinants in which ICP27 was either N-terminally tagged or C-terminally tagged with yellow fluorescent protein (YFP). N-terminally tagged ICP27 was seen to be exported to the cytoplasm around 5 h after infection, and Aly/REF was recruited by N-terminally tagged ICP27 into large globular structures resembling replication compartments, as we determined previously by staining fixed cells that had been infected with wild-type (WT) HSV-1 (6, 7). In contrast, C-terminally tagged ICP27 remained nuclear, and export to the cytoplasm was seen only when TAP/NXF1 was overexpressed, suggesting that the C-terminal tag, but not the N-terminal tag, interfered with the interaction of ICP27 and TAP/NXF1. To determine the role of Aly/REF and TAP/NXF1 in RNA export during viral infection, these factors were knocked down using small interfering RNA (siRNA). These studies showed that TAP/NXF1, but not Aly/REF, is required for RNA export during HSV-1 infection.

MATERIALS AND METHODS

Cells, viruses, and recombinant plasmids.

African green monkey kidney cells (Vero cells), HeLa R19 cells, and rabbit skin fibroblast (RSF) cells were grown on minimal essential medium containing 10% fetal calf serum. WT HSV-1 strain KOS and the ICP27 null mutant virus 27-LacZ were previously described (43). The recombinant viruses with an N-terminal YFP tag (vN-YFP-ICP27) and C-terminal YFP tag (vC-YFP-ICP27) were constructed by homologous recombination of EYFP-ICP27 into the ICP27 locus. To construct the enhanced YFP (EYFP) fusion protein constructs, an EcoRI site was first engineered either upstream or downstream of the ICP27 coding regions in plasmid pSG130B/S (15) using a Quick-Change II site-directed mutagenesis kit (Stratagene). The EYFP coding sequence (pCMV-EYFP-C1 vector; Clontech) was amplified by PCR and fused in frame into the plasmid pSG130B/S with the EcoRI site. Linearized plasmid DNA was cotransfected into Vero cells with WT HSV-1 KOS genomic DNA. Yellow plaques were picked and subjected to five rounds of plaque purification. The insertion of YFP-ICP27 was confirmed by DNA sequencing, and expression of the full-length YFP-ICP27 fusion protein was confirmed by Western blot analysis. Plasmids pCFP-Aly/REF (where CFP is cyan fluorescent protein) and pCFP-TAP/NXF1 were constructed by ligating Aly/REF and TAP/NXF1 coding sequences (6) in frame into pECFP-C1 vector (Clontech). Plasmid pCFP-9G8 was constructed by ligating 9G8 cDNA in frame into the pECFP-C1 vector (Clontech).

Virus infection and transfection procedures.

Cells were infected with WT or mutant virus for the times indicated in the figure legends at a multiplicity of infection (MOI) of 10 and incubated at 37°C unless otherwise indicated. Transfection of plasmid DNA was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells were infected 24 h after transfection.

Western blot analysis.

Cells were transfected and/or infected and then washed with cold phosphate-buffered saline and harvested in 2× ESS loading buffer (20 mM Tris, 5 mM EDTA, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol) at the times indicated after infection, as described previously (39). Cell lysates were fractionated on 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose filters. Primary antibodies used for immunoblotting were as follows: anti-ICP4 antibody P1101 (Virusys); anti-ICP0 antibody P1112 (Virusys); anti-ICP27 antibody P1113 or P1119 (Virusys); anti-glycoprotein C (gC) antibody P1104 (Virusys); anti-gD antibody P1103 (Virusys); anti-gB antibody P1123 (Virusys); anti-YY1 antibody (Abcam); anti-RNA polymerase II (RNAP II) phosphoserine-2 form H5 (Covance Research Products); anti-Aly/REF antibody 11G5 (Sigma); anti-TAP antibody (Sigma); and anti-β-tubulin antibody (Zymed).

Immunofluorescence microscopy and in situ hybridization.

Cells were grown on coverslips and transfected and/or infected as indicated in the figure legends. Cells were fixed at the indicated times after infection in 3.7% formaldehyde. Immunofluorescence staining was performed as described previously (6, 10) with anti-ICP4 antibody P1101 at 1:500 (Virusys), anti-ICP8 P1115 at 1:50 (Virusys), anti-RNAP II antibody ARNA3 at 1:50 (Research Diagnostics), anti-Aly/REF 11G5 at 1:500 (Sigma), anti-TAP 53H8 at 1:50 (Sigma), anti-ICP27 P1119 at 1:500 (Virusys), and anti-bromodeoxyuridine (BrdU) Ab-3 at 1:100 (Calbiochem). In situ hybridizations for poly(A)⁺ RNA were performed as previously described (19). Cells were viewed by fluorescence microscopy at a magnification of ×100 with a Zeiss Axiovert S100 microscope. Images were pseudocolored and merged using Adobe Photoshop software. Cells in all immunofluorescence images shown are representative of greater than 75% of the cells visualized and are shown at a higher magnification to allow observation of intracellular structures.

Time-lapse microscopy of live cells.

RSF and Vero cells were seeded into 35-mm glass-bottom dishes and were incubated at 37°C on a temperature-controlled stage (Zeiss). Cells were incubated in phenol red-free medium containing 10% fetal calf serum, 14 g of Dulbecco's modified Eagle's medium powder (Meditech Inc.), 1.85 g of sodium bicarbonate (Meditech, Inc.), 10 ml of 200 mM l-glutamine (Meditech, Inc.), 5 ml of 100 mM sodium pyruvate (Meditech, Inc.), 1.1 ml of 45% glucose (Meditech, Inc.), and 25 mM HEPES (Meditech, Inc.) into a 500-ml volume, and pH was adjusted to 7.2. Cells were infected at an MOI of 10. Live infected cells were observed with a Zeiss Axiovert S100 inverted fluorescence microscope equipped with a 100× oil immersion objective lens and a Hamamatsu Orca 2 digital camera. The excitation wavelength was controlled by mercury lamp illumination and a motorized filter wheel equipped with filters specific for enhanced CFP and EYFP. The filter wheel, shutters on fluorescence, bright-field illumination light paths, and camera image acquisition were controlled by AxioVision software (Zeiss). Images were exported from AxioVision, and individual frames were prepared using Adobe Photoshop.

siRNA transfection.

Specific On-Target Plus pools of siRNAs directed at Aly/REF (Thoc4; 012078), TAP/NXF1 (013680), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control pool (D-001830-10) or nontargeting pool (D-001810-10) were synthesized by Dharmacon, Inc. The siRNA pools represent a mixture of four different siRNAs specific for their mRNA target. Transfections were performed with a 2 mM concentration of the siRNA pools into HeLa R19 cells using the DharmaFECT 1 transfection reagent (Dharmacon, Inc.) according to the manufacturer's protocol. Cells were either mock infected or infected with WT HSV-1 KOS for 8 h at the times after transfection indicated in the figure legends. Knockdown was confirmed at both the protein level by Western blot analysis and immunofluorescent staining and at the RNA level by real-time PCR. RNA from siRNA-treated cells was isolated using a Bio-Rad Aurum Total RNA Mini Kit according to the manufacturer's instructions and then reverse transcribed into cDNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories). Quantitative real-time PCR was performed using primers designed by Beacon designer software that are specific to Aly/Ref (forward, 5′-CGTGGAGACAGGTGGGAAAC-3′; reverse, 5′-GCACAGCCGCCTTCTTCAG-3′) or TAP/NXF1 (forward, 5′-CTTGAGGAAGATGATGGAGATG-3′; reverse, 5′-GTTAGGTCGGGTGGTA TAGG-3′) (Operon) in iQ SYBR Green Supermix (Bio-Rad Laboratories) as previously described (19). The data were analyzed with iCycler software (Bio-Rad Laboratories) and Microsoft Excel. Cell viability was determined by staining cells with the vital dye trypan blue solution (Sigma), followed by cell counting using a hemacytometer. When indicated in Fig. 7 to 9, cells were cotransfected with the Dharmacon siGLO green fluorescent transfection indicator reagent (D-001630-01) according to the manufacturer's protocol.

In vitro export assay.

Cells were grown on coverslips and transfected with siRNAs for 24 h (TAP/NXF) or 48 h (Aly/REF and negative control siRNA) and then infected with WT KOS for 8 h. In vitro nuclear export assays were performed as previously described (6). Cytoplasmic membranes were permeabilized with 40 μl/ml digitonin (Calbiochem) in transport buffer (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EDTA, and 2 mM dithiothreitol). Protease inhibitors, aprotinin, leupeptin, pepstatin A, and trypsin inhibitor (at 1 μg/ml; Sigma) were added at the time of permeabilization and were present throughout the assay. Cell monolayers were washed three times with cold transport buffer after treatment with digitonin to allow cytoplasmic factors to leak out. After a washing step, 50% rabbit reticulocyte lysate and an ATP regeneration system (1 mM ATP, 0.2 mM GTP, 5 mM creatine phosphate, and 20 U/ml creatine phosphokinase) in transport buffer was added, and the assay was performed at 30°C in a humidified chamber. Assays were stopped by the addition of cold transport buffer. Exported proteins were washed away with two ice-cold washes. Proteins retained in the nucleus were harvested by direct lysis of the cells in 2× SDS-polyacrylamide gel electrophoresis loading buffer. Protein samples were analyzed by Western blot analysis as described previously (6, 7) and quantified using Quantity One software.

Bromouridine labeling.

Cells were transfected with siRNA for the indicated times then either mock infected or infected with WT KOS for 8 h. For the pulse-labeled samples, 2 mM 5-bromouridine (Sigma) was added to the cell culture medium at 7.5 h after infection, and cells were incubated at 37°C for 30 min (23). The pulse-chase labeled samples were treated with 2 mM 5-bromouridine at 7 h after infection and incubated at 37°C for 30 min, at which time cells were washed with culture medium without BrdU and incubated for 30 min longer at 37°C. When indicated in the legend to Fig. 10, 10 μg/ml actinomycin D (Sigma) was added with 5-bromouridine to stall transcription. Cells were fixed and immunostained as described above.

RESULTS

N-terminally tagged ICP27 recombinant virus replicates as efficiently as WT HSV-1, whereas the C-terminally tagged virus is severely compromised.

During WT HSV-1 infection, ICP27 begins to shuttle between the nucleus and cytoplasm beginning about 5 h after infection, as seen in fixed stained cells (7, 38, 44). To directly observe the dynamics of ICP27's intracellular movements during infection in living cells, ICP27 viral recombinants were constructed in which a YFP tag was fused in frame to the N terminus or C terminus of ICP27 (Fig. 1A). vN-YFP-ICP27 virus replicated as efficiently as WT HSV-1 KOS in one-step growth curves (Fig. 1B); however, the replication cycle of vC-YFP-ICP27 was delayed compared to HSV-1 KOS, and final titers were approximately two logs lower (Fig. 1B). Expression of the immediate-early proteins ICP4 and ICP27 was delayed during infection with vC-YFP-ICP27, and ICP0 levels were reduced about fivefold compared to WT KOS and vN-YFP-ICP27 (Fig. 1C). Levels of the late proteins gC and gD were also greatly reduced (Fig. 1C). The formation of viral replication compartments, marked by staining with antibodies to ICP4 and ICP8, was restricted to prereplication sites by 8 h after infection with vC-YFP-ICP27, whereas full-blown replication compartments were observed with vN-YFP-ICP27 (Fig. 2A). Similarly, cellular RNAP II was redistributed to viral replication compartments in infections with KOS and vN-YFP-ICP27, as reported previously for WT HSV-1 (10, 36), but during infection with vC-YFP-ICP27, RNAP II was seen to be diffusely distributed throughout the nucleus in a distribution pattern similar to what was seen in mock-infected cells (Fig. 2A).

FIG. 1.

Characterization of YFP-ICP27 recombinant viruses. (A) A schematic representation of the YFP-ICP27 fusion protein constructs that were recombined into the HSV-1 genome. The resulting viruses were termed vN-YFP-ICP27 for the N-terminal tagged version and vC-YFP-ICP27 for the C-terminal tagged version. (B) A one-step virus growth curve was performed by infecting Vero cells with either WT KOS, vN-YFP-ICP27, or vC-YFP-ICP27 at an MOI of 1. Cells were harvested at 0, 4, 8, 16, and 24 h after infection, and samples were assayed on Vero cells. The data shown were derived from a single experiment but are representative of four experiments. (C) HeLa cells were infected with WT KOS, vN-YFP-ICP27, or vC-YFP-ICP27 at an MOI of 10 for the indicated times. Whole-cell extracts were prepared and fractionated on 10% SDS-polyacrylamide gels, followed by Western blot analysis with anti-ICP4 antibody, anti-ICP0 antibody, anti-ICP27 antibody P1113, anti-gC antibody, and anti-gD antibody, as indicated. Blots were also probed with anti-YY1 antibody as a loading control.

FIG. 2.

Fusion of YFP to the C terminus of ICP27 impairs ICP27's functions. (A) RSF cells were infected with WT KOS, vN-YFP-ICP27, or vC-YFP-ICP27 at an MOI of 10 for 8 h. Cells were fixed and stained with anti-ICP27 P1119 (in WT infection), anti-ICP4 antibody, anti-ICP8 antibody, and anti-RNAP II antibody ARNA3. YFP-ICP27 expression was detected by direct fluorescence. Arrows point to HSV-1 replication compartments (middle panels) or prereplication sites (right-hand panels). (B) HeLa cells were mock infected (Mock) or were infected with WT KOS, vN-YFP-ICP27, vC-YFP-ICP27, or ICP27 null mutant 27-LacZ as indicated. Nuclear extracts were prepared at the times indicated. Western blot analysis was performed with anti-RNAP II phosphoserine-2 form antibody H5, anti-ICP27 antibody P1113, and anti-YY1 antibody as a loading control. The upper arrow indicates the fusion proteins N-YFP-ICP27 and C-YFP-ICP27 while the lower arrow indicates the WT ICP27 protein.

We have previously reported that beginning about 6 h after infection, when viral transcription is very robust, the phosphoserine-2 form of RNAP II, which is found in the elongating transcription complex, is subject to ubiquitination and proteasomal degradation, probably due to collisions or pile-ups between rapidly transcribing complexes (10, 27). A much smaller reduction in phosphoserine-2 RNAP II levels was seen in infections with an ICP27 null mutant or with ICP27 mutants that are unable to interact with and recruit RNAP II to viral replication compartments because viral transcription is greatly reduced in these ICP27 mutant infections (10). Therefore, collision or stalling of transcription complexes is less likely to occur (10). Thus, degradation of the phosphoserine-2 form of RNAP II can be used as a measure of the robustness of viral transcription during HSV-1 infection (10, 27). Phosphoserine-2 levels remained relatively constant, as expected in mock-infected cells, and a decrease was not observed in cells infected with the null mutant 27-LacZ, as we reported previously (10); but there was a clear reduction evident between 4 and 8 h after infection with both WT KOS and vN-YFP-ICP27 (Fig. 2B). In contrast, no reduction was seen by 8 h after infection with vC-YFP-ICP27 (Fig. 2B). Taken together, these results indicate that vN-YFP-ICP27 behaves equivalently to WT HSV-1 KOS, whereas vC-YFP-ICP27 is compromised in its ability to replicate, which is manifested in delayed replication compartment formation, inefficient recruitment of RNAP II to viral replication sites, and decreased degradation of the elongating form of RNAP II, which is indicative of decreased viral transcription. We have reported that both the N and C termini of ICP27 must be intact for ICP27 to interact with RNAP II (10). These findings suggest that while the N-terminal YFP tag does not appear to affect ICP27's ability to interact with RNAP II, the C-terminal YFP tag hinders the interaction between ICP27 and RNAP II.

C-terminally tagged ICP27 is confined to the nucleus.

To observe the intracellular movement of ICP27 in living cells, HeLa cells were infected with vN-YFP-ICP27 or vC-YFP-ICP27, and fluorescent images were taken at 30-min intervals. ICP27 was nuclear at 3 h after infection with vN-YFP-ICP27, but cytoplasmic fluorescence could be visualized by 5.5 h after infection (Fig. 3), in accordance with what we have observed previously in fixed cells. In contrast, ICP27 remained exclusively nuclear up to 7 h after infection with vC-YFP-ICP27 (Fig. 3). This result suggests that the C-terminal YFP tag hampers the interaction of ICP27 with TAP/NXF1.

FIG. 3.

The C-terminal YFP tag interferes with ICP27 shuttling, while the N-terminal tag does not. RSF cells were infected with vN-YFP-ICP27 or vC-YFP-ICP27 and were analyzed by live-cell microscopy. Images were captured under ×100 oil lens magnification (vN-YFP-ICP27) or ×40 magnification (vC-YFP-ICP27). The times after infection are indicated in each panel. The arrow at 330 min (upper panels) points to cytoplasmic vN-YFP-ICP27.

To determine the effect of overexpression of TAP/NXF1 on the export of ICP27, HeLa cells were first transfected with plasmid DNA encoding CFP-TAP/NXF1 and were subsequently infected with vN-YFP-ICP27 or vC-YFP-ICP27. In vN-YFP-ICP27 infection, ICP27 was more rapidly exported in the cell expressing CFP-TAP/NXF1 than in other cells in the field, with cytoplasmic fluorescence observed by 150 min after infection (Fig. 4, upper panels). Overexpression of TAP/NXF1 enabled the export of C-terminally tagged ICP27 in that the cell expressing CFP-TAP/NXF1 showed some cytoplasmic fluorescence starting at around 5.5 h after infection with vC-YFP-ICP27, whereas ICP27 remained nuclear up to 7 h after infection in the other cells in the field (Fig. 4). These results lend further support to the conclusion that the C-terminal tag interferes with ICP27's ability to interact with TAP/NXF1 effectively.

FIG. 4.

Overexpression of TAP/NXF1 stimulates the export of vN-YFP-ICP27 and vC-YFP-ICP27. RSF cells were transfected with pCFP-TAP/NXF1 and 24 h later infected with either vN-YFP-ICP27 or vC-YFP-ICP27 at an MOI of 10. Images were taken beginning at 150 min after infection under ×40 magnification. The same field of cells was monitored over the time course, and images were taken with CFP and YFP filters to visualize CFP-TAP/NXF1 (blue) and YFP-ICP27 (yellow), respectively. The times after infection are indicated in each panel.

YFP-tagged ICP27 colocalizes with Aly/REF and recruits it to structures resembling viral replication compartments.

We showed previously that during WT HSV-1 infection, ICP27 interacts with the export adaptor protein Aly/REF and recruits Aly/REF away from splicing speckles, its predominant location in uninfected cells (52), to viral replication compartments (6). To monitor the interaction of Aly/REF and ICP27 in living infected cells, HeLa cells were transfected with CFP-Aly/REF and then infected with vN-YFP-ICP27. At early times after infection, YFP-ICP27 was seen in speckled structures resembling splicing speckles (Fig. 5), as we have reported previously for WT ICP27 (41), and YFP-ICP27 and CFP-Aly/REF were largely colocalized, as can be seen more clearly in the merged images (Fig. 5). As infection proceeded, a more coalesced pattern emerged, with both YFP-ICP27 and CFP-Aly/REF colocalized in globular structures resembling viral replication compartments (Fig. 5). These results are in agreement with those we reported previously in fixed cells (6, 7) and further demonstrate that ICP27 colocalizes with Aly/REF and recruits Aly/REF to viral replication sites.

FIG. 5.

Aly/REF colocalizes with N-YFP-ICP27 in live cells. RSF cells were transfected with pCFP-Aly/REF, and 24 h later they were infected with vN-YFP-ICP27 at an MOI of 10. Live-cell images were taken beginning at 170 min after infection under ×100 magnification. The same field of cells was monitored over the time course, and images were taken with CFP and YFP filters to visualize CFP-Aly/REF (blue) and YFP-ICP27 (yellow), respectively. The times after infection are indicated in each panel.

siRNA knockdown of Aly/REF and TAP/NXF1.

ICP27 interacts directly with both Aly/REF and TAP/NXF1 (6, 7). In studies with ICP27 viral mutants that are unable to interact with TAP/NXF1, we found that ICP27 was confined to the nucleus and that export of early and late viral transcripts was impaired, indicating that ICP27 accesses the TAP/NXF1 pathway to facilitate the export of viral RNA (19). These studies did not address the role of Aly/REF in viral export, and, further, because ICP27 mutants that do not interact with TAP/NXF1 are also unable to interact with RNAP II, it is difficult to cleanly separate the roles of ICP27 in stimulating viral transcription from its role in RNA export. To address the roles of Aly/REF and TAP/NXF1 in the export of RNA during HSV-1 infection directly, we used siRNA to knock down levels of these proteins. A mixture of four targeted siRNAs for Aly/REF and TAP/NXF1 was transfected into cells, and protein levels were measured by Western blot analysis at 24 and 48 h after transfection (Fig. 6A). About an 80% reduction in Aly/REF protein levels was seen 48 h after transfection (Fig. 6A), and an even greater decrease was seen in Aly/REF RNA levels (Fig. 6B), with less than a 30% reduction in cell viability (Fig. 6C). In contrast, although a substantial knockdown of TAP/NXF1 protein and RNA levels was achieved after 48 h, there was a greater than 60% decrease in cell viability (Fig. 6A to C). This likely reflects the fact that TAP/NXF1 is required for cellular mRNA export and is an essential mammalian protein (12, 16). Therefore, in subsequent experiments, siRNA knockdown of Aly/REF was done for 48 h, and knockdown of TAP/NXF1 was for 24 h. This achieved a greater than 90% knockdown of Aly/REF and a significant reduction in TAP/NXF1 levels (Fig. 6D).

FIG. 6.

siRNA knockdown of the cellular export factors Aly/REF and TAP/NXF1. (A) HeLa cells were mock transfected or transfected with negative control siRNAs or a pool of siRNAs specific to Aly/REF or TAP/NXF1 for 24 or 48 h. Whole-cell lysates were collected and subjected to Western blot analysis with anti-Aly/REF and anti-TAP/NXF1 antibodies as indicated. The Aly/REF blot was probed with anti-β-tubulin antibody, and the TAP/NXF1 blot was probed with anti-GAPDH antibody as loading controls. (B) HeLa cells were either mock transfected or transfected with a pool of siRNAs specific to Aly/REF or TAP/NXF1 for 24 or 48 h. Total cellular RNA was isolated and reverse transcribed into cDNA. Specific primers to Aly/REF (Aly siRNA-treated cells) or TAP/NXF1 (TAP siRNA-treated cells) were used to amplify and quantify the number of copies of the respective transcript. The Aly/REF copy number detected at 24 h was below the scale shown. (C) HeLa cells were mock transfected, transfected with negative control siRNAs, or transfected with siRNAs specific to Aly/REF or TAP/NXF1 for 24 or 48 h. Cells were harvested and stained with the trypan blue vital dye. The number of blue cells (not viable) counted was divided by the total number of cells counted to determine percentage of cell death. (D) HeLa cells were transfected with negative (Neg) control siRNAs or Aly/REF siRNAs for 48 h or TAP/NXF1 for 24 h, followed by infection with WT KOS for 8 h. Cells were fixed and then stained with anti-Aly/REF or anti-TAP/NXF1, respectively.

Knockdown of TAP/NXF1 retains ICP27 in the nucleus.

To determine the effect of knockdown of Aly/REF and TAP/NXF1 on the export of ICP27 to the cytoplasm, we performed immunofluorescence assays and in vitro export assays as described previously (6, 7). In the immunofluorescence experiments, siGLO, a transfection marker, was cotransfected with siRNAs to mark cells that were transfected with siRNA. In the cell transfected with Aly/REF siRNA, ICP27 was seen to accumulate in a perinuclear pattern but was also seen in the cytoplasm, and the nucleus was nearly devoid of ICP27 (Fig. 7A). This suggests that Aly/REF is not required for ICP27 export, but it may contribute to the efficiency of ICP27 export. This was substantiated by in vitro export assays (Fig. 7B). In these assays, the amount of ICP27 protein remaining in the nucleus after the assay is initiated is measured (6), as outlined in the schematic in Fig. 7B. ICP27 in untreated infected cells was found to exit the nucleus rapidly, with less than 10% of the protein found in the nucleus by 10 min after the start of the assay, in accord with our previous findings (6, 7). In cells treated with Aly/REF siRNAs, export of ICP27 was somewhat delayed, but by 20 min greater than 80% of the protein exited the nucleus, and by 30 min virtually all of the ICP27 was gone from the nucleus (Fig. 7B). The delay in export may reflect the perinuclear staining observed in the immunofluorescence assay (Fig. 7A). In contrast, in cells transfected with siRNAs to TAP/NXF1, ICP27 was confined to the nucleus, as observed by immunofluorescent staining (Fig. 7A), and export was also found to be curtailed in the in vitro export assay (Fig. 7B). This result is similar to what we have reported previously using a TAP/NXF1 dominant negative mutant (6, 19). We conclude that TAP/NXF1 is essential for the export of ICP27 to the cytoplasm, whereas Aly/REF is not essential but contributes to the efficiency of ICP27 export.

FIG. 7.

Knockdown of Aly/REF and TAP/NXF1 and the effect on ICP27 localization and nuclear export. (A) HeLa cells were cotransfected with the siGLO transfection indicator (Dharmacon) and a pool of siRNAs specific to Aly/REF for 48 h, TAP/NXF1 for 24 h, or GAPDH for 48 h, followed by infection with WT KOS for 8 h. Cells were fixed and stained with anti-ICP27 antibody (red). siGLO fluorescence (green) was directly visualized. (B) HeLa cells were transfected with the negative (Neg) control siRNA pool for 48 h or pools of siRNA specific to TAP/NXF1 for 24 h or Aly/REF for 48 h, followed by infection with WT KOS for 8 h, at which time cells were placed on ice, and in vitro export assays were performed as previously described (6, 7, 19) and as detailed in Materials and Methods. Western blot analysis was performed with anti-ICP27 antibody and anti-YY1 antibody as a loading control. The amount of ICP27 protein in each lane was quantified by densitometry, and the value at time zero was set to 100%. The percent reduction of nuclear ICP27 at each time point was calculated and subtracted from 100% to yield the percentage of ICP27 protein remaining in the nucleus, which was then plotted relative to time. The data shown are from a single experiment but are representative of the results obtained from three independent experiments. RRL, rabbit reticulocyte lysate.

Knockdown of TAP/NXF1 decreases viral late gene expression, but knockdown of Aly/REF does not.

To determine what effect knockdown of Aly/REF and TAP/NXF1 would have on HSV-1 replication and gene expression, we monitored the formation of viral replication compartments marked by staining for ICP4 (Fig. 8A), and we measured expression of ICP27 and two late proteins, gB and gC, by Western blot analysis (Fig. 8B). Full-blown replication compartments were visualized in WT HSV-1 KOS-infected cells at 8 h after infection after knockdown of Aly/REF (Fig. 8A). In the TAP/NXF1 siRNA-treated cells, one of the two cells, which was positive for transfection as shown with the siGLO marker, showed diffuse nuclear staining of ICP4. The other siGLO-positive cell showed the formation of replication compartments. Thus, replication compartment formation may be somewhat delayed when TAP/NXF1 levels are reduced. ICP27 protein levels were not affected by Aly/REF knockdown and were reduced only about twofold after TAP/NXF1 knockdown. In contrast, gB was not detectable, and gC was barely detectable in cells in which TAP/NXF1 was knocked down (Fig. 8B), but Aly/REF knockdown did not reduce gB or gC levels. The knockdown of TAP/NXF1 also reduced Aly/REF and β-tubulin levels to some extent, which likely reflects the general requirement for TAP/NXF1 for export of mRNA. These results suggest that export of viral RNAs that have been found previously to require ICP27 and TAP/NXF1 for efficient export (19) was impaired by the reduction of TAP/NXF1. This is further supported by the finding that in one-step growth curves, viral titers of HSV-1 KOS were reduced about sevenfold in cells in which TAP/NXF1 levels were reduced (Fig. 8C). Because only about 50% of the cells were successfully transfected with TAP/NXF1 siRNA, the reduction in virus yields in those cells in which TAP/NXF1 levels were reduced was probably even greater. In contrast, reduction in Aly/REF levels did not affect gB or gC expression (Fig. 8B), and viral titers were only slightly reduced, as seen in one-step growth curves (Fig. 8C).

FIG. 8.

Knockdown of TAP/NXF1 but not Aly/REF hinders viral late protein expression. (A) HeLa cells were cotransfected with siGLO and either the negative (Neg) control siRNA pool for 48 h, Aly/REF siRNA pool for 48 h, or TAP/NXF1 siRNA pool for 24 h, followed by infection with WT KOS for 8 h. Cells were fixed and stained with anti-ICP4 antibody (red). siGLO fluorescence (green) was visualized directly. (B) HeLa cells were transfected with the negative siRNA pool for 48 h, TAP siRNA pool for 24 h, Aly siRNA pool for 48 h, or GAPDH siRNA pool for 48 h, followed by infection with WT KOS for 8 h. Whole-cell extracts were harvested and subjected to Western blot analysis with anti-TAP/NXF1 antibody, anti-Aly/REF antibody, anti-ICP27 antibody, anti-gB antibody, anti-gD antibody, or anti-β-tubulin antibody as a loading control. (C) One-step growth curves were performed in cells that were treated with negative control siRNA or with siRNA specific for TAP/NXF1 or Aly/REF. Virus yields were assayed at the times indicated. The experiment was performed three times, and error bars indicating the standard deviations are shown.

Knockdown of Aly/REF does not impair poly(A)⁺ RNA export during viral infection; however, knockdown of TAP/NXF1 does.

To determine the effect of knockdown of Aly/REF and TAP/NXF1 on export of RNA during WT HSV-1 KOS infection, HeLa cells were transfected with siRNAs to Aly/REF or TAP/NXF1 for 48 h and 24 h, respectively, and then mock infected or infected with HSV-1 KOS. Eight hours later, cells were fixed, and in situ hybridization was performed with an oligo(dT) probe to visualize poly(A)⁺ RNA, as described previously (19). In mock-infected cells transfected with the negative control siRNA, poly(A)⁺ was seen diffusely distributed throughout the nucleus and cytoplasm, indicating active RNA export (Fig. 9A). In mock-infected cells that were transfected with Aly/REF siRNA, cytoplasmic poly(A)⁺ RNA was clearly seen (Fig. 9A), indicating that Aly/REF is not essential for cellular mRNA export. This result has been reported previously using knockdown of Aly/REF (12, 29). We did, however, note a difference in the distribution of poly(A)⁺ RNA in the nucleus in the Aly/REF siRNA sample compared to the negative control siRNA sample. Instead of a diffuse nuclear distribution, poly(A)⁺ RNA was seen to be prominently localized to speckled-like structures that resembled splicing speckles (Fig. 9A). A similar pattern was observed in WT KOS-infected cells treated with the negative control siRNA as well as in cells transfected with Aly/REF siRNA (Fig. 9B), in that poly(A)⁺ RNA in the nucleus was also found in speckled-like structures, and poly(A)⁺ RNA was clearly seen in the cytoplasm. We have reported previously that ICP27 recruits Aly/REF to viral replication compartments (6), in effect depleting Aly/REF from cellular transcription-processing sites. Based upon the results that we found after depleting Aly/REF in mock- or KOS-infected cells with siRNA (FIG. 9), it appears that reducing Aly/REF levels has little effect on export of poly(A)⁺ RNA to the cytoplasm but, instead, appears to sequester nuclear poly(A)⁺ RNA to speckled structures. A similar pattern was seen for nuclear poly(A)⁺ RNA in mock- and KOS-infected cells treated with TAP/NXF1 siRNA, but a major difference was that cytoplasmic poly(A)⁺ RNA was not detected (Fig. 9). These results indicate that TAP/NXF1 is required for poly(A)⁺ RNA export in uninfected and HSV-1-infected cells, whereas Aly/REF is not required for RNA export to the cytoplasm.

FIG. 9.

siRNA knockdown of TAP/NXF1 inhibits the export of poly(A)⁺ RNA in uninfected and infected cells. (A) HeLa cells were cotransfected with siGLO, and the negative (Neg) control siRNA pool or Aly/REF-specific siRNA pool for 48 h or the TAP/NXF1 siRNA pool for 24 h. Cells were fixed, and in situ hybridization was performed using a biotinylated oligo(dT) probe to detect poly(A)⁺ RNA. Poly(A)⁺ RNA was visualized using a streptavidin-Texas Red secondary antibody, and siGLO fluorescence was visualized directly (green). (B) HeLa cells were cotransfected with siGLO and the negative control siRNA pool for 48 h, Aly/REF siRNA for 48 h, or TAP/NXF1 siRNA for 24 h, followed by infection with WT KOS for 8 h. Cells were fixed, and in situ hybridization was performed with a biotinylated oligo(dT) probe, which was visualized with a streptavidin-Texas Red secondary antibody. siGLO fluorescence was directly visualized (green).

TAP/NXF1 is required for the export of newly transcribed RNA during HSV-1 infection.

In the in situ hybridization experiments, we looked at poly(A)⁺ RNA at 8 h after infection when viral transcription is very active and a large portion of the mRNA is viral, as reported previously (19). To explicitly follow the fate of newly transcribed RNA, we performed pulse-chase experiments with 5-BrU, which is specifically incorporated into newly transcribed RNA (23). Labeled RNA was detected with an antibody specific to 5-BrU. Mock- and HSV-1 KOS-infected cells were pulse-labeled with 5-BrU at 7 h after infection for 30 min. Cells were either fixed and stained at this point or were chased with label-free medium and then fixed and stained 30 min later. In mock-infected cells, the BrU-labeled RNA was nuclear after the 30-min pulse but was predominantly cytoplasmic after the chase, indicating efficient RNA export (Fig. 10A). Similarly, in HSV-1 KOS-infected cells treated with the negative control siRNA and labeled with BrU at 7 h after infection, the label was mainly detected in the nucleus after the pulse but was found in the cytoplasm after the chase (Fig. 10A). HSV-1 KOS-infected cells treated with Aly/REF siRNA showed a predominantly nuclear fluorescence during the pulse, but BrU-labeled RNA was efficiently chased into the cytoplasm (Fig. 10A), lending strong support to our conclusion that Aly/REF is not required for viral RNA export. On the other hand, even after the chase, BrU-labeled RNA remained nuclear in KOS-infected cells treated with TAP/NXF1 siRNA (Fig. 10A), further illustrating the essential role of TAP/NXF1 in RNA export. That the BrU was specifically incorporated into newly transcribed RNA was verified by treating mock- and HSV-1-infected cells with the RNAP II transcriptional elongation inhibitor actinomycin D (Fig. 10B). Label was not incorporated in the presence of actinomycin D.

FIG. 10.

Knockdown of TAP/NXF1 but not Aly/REF interferes with the nuclear export of newly transcribed mRNA. (A) HeLa cells were transfected with a negative (Neg) control siRNA pool for 48 h, an Aly/REF siRNA pool for 48 h, or a TAP/NXF siRNA pool for 24 h as indicated and then mock infected or infected with HSV-KOS. At 7 h, cells were incubated with culture medium containing 2 mM BrU for 30 min and then either fixed and stained using an anti-BrdU antibody (pulse) or washed and incubated in culture medium without BrU for 30 min, followed by fixation and staining with anti-BrdU antibody (pulse-chase). (B) HeLa cells were transfected with CFP-9G8 for 24 h to mark the nucleus because the cellular protein 9G8 is predominantly nuclear (5) and then either mock infected or infected with WT KOS for 8 h. At 7.5 h the cells were treated with BrU alone or BrU and actinomycin D (ActD) and incubated for 30 min at 37°C. Cells were fixed and stained with anti-BrdU antibody (red). CFP-9G8 fluorescence was visualized directly (blue).

DISCUSSION

In this study, we tracked the intracellular movement of HSV-1 ICP27 in living infected cells and observed its interactions with two cellular export proteins, Aly/REF and TAP/NXF1. The important conclusions that arose were that ICP27 traffics to the cytoplasm between 5 and 6 h after infection, as we found previously in fixed stained cells (6, 7); that ICP27 interacts with Aly/REF and recruits it to globular compartments resembling viral replication compartments; and that the N-terminal YFP tag on ICP27 did not affect its functions, but the YFP tag on the C terminus of ICP27 interfered with its ability to recruit RNAP II to viral replication sites as well as ICP27's ability to interact with TAP/NXF1. As a result, C-terminally tagged ICP27 remained nuclear, and export to the cytoplasm occurred only when TAP/NXF1 was overexpressed.

To determine the role of Aly/REF and TAP/NXF1 in the export of mRNA during viral infection, we knocked down these factors using siRNAs. A reduction in Aly/REF levels of greater than 80% had no discernible effect on the export of poly(A)⁺ RNA in mock- or HSV-1 KOS-infected cells. It has been reported that Aly/REF is not essential for cellular mRNA export (12, 29). Recent studies have shown that Aly/REF forms part of the TREX complex of proteins that associate with the 5′ end of mRNA through an interaction with cap-binding protein CBP80 (33). The TREX complex has been shown to be required for the export of both spliced and intronless mRNAs in mammalian cells (8, 33), and this has also been demonstrated for the export of a Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNA (3). The only effect that we observed upon reduction of Aly/REF levels was a change in the distribution of nuclear poly(A)⁺ RNA in both mock- and HSV-1-infected cells from a diffuse nuclear distribution to a coalescence into speckled-like structures that resembled splicing speckles. The significance of this finding is unclear at this point, but, importantly, decreasing Aly/REF did not prevent mRNA export.

The question then arises as to why ICP27 interacts with Aly/REF and recruits it to viral replication compartments. In fact, the ICP27 homologues SM protein in Epstein-Barr virus and ORF57 in KSHV also interact with Aly/REF (17, 32). Aly/REF has been reported to interact with UAP56, another component of the TREX complex, and UAP56 has been shown to recruit Aly/REF to mRNA (13, 30, 31). Further, it has been shown that the cytomegalovirus homologue of ICP27, termed UL69, interacts with UAP56 (28), and both Aly/REF and UAP56 were recruited to a KSHV mRNA, as well as other components of the TREX complex (3). Recent studies in our lab have shown that ICP27 interacts with UAP56 both in vivo and in vitro and that Aly/REF appears to stabilize this interaction (L.A. Johnson and R. M. Sandri-Goldin, unpublished results). Thus, although the TREX complex may be essential to guide mRNA through the NPC in a 5′ to 3′ fashion (8), it is possible that the TREX complex can form with one of the components missing. Knockdown of UAP56 has been reported to impair cellular mRNA export (13, 31), and studies are in progress to determine if this is the case for mRNA export during HSV-1 infection.

In contrast to the findings with Aly/REF, knockdown of TAP/NXF1 prevented export of poly(A)⁺ and newly transcribed RNA during HSV-1 KOS infection. Reduction in TAP/NXF1 levels had to be carefully modulated because TAP/NXF1 is an essential protein, and cell viability was substantially reduced when TAP/NXF1 levels were too low. Even a moderate reduction resulted in the nuclear retention of both ICP27 and mRNA in HSV-1 KOS-infected cells. These findings indicate that TAP/NXF1 is absolutely required for RNA export during HSV-1 infection.