Control of VP16 Translation by the Herpes Simplex
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病菌学杂志 2005年第7期
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
ABSTRACT
Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and ?-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.
INTRODUCTION
Lytic infection by herpes simplex virus type 1 (HSV-1) is characterized by shutoff of host protein synthesis and the temporally regulated expression of three classes of viral genes: immediate-early (IE), early (E), and late (L) (reviewed in reference 66). The infectious cycle is initiated by the virion transactivator VP16, which upon delivery into host cells acts in concert with cellular factors to induce the expression of the five IE genes. Four of these encode nuclear regulatory proteins (ICP0, ICP22, ICP4, and ICP27) that orchestrate the timely expression of the E and L genes. ICP27 plays an indispensable role in the viral life cycle (41, 67). ICP27 is necessary for efficient expression of some E genes, including a subset of those that encode proteins required for DNA replication (83). Hence, ICP27-null mutants display a partial defect in viral DNA replication. ICP27 also promotes the expression of most L genes (11, 41, 45, 62, 63, 67, 75) and is required for the so-called delayed shutoff of host protein synthesis (19, 20, 67, 79). How ICP27 performs its various functions remains poorly defined; however, it is becoming increasingly evident that ICP27 modulates virtually every aspect of mRNA metabolism, including primary transcription, polyadenylation, splicing, nuclear export, and mRNA stability (reviewed below).
It was initially assumed that the effects of ICP27 on HSV-1 gene expression were mediated primarily at the transcriptional level. Indeed, ICP27 is required for the transcription of at least two viral late genes, gC and UL47 (24). Furthermore, ICP27 interacts with the RNA polymerase II holoenzyme, providing further evidence for transcriptional modulation by ICP27 (25, 89). However, a wealth of data accumulated over the past decade has demonstrated that ICP27 mediates many of its effects posttranscriptionally. ICP27 alters the specificity of the polyadenylation machinery, an effect which may enhance the expression of viral L genes bearing inherently weak poly(A) signals (42-44, 72). ICP27 also impairs pre-mRNA splicing via multiple contacts with the splicing machinery (1, 19, 20, 36, 55, 70, 71). More recently, intensive studies have shown that ICP27 enhances the efficiency of nuclear export of intronless HSV-1 mRNAs (3, 4, 30, 69, 79).
ICP27 is an RNA binding protein that shuttles between the nucleus and the cytoplasm (23, 47, 48, 56, 69, 79). It contains an arginine-rich RGG box that is required for RNA binding (48, 69), as well as two nuclear localization sequences (46) and a leucine-rich sequence that bears a strong resemblance to the nuclear export sequence (NES) of the human immunodeficiency virus (HIV) protein Rev (69). Mutations that delete the NES restrict the protein to the nucleus and severely impair the viability of the virus (34). ICP27 was originally proposed to promote export of viral intronless RNAs through the cellular export adaptor CRM1 (80) in essentially the same manner as HIV Rev (reviewed in references 7, 58, and 82). However, Koffa et al. recently reported that CRM1 is not required for ICP27-induced mRNA export in Xenopus laevis oocytes (30), and Chen et al. provided evidence that the leucine-rich NES of ICP27 does not require CRM1 for activity (3). The current model for ICP27-mediated export of viral mRNAs is that ICP27 recruits the cellular mRNA export factor REF to viral intronless mRNAs (3, 30). REF is normally deposited onto cellular mRNAs as part of a multicomponent complex (the exon junction complex, or EJC) during the process of splicing and serves as a license for nuclear export of the spliced mRNA by interacting with the export factor TAP/NFX1 (26, 32, 33, 65, 81, 90; reviewed in reference 61). ICP27 has been shown to bind to REF and is thought to recruit REF to HSV RNAs, providing them with a splicing-independent means of accessing the TAP cellular mRNA export pathway (3, 30). Although the data supporting this model are compelling, it is noteworthy that a deletion (d3-4) that abolishes the ICP27-REF interaction (30) has little or no effect on virus growth or gene expression in Vero cells (34). In contrast, a deletion that removes the leucine-rich NES has much more serious consequences for the viability of the virus (34). Thus, the ICP27-REF interaction appears to be largely dispensable, at least in some cell types.
Given the multifunctional nature of the ICP27 protein, it has been relatively difficult to decipher at what level of gene expression ICP27 acts to regulate individual viral genes. In this study, we have examined how ICP27 modulates the expression of the essential viral transactivator, VP16. We find that ICP27 enhances VP16 expression primarily by increasing the translational efficiency of VP16 mRNA. Thus, we have uncovered a novel role for ICP27 in the translational control of viral gene expression.
MATERIALS AND METHODS
Cells and viruses. Vero cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. The HSV-1 wild-type strain used in this study was KOS1.1 (22). The ICP27-null strain d27-1 contains a 1.6-kb deletion that removes the ICP27 promoter and most of the open reading frame (63, 64). The 5dl1.2 virus is an independently derived ICP27 deletion mutant (41). The d27-lacZ1 virus bears an ICP27-lacZ fusion gene in place of the wild-type ICP27 gene and has a defect in ICP27 function (63). The ICP27 mutant viruses were propagated in V27 cells, a derivative of Vero cells engineered to express ICP27 upon infection with HSV (63). V27 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 100 μg of Geneticin (G418; Invitrogen)/ml.
Isolation of RNA and Northern blot analysis. Nuclear and cytoplasmic RNA fractions were isolated from infected Vero cells in 100-mm-diameter dishes by using the RNeasy purification kit (QIAGEN) as previously described (10). Total RNA was harvested from infected Vero cells by using the Trizol reagent (Invitrogen). Probes and hybridization conditions for Northern blot analysis were as follows. The VP16 probe was a fragment generated by PCR using plasmid pVP16-KOS as a template (86). To probe for the U3 snoRNA, a radiolabeled oligonucleotide specific for U3 was used as previously described (4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ?-actin transcripts were detected with 32P-labeled oligonucleotide probes (for GAPDH, 5'-TTGACTCCGACCTTCACCTTCCCCAT-3'; for ?-actin, 5'-GACGACGAGCGCGGCGATATCATCATCCATG-3'). Hybridizations using the GAPDH and ?-actin probes were carried out in modified Westneat solution (6.6% sodium dodecyl sulfate [SDS], 250 mM morpholinepropanesulfonic acid [MOPS; pH 7.0], 5x Denhardt's solution, 1 mM EDTA) at 55°C. To detect thymidine kinase (TK) transcripts, a 662-bp SstI/SmaI fragment from plasmid pTK173 (84) was used. The ICP8 probe was a 1,857-bp BamHI/EcoRI restriction fragment isolated from plasmid pE/3583 (16). Hybridizations with double-stranded DNA probes were carried out either in Church buffer (9) at 65°C or in ExpressHyb (Clontech) at 68°C. Quantification of bands on all Northern blots was carried out using a Storm 860 PhosphorImager (Molecular Dynamics) with ImageQuant software.
Western blot analysis. To detect VP16 protein, 1.1 x 106 Vero cells in 6-well plates were mock infected or infected with HSV-1 at a multiplicity of 10 and at various times postinfection were harvested for total protein by lysing the cells in 200 μl of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Proteins (8 μl of the cell lysate) were separated on an SDS-12% polyacrylamide gel and blotted for Western blot analysis. The anti-VP16 monoclonal antibody LP1 (obtained from A. Minson) was used at a 1:15,000 dilution in 5% skim milk powder in 25 mM Tris-HCl (pH 8)-150 mM NaCl-0.1% Tween 20. Proteins were visualized by using the ECL Plus Western blotting detection reagent (Amersham Biosciences) and quantified by using the blue fluorescence-chemifluorescence scanner of a Storm 860 PhosphorImager (Molecular Dynamics). Care was taken in this analysis to ensure that the Western blotting conditions yielded accurate quantification of the VP16 protein levels. Therefore, a dilution series of the protein lysate from KOS1.1-infected cells (2, 4, 6, 8, and 10 μl) was used to construct a standard curve, which showed a linear response.
To detect Ser-51-phosphorylated or total eIF2, Vero cells were mock infected or infected with HSV-1 at a multiplicity of 10 and cell lysates were prepared at 12 h postinfection. As a positive control for Ser51 phosphorylation of eIF2, cells were treated with 1 μM thapsigargin for 1 h prior to harvesting. Proteins were separated on an SDS-12% polyacrylamide gel and blotted for Western blot analysis. Polyclonal antibodies against eIF2 and phospho-eIF2 (Ser51) were obtained from Cell Signaling Technology and used at a 1:1,000 dilution.
Association of mRNAs with polyribosomes. Sucrose density gradient fractionation of polyribosomes in extracts prepared from mock-infected or HSV-1-infected Vero cells was performed 6 or 12 h postinfection essentially as described by Greco et al. (18). Briefly, postmitochondrial supernatants prepared from 3 x 107 infected or uninfected Vero cells were layered onto 10 to 42% sucrose gradients and spun in an SW40 rotor for 105 min at 38,000 rpm and 4°C. In some experiments, the gradients were 10 to 50% sucrose and were spun at 37,000 rpm. Fractions were collected by hand from the top of the gradient, and their absorbances at 254 nm were measured. RNA was extracted from each fraction as described elsewhere (18), and Northern blot analysis was performed as described above to determine the distribution of selected RNA species across the gradient.
RESULTS
ICP27 is not required for nuclear export of VP16 mRNA. ICP27 is known to enhance the efficiency of HSV mRNA nuclear export in X. laevis oocytes (30), although it has yet to be ascertained directly which individual transcripts are targets for this activity of ICP27. It has been reported that VP16 is one of the viral transcripts that requires ICP27 for its nuclear export (80), and thus it is conceivable that VP16 mRNA would be sequestered in the nucleus in cells infected with HSV variants lacking ICP27. However, Pearson et al. recently reported that there is no difference in the nuclear/cytoplasmic distribution of several HSV-1 mRNAs, including VP16, in the presence or absence of ICP27 (54). Our data, obtained in repeated experiments, are in complete accord with those of Pearson et al., as shown in Fig. 1. Vero cells were either mock infected or infected for 12 h with the wild-type KOS1.1 virus or the ICP27-null mutant d27-1. The cells were fractionated into a postmitochondrial supernatant and a nuclear pellet, and total RNA was isolated from each fraction. A Northern blot of these cytoplasmic and nuclear RNA preparations was probed for VP16 mRNA (Fig. 1). The majority of the VP16 mRNA was recovered in the cytoplasmic fraction in each infection (73% in the KOS1.1 infection and 77% in the d27-1 infection). In order to verify that the postmitochondrial supernatant was a clean preparation of cytoplasmic and not nuclear contents, the Northern blot was subsequently probed for the U3 snoRNA, which is exclusively nuclear in localization. As expected, the majority (83 to 97%) of the U3 RNA was recovered in the nuclear fraction (Fig. 1), showing that the postmitochondrial supernatant was not contaminated with nuclear contents to any significant degree. These data demonstrate that nuclear export of VP16 RNA is not affected by the presence or absence of ICP27, confirming the observations of Pearson et al. (54). This conclusion is also consistent with the earlier observation of Phelan et al. (57) that both intronless and intron-containing viral RNAs are present in the cytoplasm in cells infected with the ICP27-null mutant virus 27-lacZ.
In the experiment for which results are shown in Fig. 1, the total quantities of VP16 mRNA (nuclear plus cytoplasmic) were approximately equivalent in cells infected with either the wild-type virus or the ICP27 mutant (the d27-1 sample had 107% of the levels in the KOS1.1 sample). We have observed significant variation in this value between experiments: as noted below, in eight trials, the levels of VP16 mRNA observed at 12 h after infection with d27-1 ranged from 25 to 116% of wild-type levels, with a mean of 68%. We suggest a possible explanation for this variation in the next section.
The translational yield of VP16 mRNA is greatly decreased in cells infected with viruses lacking ICP27. In order to determine whether and how ICP27 modulates VP16 gene expression, we undertook a series of experiments that carefully compared the levels of VP16 protein and mRNA in cells infected with HSV strains containing or lacking ICP27. First, we monitored VP16 protein levels by Western blot analysis over a 12-h time course in cells infected with the wild-type virus or the ICP27-null mutant, d27-1 (Fig. 2A). Note that the Western blotting conditions we used were empirically determined to yield quantitative data, in which the VP16 protein levels were in a linear range compared to a standard curve. VP16 protein was detectable at 6 h postinfection in wild-type-infected cells and accumulated to high levels by 12 h. In d27-1-infected cells, however, VP16 protein accumulation was greatly decreased, yielding less than 3% of wild-type levels by 12 h postinfection. Clearly, therefore, ICP27 is required at some stage for VP16 gene expression. Next, VP16 mRNA levels were determined by Northern blot analysis over a duplicate 12-h time course on the same day (Fig. 2B). In wild-type-infected cells, VP16 RNA began to accumulate at 4 h postinfection and levels rose rapidly thereafter, reaching a plateau at 8 to 10 h. Accumulation of VP16 RNA in d27-1-infected cells was delayed, and RNA levels were reduced, relative to that for the wild-type infection at all time points analyzed. However, as the infection proceeded, VP16 mRNA continued to accumulate in d27-1-infected cells rather than reaching a plateau, and hence at 12 h postinfection the levels were approximately 50% of those for the wild-type infection, compared to only 10% at 6 h postinfection. Although significant, these reductions in VP16 mRNA levels were not sufficient to explain the striking decrease in VP16 protein accumulation (compare the graphs in Fig. 2A and B). That is, at each time analyzed, VP16 protein levels were decreased far more than the corresponding mRNA levels.
We have conducted eight independent trials in which VP16 mRNA and protein levels were compared at 12 h postinfection, and we found that VP16 mRNA levels in d27-1-infected cells were either modestly reduced from, or very similar to, wild-type levels (range, 25 to 116% of wild-type levels; mean, 68%). We consider it likely that much of this variation stems from differences in the rate of progression of the lytic cycle between experiments, predicted to yield variable mutant/wild-type mRNA ratios (see Fig. 2B). In contrast, VP16 protein levels were reduced to less than 8% of the quantities in KOS1.1-infected cells (range, 1.4 to 7.8% of wild-type levels; mean, 4.7%). Taken together, these data suggest that ICP27 strongly stimulates efficient accumulation of VP16 protein and argue that it does so primarily by acting downstream of the production of cytoplasmic mRNA.
We wanted to confirm that the defect in VP16 protein accumulation was due to the lack of ICP27 and not to another peculiarity of the d27-1 virus. Thus, we quantified VP16 RNA and protein levels at 12 h postinfection in cells infected with either the wild-type virus KOS1.1, the d27-1 mutant (using several independently prepared stocks), d27-lacZ1 (a precursor of the d27-1 virus), or, most importantly, a completely independent ICP27-null virus, 5dl1.2. Table 1 shows the relative VP16 RNA-to-protein ratios normalized to the ratios in KOS1.1-infected cells. In all cases, the RNA-to-protein ratio was significantly higher in cells infected with viruses lacking ICP27 (9- to 81-fold). These data demonstrate that compared to that with the wild-type virus, there is a large defect in the accumulation of VP16 protein in cells infected with viruses lacking ICP27 that cannot be accounted for by a drop in mRNA levels, and this effect is not unique to the d27-1 virus.
The decrease in VP16 protein levels in the absence of ICP27 could reflect a decrease in the translation rate of VP16 mRNA or, alternatively, an enhanced turnover rate of VP16 protein, or a combination of both effects. To determine if the stability of VP16 protein was decreased in d27-1-infected cells, Vero cells were infected with KOS1.1 or d27-1 for 12 h to allow VP16 protein to accumulate, and the cells were then treated with cycloheximide to arrest further protein synthesis. Cells were lysed at 2, 4, and 6 h post-cycloheximide treatment, and VP16 protein levels were analyzed by Western blotting (Fig. 3). The results showed no significant decrease in VP16 levels following cycloheximide treatment in either KOS1.1- or d27-1-infected cells, indicating that VP16 protein stability is unaltered in the absence of ICP27. Thus, the defect in VP16 protein accumulation in cells infected with viruses lacking ICP27 likely is due to decreased synthesis. We therefore conclude that the translational yield of VP16 mRNA, defined as the amount of VP16 protein translated per mRNA molecule (49, 50), is severely impaired in the absence of ICP27.
Polyribosomal distribution of VP16 mRNA. To further analyze the effects of ICP27 on the translation of VP16 mRNA, we examined the distribution of the VP16 transcript on polyribosomes in HSV-infected cells. In this experiment, cells were infected with KOS1.1 or d27-1, and postmitochondrial supernatants, prepared at 12 h postinfection, were fractionated by velocity sedimentation through sucrose density gradients. RNA was isolated from 20 equal fractions collected from the top of the gradient, and these fractions were subjected to Northern blot analysis to determine the distribution of VP16 mRNA across the gradient. The UV absorbance profiles across each gradient are shown in Fig. 4A. The two profiles are very similar, although the d27-1 profile shows a higher peak in fractions 6 to 8, corresponding to the ribosomal subunits, and slightly less absorbance in the fractions at the bottom of the gradient, where the large polyribosomes sediment (fractions 11 to 20). Figure 4B shows the RNA extracted from each fraction of the gradients, separated on an agarose gel and stained with ethidium bromide. Note that the first four fractions contain only 5 or 18S RNA and thus lack intact ribosomes. Therefore, any mRNAs that sediment in these fractions are not undergoing translation. Figure 4C shows the distribution of VP16 mRNA in these gradients. The KOS1.1 Northern blot showed considerable smears of radioactivity above and below the band corresponding to VP16 mRNA. This is most likely due to the read-through transcription of both strands of the viral genome that occurs at late times postinfection (87). This smear is largely absent in d27-1-infected cells, presumably due to the impairment of DNA replication. The distribution of KOS1.1 VP16 mRNA on the sucrose gradient showed substantial amounts associated with large polyribosomes (40% in fractions 17 to 20) and the remainder of the RNA spread more or less evenly throughout the upper portion of the gradient (Fig. 4D). To verify that the VP16 mRNA was indeed associated with active translation complexes, infected cells were treated with puromycin, which causes nascent peptide release and dissociation of polysomes. This treatment induced a shift in VP16 RNA from the bottom of the gradient (fractions 17 to 20) to fractions higher up in the gradient (fractions 9 to 15) (data not shown). Similarly, disruption of polysomes in the postmitochondrial supernatant by EDTA caused all of the VP16 mRNA to fractionate at the top of the gradient with ribosomal subunits (data not shown). Thus, VP16 RNA is associated with actively translating polyribosomes in cells infected with KOS1.1.
The ribosomal distribution of VP16 mRNA in cells infected with d27-1 differed dramatically (Fig. 4C and D). More than 50% of the mRNA was recovered in fractions 3 to 6, with the majority associated with the 40S ribosomal subunit in fraction 4 (see the ethidium bromide-stained gel in Fig. 4B). Only a minor fraction of the VP16 mRNA was associated with polyribosomes in d27-1-infected cells. These results, taken together with the RNA/protein ratios (Table 1), demonstrate that ICP27 is required for efficient translation of VP16 mRNA in HSV-infected cells, and they provide strong evidence that translation is blocked at the level of initiation in the absence of ICP27.
Infection with the ICP27 mutant virus does not alter the phosphorylation of the translation factor eIF2. Translational arrest mediated by PKR-induced Ser51 phosphorylation of the translation initiation factor eIF2 is widely observed in virus-infected cells (reviewed in reference 15). Phosphorylation of the eIF2 subunit is reversed in HSV-1 infection by the action of the 134.5 gene product, which binds to protein phosphatase 1 and promotes the dephosphorylation of eIF2 (21). In view of the effects of ICP27 on the translation of VP16 mRNA, we asked whether Ser51 phosphorylation of eIF2 was induced in cells infected with the d27-1 virus. The contents of total eIF2 and phospho-Ser51-eIF2 were determined by Western blotting (Fig. 5). The results showed that HSV infection with either KOS1.1 or d27-1 did not increase the extent of Ser51 phosphorylation of eIF2 above that present in mock-infected cells. As a positive control, uninfected cells were treated with thapsigargin, a compound that depletes calcium levels in the endoplasmic reticulum, thereby inducing Ser51 phosphorylation of eIF2 by the PERK/PEK kinase (60). As shown in Fig. 5, thapsigargin treatment readily induced Ser51 phosphorylation of eIF2. These data argue that the reduced translational efficiency of VP16 mRNA in the absence of ICP27 is not due to increased phosphorylation of eIF2.
Polyribosomal distribution of cellular RNAs in the absence of ICP27. The observed translational defect of VP16 RNA in cells infected with viruses lacking ICP27 is striking, because it is well known that ICP27 knockout viruses fail to shut off cellular protein synthesis. This seems to imply that there is no particular global arrest of protein synthesis in infected cells lacking ICP27. To verify this, we analyzed the polyribosomal distribution of two cellular RNAs, GAPDH and ?-actin, in uninfected cells or cells infected with d27-1. The wild-type virus KOS1.1 was not analyzed, because virtually no cellular mRNA remains at 12 h postinfection. The UV absorbance profiles of the sucrose gradients (Fig. 6A) and the ethidium bromide-stained gels of total RNA extracted from each fraction (Fig. 6B) show a considerable loss of polyribosomes in d27-1-infected cells compared to uninfected cells. This most likely reflects the activity of the virion host shutoff protein, vhs, which has previously been documented to induce the disruption of polyribosomes (12). Nonetheless, the distribution of the GAPDH and ?-actin mRNAs in the d27-1 sucrose gradients was very similar to that in uninfected cells (Fig. 6C and D). Approximately two-thirds of the total GAPDH (Fig. 6C) or ?-actin (Fig. 6D) mRNA in mock-infected cells sedimented with large polyribosomes in fractions 18 to 20, demonstrating that the mRNAs were undergoing active translation. The abundance of the cellular mRNAs in d27-1-infected cells was reduced to approximately 40% of uninfected levels, presumably due to vhs-induced loss of cytoplasmic mRNAs. However, the polyribosomal distribution of GAPDH and ?-actin transcripts was virtually unaltered, with approximately 70% of the transcripts in fractions 18 to 20. Collectively, these data demonstrate that these two cellular transcripts are translated efficiently in HSV-infected cells lacking ICP27. Thus, the requirement for ICP27 for efficient translation in HSV-infected cells is not a global phenomenon. These results have implications for the role of ICP27 in host shutoff (see Discussion).
Comparison of polyribosomal distributions of VP16, TK, and ICP8 mRNAs. ICP27 is known to influence the expression of many HSV genes, both positively and negatively, and it was of interest to assess which other viral mRNAs, if any, have reduced translational efficiencies in cells infected with viruses lacking ICP27. We therefore compared the polyribosomal distributions of VP16, TK, and ICP8 mRNAs in KOS1.1- and d27-1 infected cells at both 6 and 12 h postinfection. To obtain higher resolution at the bottom of the sucrose gradients, we used conditions under which the polysomes sedimented higher up in the gradient, with a peak in fractions 15 to 17. RNA was also recovered from the pellet for analysis. Figure 7A shows the distribution of VP16 RNA under these conditions. In KOS1.1-infected cells, a significant percentage of the VP16 RNA was recovered in the pellet (10 to 20%; fraction 21), particularly at 6 h postinfection. This suggests that a proportion of VP16 mRNA is associated with extremely large polysomes, indicating very active translation. It is interesting that the polysome peak in fractions 15 to 17 is significantly larger at 6 than at 12 h postinfection, with a concomitant shift to polysomes with a smaller average size, suggesting that translation efficiency is declining at later times of infection. In contrast, the amount of VP16 mRNA recovered in the pellet in the d27-1 samples was reduced two- to fourfold, and the great majority at both 6 and 12 h was associated with individual ribosome subunits and small polysomes in fractions 3 to 6. We conclude that the VP16 transcript requires ICP27 for efficient translation throughout infection.
The distribution profiles of the TK transcript are shown in Fig. 7B. At 6 h postinfection, the distribution profiles of the TK transcript in cells infected with KOS1.1 and cells infected with d27-1 were virtually identical: approximately 40% was associated with large polyribosomes (fractions 16 to 18), and 10 to 15% was associated with the pellet (fraction 21). This suggests that ICP27 is not required for efficient translation of the TK transcript at this early time postinfection. Lending support to this conclusion, we found that the TK RNA/protein ratios at 4 and 6 h postinfection were very similar in KOS1.1- and d27-1-infected cells (less than a twofold difference [data not shown]). The TK polysomal distribution profiles at 12 h postinfection were quite different, with most of the RNA in d27-1-infected cells sedimenting at the top of the gradient (55% in fractions 3 to 6) and a minor proportion in the polysomal fractions (15% in fractions 16 to 18). The TK RNA abundance in wild-type-infected cells was down-regulated to 20% of the levels in d27-1-infected cells by 12 h postinfection (data not shown). The remaining transcript was diffusely distributed through the upper half of the sucrose gradient, and a small fraction (7%) was associated with polysomes (Fig. 7B). We conclude that the TK transcript is actively translated early in infection in an ICP27-independent manner but that the translational efficiency decreases as infection proceeds.
Finally, the polysomal distribution profiles of ICP8 mRNA are shown in Fig. 7C. At 6 h postinfection, approximately 40% of the transcript sedimented in polysomal fractions in both KOS1.1- and d27-1-infected cells, suggesting that the translational efficiency of ICP8 is not affected by ICP27. In accord with this conclusion, it has been documented that expression of ICP8 does not require ICP27 (83). It was somewhat surprising that 40 to 50% of the mRNA sedimented in fractions 6 to 10 in both KOS1.1- and d27-1-infected cells. Since ICP8 is a large transcript of 4 kb, these fractions may represent small polyribosomes. The data seem to suggest that ICP8 mRNA is not assembled efficiently into large polyribosomes. It is not clear why this is the case, but the result may indicate that translation of ICP8 mRNA is subject to ICP27-independent differential regulation during infection. Interestingly, the proportion of total RNA in these fractions was even greater at 12 h (65%), which again suggests a progressive decline in translational efficiency as infection proceeds. Taken together, these data strongly suggest that neither the TK nor the ICP8 transcript exhibits the striking dependence on ICP27 for efficient translation that we observe for VP16 mRNA. However, the efficiencies of translation of all three mRNAs seem to decline at late times, and this effect is independent of ICP27. This finding is consistent with the progressive decrease in translational efficiency of HSV-1 mRNAs previously documented by Laurent et al. (31).
DISCUSSION
We report here a previously unrecognized function of ICP27: translational regulation of viral gene expression. As reviewed in the introduction, ICP27 modulates many of the posttranscriptional events that are required for gene expression, including polyadenylation, pre-mRNA splicing, and nuclear RNA export. More recently, ICP27 has been shown to increase the expression of two late genes by increasing their rates of transcription. Now, translation joins the list of the many cellular pathways influenced by ICP27.
We have analyzed the effects of ICP27 on nuclear/cytoplasmic RNA distribution, mRNA abundance, protein accumulation, and mRNA polyribosomal distribution of the essential HSV-1 transactivator VP16. We provide strong evidence that ICP27 is not required for cytoplasmic accumulation of the VP16 transcript (Fig. 1). This conclusion is in complete agreement with the results of Pearson et al. (54), who observed that the nuclear/cytoplasmic distributions of several HSV-1 mRNAs (gB, gC, VP16, UL42, UL26.5, and the short UL24 transcript) were not affected by the presence or absence of ICP27. In fact, the only transcripts in which cytoplasmic localization was found to be defective in the absence of ICP27 were the long UL24 transcripts. These observations led Pearson et al. to suggest that ICP27-independent modes of HSV-1 RNA export must exist. In this context, it is noteworthy that REF, the ICP27 binding partner thought to promote ICP27-dependent RNA export, is dispensable for RNA nuclear export in Drosphila melanogaster and Caenorhabditis elegans (17, 37).
The main effects of ICP27 on VP16 gene expression were found at the levels of mRNA abundance and translational efficiency. VP16 RNA levels were somewhat reduced in the absence of ICP27 at all times of infection (see Fig. 2). However, VP16 protein levels were reduced to a far greater extent. The most striking and novel finding we report here is that ICP27 greatly enhances the translational efficiency of the cytoplasmic mRNA, resulting in VP16 protein yields that are 9- to 80-fold greater per RNA molecule than those in the absence of ICP27. The increased accumulation of VP16 protein was not due to changes in its turnover rate but rather correlated with a dramatic mobilization of VP16 mRNA from 40S ribosomal subunits into actively translating polysomes. These effects appear to be mRNA specific, since two cellular RNAs and two early HSV RNAs did not show the same dependence on ICP27 for translational stimulation. Pearson et al. recently reported a large defect in the accumulation of UL24 protein in the absence of ICP27, compared to a minor reduction in short UL24 transcript levels, leading the authors to speculate that the translation of these transcripts is impaired in the absence of ICP27 (54). Thus, it is likely that the requirement of ICP27 for efficient translation is not unique to the VP16 message. It is possible that identification of additional mRNA targets of such regulation might be facilitated by examining infections conducted under more-restrictive conditions, such as low-multiplicity infection of primary cells. In this context, it will be important to learn if the requirement is manifest during infection of other cell types.
Our experiments have not addressed whether the effects of ICP27 on the translational enhancement of VP16 mRNA are direct or indirect. ICP27 stimulates the expression of some of the E genes that participate in DNA replication, and as a consequence the ability of ICP27 mutant viruses to replicate DNA is defective. The expression of many L genes is compromised in d27-1 infection because of the impairment of DNA replication, and furthermore, several late genes strictly require ICP27 directly for their expression. Thus, it is not clear if the requirement for ICP27 for efficient VP16 translation is direct or rather reflects reduced levels of one or more other viral proteins. Several considerations make it tempting to speculate that ICP27 is acting directly. These are outlined below.
Our findings that ICP27 increases the abundance and translational efficiency of VP16 mRNA, but not its nuclear export, are reminiscent of several recent studies that have analyzed how the process of splicing enhances gene expression (38, 49, 50, 88). It has been noted for many years that the presence of an intron in an mRNA can dramatically boost the levels of gene expression. It is now recognized that the process of splicing does not merely serve to remove unwanted sequences from RNA but also enhances other vital mRNA-processing events such as polyadenylation and nuclear export. Many of these effects may be attributable to the EJC, a protein "mark" that is deposited 20 to 24 nucleotides upstream of exon-exon junctions during the process of splicing (26, 32, 33, 65, 81, 90; reviewed in reference 61). At least seven proteins comprise the EJC, some of which (such as REF) are thought to promote nuclear export of the spliced RNA by recruiting the TAP/NXF1 nuclear export factor. Furthermore, several EJC proteins remain bound to the RNA in the cytoplasm, and at least one of these, Y14, remains associated with mRNA until it engages the ribosome and is not removed until the mRNA has undergone translation (8, 27, 28). It has been suggested that this is the means by which the splicing history of an RNA is communicated to the translation machinery in the process of nonsense-mediated decay, a degradation pathway for RNAs that bear premature termination codons (8, 29, 32, 40; reviewed in reference 39). The laboratories of Moore and Cullen have recently reported that splicing-dependent enhancement of gene expression occurs primarily at the levels of mRNA abundance and increased translational efficiency rather than nuclear RNA export, and these effects are due to the action of the EJC proteins RNPS1, Y14, and Magoh (38, 49, 50, 88). Factors involved in nonsense-mediated decay were also found to stimulate translation when tethered to an intronless reporter mRNA (49). The similarities between our data and these reports are intriguing. In particular, we note that splicing was found to enhance the translational utilization of ?-globin mRNA, a highly intron-dependent gene, more than 30-fold (38, 88). This is very similar to the magnitude of the ICP27-mediated translational enhancement of the VP16 transcript. These considerations may give some clues as to how ICP27 stimulates the translation of VP16 mRNA. Being intronless, the VP16 transcript could not acquire EJC proteins via splicing. Perhaps ICP27 binds to VP16 mRNA and recruits the translation-stimulatory EJC proteins in a splicing-independent manner, possibly through its documented interaction with REF. Indeed, the VP16 transcript is one of several HSV-1 mRNAs that have been shown to contain a binding site for ICP27 protein (78). However, the ICP8 mRNA also carries an ICP27 binding site (78), yet we saw no alterations in the polyribosomal distribution profiles of ICP8 mRNA in the presence or absence of ICP27. Thus, it is not clear to what extent, if any, the translational effects of ICP27 are mediated through direct binding to RNA.
Other links between the EJC, splicing, and translation have recently been uncovered. A protein termed eIF4AIII, which is highly similar to translation initiation factors eIFAI and eIFAII, has been shown to be an integral part of the EJC (2, 13, 53, 76). eIF4AIII is a nucleocytoplasmic shuttling protein, interacts with Y14 and Magoh, and has been shown to inhibit translation in vitro by binding eIF4G (35). It is interesting to speculate that ICP27 could modulate the function of this protein in HSV-infected cells and thereby influence the translational efficiencies of either viral or cellular transcripts. A further example that illustrates how nuclear events can regulate translation is the recent finding that shuttling SR proteins, which play important roles in constitutive and alternative splicing, associate with polyribosomes and stimulate translation when tethered to a reporter RNA (73). This observation is intriguing in view of the many known interactions between ICP27 and the splicing machinery: (i) ICP27 colocalizes with and redistributes splicing factors (55, 70, 71); (ii) ICP27 binds the SAP145 splicing factor (1); and (iii) extracts of infected cells containing ICP27 show defective splicing in vitro (1, 20, 36). Recently, Sciabica et al. showed that ICP27 interacts with and alters the phosphorylation of some SR proteins (74). Thus, it is conceivable that ICP27 might modulate translation via alterations in the activity of shuttling SR proteins.
Our data suggest that VP16 mRNA encounters a barrier to translational initiation in infected cells in the absence of ICP27. Since the translational inhibition is mRNA specific (?-actin, GAPDH, TK, and ICP8 transcripts are exempt), it seems possible that VP16 mRNA bears specific cis-acting sequences that hinder translation, and that ICP27 acts to overcome this negative effect. Such cis-acting sequences might be structural features that act as a translational impediment or might correspond to binding sites for trans-acting factors that repress translation. In the latter scenario, ICP27 could act to antagonize the function of the factors that bind the repression element. One example of this type of regulation is the control of translational silencing of the cellular 15-lipoxygenase (LOX) and human papillomavirus type 16 L2 mRNAs (6, 51, 52). These RNAs are silenced at the level of translational initiation in undifferentiated erythroid and epithelial cells, respectively, by binding of cellular hnRNP proteins K and E1/E2 to negative elements located in their 3' untranslated regions (6, 51). This repression system is inactivated during differentiation, in part through phosphorylation of hnRNP K by src-family kinases, leading to translational activation (52). Perhaps a similar type of silencing reversal is the mechanism by which ICP27 promotes the translation of VP16 mRNA. Indeed, ICP27 has been shown to bind hnRNP K (85). Furthermore, the block to the translational initiation of the LOX and L2 mRNAs occurs after recruitment of the 40S ribosomal subunit to the mRNAs but prior to joining of the 60S subunit (51). Our data are consistent with a similar block to VP16 translation in cells infected with d27-1, because most of the mRNA sediments with the 40S subunit.
Another mechanism by which ICP27 might act to regulate the translation of VP16 mRNA is correct localization of the RNA to a subcellular compartment that is highly active in translation. A precedent for this idea comes from the HIV Rev protein, which promotes the cytoplasmic accumulation of unspliced and partially spliced HIV RNAs that possess a Rev binding site. Rev interacts with a cellular protein, termed hRIP, and it has recently been shown that this interaction is necessary for releasing the HIV RNAs from the nuclear periphery into the cytoplasm (68). While this study did not address whether the movement of the HIV RNAs from the nuclear periphery to the cytoplasm correlated with an enhanced translational yield, it raises the possibility that VP16 mRNA could be similarly mislocalized in the absence of ICP27, resulting in sequestration away from the translational apparatus.
Our data have not addressed whether the translation of VP16 mRNA is impaired in uninfected cells or whether, instead, HSV infection induces a translational barrier that is overcome by the expression of ICP27. The best-documented virus-induced translational repression system is triggered by PKR-mediated phosphorylation of the translation initiation factor eIF2 (14, 15). This translational inhibition is counteracted in HSV infection by the action of the 134.5 gene product, which binds to protein phosphatase 1 and redirects it to dephosphorylate eIF2 (21). Because 134.5 is encoded by a late gene, it seemed possible that its expression would be reduced during infection with d27-1, leading to increased accumulation of phosphorylated eIF2. However, we did not detect an increase in phospho-Ser51 eIF2 levels in cells infected with d27-1 (Fig. 5). Thus, it is very unlikely that phosphorylation of eIF2 underlies the ICP27-dependent translational regulation that we have uncovered. Consistent with this conclusion, the phenotype of 134.5 mutants is characterized by global translational arrest (5), while the effects of deleting ICP27 are mRNA specific. In addition, Vero cells are fully permissive for 134.5 mutants (5, 59).
We find that the cellular mRNAs for GAPDH and ?-actin are associated with polyribosomes in cells infected with the ICP27-null mutant, a distribution similar to that in uninfected cells. This observation is interesting because it has been recognized for several years that translational control plays an important role in the down-regulation of cellular gene expression during HSV-1 infection. Simonin et al. (77) and Greco et al. (18) reported that while the levels of the cellular mRNAs encoding actin and ribosomal proteins decrease in parallel in HSV-infected cells, the translation rates of these RNAs are differentially controlled, such that the synthesis of ribosomal proteins persists while actin synthesis is strongly inhibited. These authors also reported that the actin mRNA is progressively shifted from polyribosomes to an association with the 40S ribosomal subunit, indicating that translation is inhibited at a step prior to binding of the 60S subunit. Our data suggest that this shift does not occur in cells infected with viruses lacking ICP27, implying that ICP27 or an ICP27-dependent gene product is responsible. These observations raise the possibility that ICP27 impairs the translation of cellular mRNAs, contributing to the ICP27-dependent shutoff of host protein synthesis.
Finally, it is clear that not all HSV mRNAs require ICP27 in order to be translated efficiently. The TK and ICP8 transcripts exhibited no obvious changes in their polyribosomal association in the presence and absence of ICP27. Preliminary data suggest that the same is true for the UL30 and gB mRNAs (K. S. Ellison, R. M. Maranchuk, and J. R. Smiley, unpublished data). Thus, it remains to be seen which other HSV transcripts, if any, are targets for this function of ICP27. Interestingly, our polysomal analysis of VP16, TK, and ICP8 RNAs suggests that the translation rates of all three decline at late times of infection, and this decline appears to be independent of ICP27. This finding is consistent with those of Laurent et al. (31), who showed that the rates of synthesis of several viral proteins from all three temporal classes decline progressively, even though the corresponding mRNAs are maintained at high levels. The transcripts encoding these proteins were shown to be partially displaced from polyribosomes to particles that cosediment with the 40S ribosomal subunit, exactly as we find for the VP16, TK, and ICP8 mRNAs. Thus, our results, in combination with those of Laurent et al., suggest that the shutoff of viral protein synthesis results from a mechanism that represses translation at the initiation step. Unlike the shutoff of cellular proteins, however, the down-regulation of viral protein synthesis apparently does not require ICP27.
In summary, we have uncovered a previously unknown role for ICP27 in the translational regulation of gene expression and have identified a natural viral target for this activity. Not surprisingly, many questions remain outstanding. First, are the effects of ICP27 on translation direct or, instead, mediated by another ICP27-induced viral protein? Second, what is the mechanism that restricts the translation of VP16 mRNA in the absence of ICP27, and is it induced by virus infection? Third, which other HSV mRNAs require ICP27 for efficient translation? Fourth, what domains of the ICP27 protein are required for this activity? The answers to these and other questions will generate many important insights into the complex interactions between HSV-1 and the host cell.
ACKNOWLEDGMENTS
We thank Holly Saffran for expert technical help, support, and comments on the manuscript and Sarah Richart for comments on the manuscript.
This research was supported by a grant from the Canadian Institutes of Health Research to J.R.S.
REFERENCES
Bryant, H. E., S. E. Wadd, A. I. Lamond, S. J. Silverstein, and J. B. Clements. 2001. Herpes simplex virus IE63 (ICP27) protein interacts with spliceosome-associated protein 145 and inhibits splicing prior to the first catalytic step. J. Virol. 75:4376-4385.
Chan, C. C., J. Dostie, M. D. Diem, W. Feng, M. Mann, J. Rappsilber, and G. Dreyfuss. 2004. eIF4A3 is a novel component of the exon junction complex. RNA 10:200-209.
Chen, I.-H. B., K. S. Sciabica, and R. M. Sandri-Goldin. 2002. ICP27 interacts with the RNA export factor Aly/REF to direct herpes simpex virus type 1 intronless mRNAs to the TAP export pathway. J. Virol. 76:12877-12889.
Cheung, P., K. S. Ellison, R. Verity, and J. R. Smiley. 2000. Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha-globin pre-mRNA in infected HeLa cells. J. Virol. 74:2913-2919.
Chou, J., J. J. Chen, M. Gross, and B. Roizman 1995. Association of a Mr 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 and premature shutoff of protein synthesis after infection with 134.5– mutants of herpes simplex virus 1. Proc. Natl. Acad. Sci. USA 92:10516-10520.
Collier, B., L. Goobar-Larsson, M. Sokolowski, and S. Schwartz. 1998. Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2. J. Biol. Chem. 273:22648-22656.
Cullen, B. R. 1998. Retroviruses as model systems for the study of nuclear RNA export pathways. Virology 249:203-210.
Dostie, J., and G. Dreyfuss. 2002. Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr. Biol. 12:1060-1067.
Elgadi, M. M., and J. R. Smiley. 1999. Picornavirus internal ribosome entry site elements target RNA cleavage events induced by the herpes simplex virus virion host shutoff protein. J. Virol. 73:9222-9231.
Ellison, K. S., S. A. Rice, R. Verity, and J. R. Smiley. 2000. Processing of -globin and ICP0 mRNA in cells infected with herpes simplex virus type 1 ICP27 mutants. J. Virol. 74:7307-7319.
Everett, R. D. 1986. The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2, and 3 can activate HSV-1 gene expression in trans. J. Gen. Virol. 67:2507-2513.
Fenwick, M. L., and R. D. Everett. 1990. Inactivation of the shutoff gene (UL41) of herpes simplex virus types 1 and 2. J. Gen. Virol. 71:2961-2967.
Ferraiuolo, M. A., C.-S. Lee, L. W. Ler, J. L. Hsu, M. Costa-Mattioli, M.-J. Luo, R. Reed, and N. Sonenberg. 2004. A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc. Natl. Acad. Sci. USA 101:4118-4123.
Gale, M., and M. G. Katze. 1998. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78:29-46.
Gale, M. J., S.-L. Tan, and M. G. Katze. 2000. Translational control of viral gene expression in eukaryotes. Microbiol. Mol. Biol. Rev. 64:239-280.
Gao, M., and D. M. Knipe. 1993. Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants. J. Virol. 67:876-885.
Gatfield, D., and E. Izaurralde. 2002. REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. J. Cell Biol. 159:579-588.
Greco, A., A.-M. Laurent, and J.-J. Madjar. 1997. Repression of beta-actin synthesis and persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1 infection are under translational control. Mol. Gen. Genet. 256:320-327.
Hardwicke, M. A., and R. M. Sandri-Goldin. 1994. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J. Virol. 68:4797-4810.
Hardy, W. R., and R. M. Sandri-Goldin. 1994. Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J. Virol. 68:7790-7799.
He, B., M. Gross, and B. Roizman. 1997. The 134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1 to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl. Acad. Sci. USA 94:843-848.
Hughes, R. G., and W. H. Munyon. 1975. Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation. J. Virol. 16:275-283.
Ingram, A., A. Phelan, J. Dunlop, and J. B. Clements. 1996. Immediate early protein ICP63 of herpes simplex virus type 1 binds RNA directly. J. Gen. Virol. 77:1847-1851.
Jean, S., K. M. LeVan, B. Song, M. Levine, and D. M. Knipe. 2001. Herpes simplex virus 1 ICP27 is required for transcription of two viral late (g2) genes in infected cells. Virology 283:273-284.
Jenkins, H. L., and C. A. Spencer. 2001. RNA polymerase II holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells. J. Virol. 75:9872-9884.
Kang, Y., and B. R. Cullen. 1999. The human TAP protein is a nuclear mRNA export factor that contains novel RNA binding and nucleocytoplasmic transport sequences. Genes Dev. 13:1126-1139.
Kataoka, N., J. Yong, V. N. Kim, F. Velazquez, R. A. Perkinson, F. Wang, and G. Dreyfuss. 2000. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell 6:673-682.
Kim, V. N., J. Jong, N. Kataoka, and G. Dreyfuss. 2001. The Y14 protein communicates to the cytoplasm the positions of exon-exon junctions. EMBO J. 20:2062-2068.
Kim, V. N., N. Kataoka, and G. Dreyfuss. 2001. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. Science 293:1832-1836.
Koffa, M. D., J. B. Clements, E. Izaurralde, S. Wadd, S. A. Wilson, I. W. Mattaj, and S. Kuersten. 2001. Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway. EMBO J. 20:5769-5778.
Laurent, A.-M., J.-J. Madjar, and A. Greco. 1998. Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the rate limiting step. J. Gen. Virol. 79:2765-2775.
Le Hir, H., D. Gatfield, E. Izaurralde, and M. J. Moore. 2001. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 20:4987-4997.
Le Hir, H., E. Izaurralde, L. E. Maquat, and M. J. Moore. 2000. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19:6860-6869.
Lengyel, J., C. Guy, V. Leong, S. Borge, and S. A. Rice. 2002. Mapping of functional regions in the amino-terminal portion of the herpes simplex virus ICP27 regulatory protein: importance of the leucine-rich nuclear export signal and RGG box RNA-binding domain. J. Virol. 76:11866-11879.
Li, Q., H. Imataka, S. Morino, G. W. J. Rogers, N. J. Richter-Cook, W. C. Merrick, and N. Sonenberg. 1999. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol. Cell. Biol. 19:7336-7346.
Lindberg, A., and J.-P. Kreivi. 2002. Splicing inhibition at the level of spliceosome assembly in the presence of herpes simplex virus protein ICP27. Virology 294:189-198.
Longman, D., I. L. Johnstone, and J. F. Caceres. 2003. The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans. RNA 9:881-891.
Lu, S., and B. R. Cullen. 2003. Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 9:618-630.
Lykke-Anderson, J. 2001. mRNA quality control: marking the message for life and death. Curr. Biol. 11:88-91.
Lykke-Anderson, J., M. D. Shu, and J. A. Steitz. 2001. Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293:1836-1839.
McCarthy, A. M., L. McMahan, and P. A. Schaffer. 1989. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J. Virol. 63:18-27.
McGregor, F., A. Phelan, J. Dunlop, and J. B. Clements. 1996. Regulation of herpes simplex virus poly(A) site usage and the action of immediate-early protein IE63 in the early-late switch. J. Virol. 70:1931-1940.
McLauchlan, J., P. C. Loney, R. M. Sandri-Goldin, and J. B. Clements. 1992. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. J. Virol. 66:6939-6945.
McLauchlan, J., S. Simpson, and J. B. Clements. 1989. Herpes simplex virus induces a processing factor that stimulates poly(A) site usage. Cell 59:1093-1105.
McMahan, L., and P. A. Schaffer. 1990. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to the C-terminal regions and are required to modulate viral gene expression very early in infection. J. Virol. 64:3471-3485.
Mears, W. E., V. Lam, and S. A. Rice. 1995. Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. J. Virol. 69:935-947.
Mears, W. E., and S. A. Rice. 1998. The herpes simplex virus immediate-early protein ICP27 shuttles between nucleus and cytoplasm. Virology 242:128-137.
Mears, W. E., and S. A. Rice. 1996. The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation. J. Virol. 70:7445-7453.
Nott, A., H. Le Hir, and M. J. Moore. 2004. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes Dev. 18:210-222.
Nott, A., S. H. Meislin, and M. J. Moore. 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA 9:607-617.
Ostareck, D. H., A. Ostareck-Lederer, I. N. Shatsky, and M. W. Hentze. 2001. Lipoxygenase mRNA silencing in erythroid differentiation: the 3' UTR regulatory complex controls 60S ribosomal subunit joining. Cell 104:281-290.
Ostareck-Lederer, A., D. H. Ostareck, C. Cans, G. Neubauer, K. Bomsztyk, G. Superti-Furga, and M. W. Hentze. 2002. c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs. Mol. Cell. Biol. 22:4535-4543.
Palacios, I. M., D. Gatfield, D. St Johnston, and E. Izaurralde. 2004. An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427:753-757.
Pearson, A., D. M. Knipe, and D. M. Coen. 2004. ICP27 selectively regulates the cytoplasmic localization of a subset of viral transcripts in herpes simplex virus type 1-infected cells. J. Virol. 78:23-32.
Phelan, A., M. Carmo-Fonseca, J. McLauchlan, A. I. Lamond, and J. B. Clements. 1993. A herpes simplex virus type-1 immediate early gene product IE63 regulates small ribonucleoprotein distribution. Proc. Natl. Acad. Sci. USA 90:9056-9060.
Phelan, A., and J. B. Clements. 1997. Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm. J. Gen. Virol. 78:3327-3331.
Phelan, A., J. Dunlop, and J. B. Clements. 1996. Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts. J. Virol. 70:5255-5265.
Pollard, V. W., and M. H. Malim. 1998. The HIV-1 Rev protein. Annu. Rev. Microbiol. 52:491-532.
Poon, A. P., and B. Roizman. 1997. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant 134.5 genes of herpes simplex virus 1. Virology 229:98-105.
Prostko, C. R., M. A. Brostrom, E. M. Malara, and C. O. Brostrom. 1992. Phosphorylation of eukaryotic initiation factor (eIF) 2 and inhibition of eIF-2B in GH3 pituitary cells by perturbants of early protein processing that induce GRP78. J. Biol. Chem. 267:16751-16754.
Reed, R., and B. Hurt. 2002. A conserved RNA export machinery coupled to pre-mRNA splicing. Cell 108:523-531.
Rice, S. A., and D. M. Knipe. 1988. Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27. J. Virol. 62:3814-3823.
Rice, S. A., and D. M. Knipe. 1990. Genetic evidence for two distinct transactivation functions of the herpes simplex virus protein ICP27. J. Virol. 64:1704-1715.
Rice, S. A., L. Su, and D. M. Knipe. 1989. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J. Virol. 63:3399-3407.
Rodrigues, J. P., M. Rode, D. Gatfield, B. J. Blencowe, M. Carmo-Fonseca, and E. Izaurralde. 2001. REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc. Natl. Acad. Sci. USA 98:1030-1035.
Roizman, B., and D. M. Knipe. 2001. Herpes simplex viruses and their replication, p. 2381-2398. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Strauss (ed.), Field's virology, 4th ed. Lippincott, Williams & Wilkins, Philadelphia, Pa.
Sacks, W. R., C. C. Greene, D. P. Ashman, and P. A. Schaffer. 1985. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J. Virol. 55:796-805.
Sanchez-Velar, N., E. B. Udofia, Z. Yu, and M. L. Zapp. 2004. hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region. Genes Dev. 18:23-34.
Sandri-Goldin, R. M. 1998. ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 12:868-879.
Sandri-Goldin, R. M., and M. K. Hibbard. 1996. The herpes simplex virus type 1 regulatory protein ICP27 coimmunoprecipitates with anti-Sm antiserum, and the C terminus appears to be required for this interaction. J. Virol. 70:108-118.
Sandri-Goldin, R. M., M. K. Hibbard, and M. A. Hardwicke. 1995. The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing. J. Virol. 69:6063-6076.
Sandri-Goldin, R. M., and G. E. Mendoza. 1992. A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Genes Dev. 6:848-863.
Sanford, J. R., N. K. Gray, K. Beckmann, and J. F. Caceres. 2004. A novel role for shuttling SR proteins in mRNA translation. Genes Dev. 18:755-768.
Sciabica, K. S., Q. J. Dai, and R. M. Sandri-Goldin. 2003. ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation. EMBO J. 22:1608-1619.
Sekulovich, R. E., K. Leary, and R. M. Sandri-Goldin. 1988. The herpes simplex virus type 1 protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. J. Virol. 62:4510-4522.
Shibuya, T., T. O. Tange, N. Sonenberg, and M. J. Moore. 2004. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11:346-351.
Simonin, D., J.-J. Diaz, T. Masse, and J.-J. Madjar. 1997. Persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1. J. Gen. Virol. 78:435-443.
Sokolowski, M., J. E. Scott, R. P. Heaney, A. H. Patel, and J. B. Clements. 2003. Identification of herpes simplex virus RNAs that interact specifically with regulatory protein ICP27 in vivo. J. Biol. Chem. 278:33540-33549.
Soliman, T. M., R. M. Sandri-Goldin, and S. J. Silverstein. 1997. Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins. J. Virol. 71:9188-9197.
Soliman, T. M., and S. J. Silverstein. 2000. Herpesvirus mRNAs are sorted for export via Crm1-dependent and -independent pathways. J. Virol. 74:2814-2825.
Stutz, F., A. Bachi, T. Doerks, I. C. Braun, B. Seraphin, M. Wilm, P. Bork, and E. Izaurralde. 2000. REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6:638-650.
Stutz, F., and M. Rosbash. 1998. Nuclear RNA export. Genes Dev. 12:3303-3319.
Uprichard, S. L., and D. M. Knipe. 1996. Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes. J. Virol. 70:1969-1980.
Varmuza, S. L., and J. R. Smiley. 1985. Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences. Cell 41:793-802.
Wadd, S., H. Bryant, O. Filhol, J. E. Scott, T.-Y. Hsieh, R. D. Everett, and J. B. Clements. 1999. The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2. J. Biol. Chem. 274:28991-28998.
Weinheimer, S. P., B. A. Boyd, S. K. Durham, J. L. Resnick, and D. R. O'Boyle II. 1992. Deletion of the VP16 open reading frame of herpes simplex virus type 1. J. Virol. 66:258-269.
Weinheimer, S. P., and S. L. McKnight. 1987. Transcriptional and post-transcriptional controls establish the cascade of herpes simplex virus protein synthesis. J. Mol. Biol. 195:819-833.
Wiegand, H. L., S. Lu, and B. R. Cullen. 2003. Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proc. Natl. Acad. Sci. USA 100:11327-11332.
Zhou, C., and D. M. Knipe. 2002. Association of herpes simplex virus type 1 ICP8 and ICP27 proteins with cellular RNA polymerase II holoenzyme. J. Virol. 76:5893-5904.
Zhou, Z., M.-J. Luo, K. Straesser, J. Katahira, E. Hurt, and R. Reed. 2000. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407:401-405.(Kimberly S. Ellison, Robe)
ABSTRACT
Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and ?-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.
INTRODUCTION
Lytic infection by herpes simplex virus type 1 (HSV-1) is characterized by shutoff of host protein synthesis and the temporally regulated expression of three classes of viral genes: immediate-early (IE), early (E), and late (L) (reviewed in reference 66). The infectious cycle is initiated by the virion transactivator VP16, which upon delivery into host cells acts in concert with cellular factors to induce the expression of the five IE genes. Four of these encode nuclear regulatory proteins (ICP0, ICP22, ICP4, and ICP27) that orchestrate the timely expression of the E and L genes. ICP27 plays an indispensable role in the viral life cycle (41, 67). ICP27 is necessary for efficient expression of some E genes, including a subset of those that encode proteins required for DNA replication (83). Hence, ICP27-null mutants display a partial defect in viral DNA replication. ICP27 also promotes the expression of most L genes (11, 41, 45, 62, 63, 67, 75) and is required for the so-called delayed shutoff of host protein synthesis (19, 20, 67, 79). How ICP27 performs its various functions remains poorly defined; however, it is becoming increasingly evident that ICP27 modulates virtually every aspect of mRNA metabolism, including primary transcription, polyadenylation, splicing, nuclear export, and mRNA stability (reviewed below).
It was initially assumed that the effects of ICP27 on HSV-1 gene expression were mediated primarily at the transcriptional level. Indeed, ICP27 is required for the transcription of at least two viral late genes, gC and UL47 (24). Furthermore, ICP27 interacts with the RNA polymerase II holoenzyme, providing further evidence for transcriptional modulation by ICP27 (25, 89). However, a wealth of data accumulated over the past decade has demonstrated that ICP27 mediates many of its effects posttranscriptionally. ICP27 alters the specificity of the polyadenylation machinery, an effect which may enhance the expression of viral L genes bearing inherently weak poly(A) signals (42-44, 72). ICP27 also impairs pre-mRNA splicing via multiple contacts with the splicing machinery (1, 19, 20, 36, 55, 70, 71). More recently, intensive studies have shown that ICP27 enhances the efficiency of nuclear export of intronless HSV-1 mRNAs (3, 4, 30, 69, 79).
ICP27 is an RNA binding protein that shuttles between the nucleus and the cytoplasm (23, 47, 48, 56, 69, 79). It contains an arginine-rich RGG box that is required for RNA binding (48, 69), as well as two nuclear localization sequences (46) and a leucine-rich sequence that bears a strong resemblance to the nuclear export sequence (NES) of the human immunodeficiency virus (HIV) protein Rev (69). Mutations that delete the NES restrict the protein to the nucleus and severely impair the viability of the virus (34). ICP27 was originally proposed to promote export of viral intronless RNAs through the cellular export adaptor CRM1 (80) in essentially the same manner as HIV Rev (reviewed in references 7, 58, and 82). However, Koffa et al. recently reported that CRM1 is not required for ICP27-induced mRNA export in Xenopus laevis oocytes (30), and Chen et al. provided evidence that the leucine-rich NES of ICP27 does not require CRM1 for activity (3). The current model for ICP27-mediated export of viral mRNAs is that ICP27 recruits the cellular mRNA export factor REF to viral intronless mRNAs (3, 30). REF is normally deposited onto cellular mRNAs as part of a multicomponent complex (the exon junction complex, or EJC) during the process of splicing and serves as a license for nuclear export of the spliced mRNA by interacting with the export factor TAP/NFX1 (26, 32, 33, 65, 81, 90; reviewed in reference 61). ICP27 has been shown to bind to REF and is thought to recruit REF to HSV RNAs, providing them with a splicing-independent means of accessing the TAP cellular mRNA export pathway (3, 30). Although the data supporting this model are compelling, it is noteworthy that a deletion (d3-4) that abolishes the ICP27-REF interaction (30) has little or no effect on virus growth or gene expression in Vero cells (34). In contrast, a deletion that removes the leucine-rich NES has much more serious consequences for the viability of the virus (34). Thus, the ICP27-REF interaction appears to be largely dispensable, at least in some cell types.
Given the multifunctional nature of the ICP27 protein, it has been relatively difficult to decipher at what level of gene expression ICP27 acts to regulate individual viral genes. In this study, we have examined how ICP27 modulates the expression of the essential viral transactivator, VP16. We find that ICP27 enhances VP16 expression primarily by increasing the translational efficiency of VP16 mRNA. Thus, we have uncovered a novel role for ICP27 in the translational control of viral gene expression.
MATERIALS AND METHODS
Cells and viruses. Vero cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. The HSV-1 wild-type strain used in this study was KOS1.1 (22). The ICP27-null strain d27-1 contains a 1.6-kb deletion that removes the ICP27 promoter and most of the open reading frame (63, 64). The 5dl1.2 virus is an independently derived ICP27 deletion mutant (41). The d27-lacZ1 virus bears an ICP27-lacZ fusion gene in place of the wild-type ICP27 gene and has a defect in ICP27 function (63). The ICP27 mutant viruses were propagated in V27 cells, a derivative of Vero cells engineered to express ICP27 upon infection with HSV (63). V27 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 100 μg of Geneticin (G418; Invitrogen)/ml.
Isolation of RNA and Northern blot analysis. Nuclear and cytoplasmic RNA fractions were isolated from infected Vero cells in 100-mm-diameter dishes by using the RNeasy purification kit (QIAGEN) as previously described (10). Total RNA was harvested from infected Vero cells by using the Trizol reagent (Invitrogen). Probes and hybridization conditions for Northern blot analysis were as follows. The VP16 probe was a fragment generated by PCR using plasmid pVP16-KOS as a template (86). To probe for the U3 snoRNA, a radiolabeled oligonucleotide specific for U3 was used as previously described (4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ?-actin transcripts were detected with 32P-labeled oligonucleotide probes (for GAPDH, 5'-TTGACTCCGACCTTCACCTTCCCCAT-3'; for ?-actin, 5'-GACGACGAGCGCGGCGATATCATCATCCATG-3'). Hybridizations using the GAPDH and ?-actin probes were carried out in modified Westneat solution (6.6% sodium dodecyl sulfate [SDS], 250 mM morpholinepropanesulfonic acid [MOPS; pH 7.0], 5x Denhardt's solution, 1 mM EDTA) at 55°C. To detect thymidine kinase (TK) transcripts, a 662-bp SstI/SmaI fragment from plasmid pTK173 (84) was used. The ICP8 probe was a 1,857-bp BamHI/EcoRI restriction fragment isolated from plasmid pE/3583 (16). Hybridizations with double-stranded DNA probes were carried out either in Church buffer (9) at 65°C or in ExpressHyb (Clontech) at 68°C. Quantification of bands on all Northern blots was carried out using a Storm 860 PhosphorImager (Molecular Dynamics) with ImageQuant software.
Western blot analysis. To detect VP16 protein, 1.1 x 106 Vero cells in 6-well plates were mock infected or infected with HSV-1 at a multiplicity of 10 and at various times postinfection were harvested for total protein by lysing the cells in 200 μl of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Proteins (8 μl of the cell lysate) were separated on an SDS-12% polyacrylamide gel and blotted for Western blot analysis. The anti-VP16 monoclonal antibody LP1 (obtained from A. Minson) was used at a 1:15,000 dilution in 5% skim milk powder in 25 mM Tris-HCl (pH 8)-150 mM NaCl-0.1% Tween 20. Proteins were visualized by using the ECL Plus Western blotting detection reagent (Amersham Biosciences) and quantified by using the blue fluorescence-chemifluorescence scanner of a Storm 860 PhosphorImager (Molecular Dynamics). Care was taken in this analysis to ensure that the Western blotting conditions yielded accurate quantification of the VP16 protein levels. Therefore, a dilution series of the protein lysate from KOS1.1-infected cells (2, 4, 6, 8, and 10 μl) was used to construct a standard curve, which showed a linear response.
To detect Ser-51-phosphorylated or total eIF2, Vero cells were mock infected or infected with HSV-1 at a multiplicity of 10 and cell lysates were prepared at 12 h postinfection. As a positive control for Ser51 phosphorylation of eIF2, cells were treated with 1 μM thapsigargin for 1 h prior to harvesting. Proteins were separated on an SDS-12% polyacrylamide gel and blotted for Western blot analysis. Polyclonal antibodies against eIF2 and phospho-eIF2 (Ser51) were obtained from Cell Signaling Technology and used at a 1:1,000 dilution.
Association of mRNAs with polyribosomes. Sucrose density gradient fractionation of polyribosomes in extracts prepared from mock-infected or HSV-1-infected Vero cells was performed 6 or 12 h postinfection essentially as described by Greco et al. (18). Briefly, postmitochondrial supernatants prepared from 3 x 107 infected or uninfected Vero cells were layered onto 10 to 42% sucrose gradients and spun in an SW40 rotor for 105 min at 38,000 rpm and 4°C. In some experiments, the gradients were 10 to 50% sucrose and were spun at 37,000 rpm. Fractions were collected by hand from the top of the gradient, and their absorbances at 254 nm were measured. RNA was extracted from each fraction as described elsewhere (18), and Northern blot analysis was performed as described above to determine the distribution of selected RNA species across the gradient.
RESULTS
ICP27 is not required for nuclear export of VP16 mRNA. ICP27 is known to enhance the efficiency of HSV mRNA nuclear export in X. laevis oocytes (30), although it has yet to be ascertained directly which individual transcripts are targets for this activity of ICP27. It has been reported that VP16 is one of the viral transcripts that requires ICP27 for its nuclear export (80), and thus it is conceivable that VP16 mRNA would be sequestered in the nucleus in cells infected with HSV variants lacking ICP27. However, Pearson et al. recently reported that there is no difference in the nuclear/cytoplasmic distribution of several HSV-1 mRNAs, including VP16, in the presence or absence of ICP27 (54). Our data, obtained in repeated experiments, are in complete accord with those of Pearson et al., as shown in Fig. 1. Vero cells were either mock infected or infected for 12 h with the wild-type KOS1.1 virus or the ICP27-null mutant d27-1. The cells were fractionated into a postmitochondrial supernatant and a nuclear pellet, and total RNA was isolated from each fraction. A Northern blot of these cytoplasmic and nuclear RNA preparations was probed for VP16 mRNA (Fig. 1). The majority of the VP16 mRNA was recovered in the cytoplasmic fraction in each infection (73% in the KOS1.1 infection and 77% in the d27-1 infection). In order to verify that the postmitochondrial supernatant was a clean preparation of cytoplasmic and not nuclear contents, the Northern blot was subsequently probed for the U3 snoRNA, which is exclusively nuclear in localization. As expected, the majority (83 to 97%) of the U3 RNA was recovered in the nuclear fraction (Fig. 1), showing that the postmitochondrial supernatant was not contaminated with nuclear contents to any significant degree. These data demonstrate that nuclear export of VP16 RNA is not affected by the presence or absence of ICP27, confirming the observations of Pearson et al. (54). This conclusion is also consistent with the earlier observation of Phelan et al. (57) that both intronless and intron-containing viral RNAs are present in the cytoplasm in cells infected with the ICP27-null mutant virus 27-lacZ.
In the experiment for which results are shown in Fig. 1, the total quantities of VP16 mRNA (nuclear plus cytoplasmic) were approximately equivalent in cells infected with either the wild-type virus or the ICP27 mutant (the d27-1 sample had 107% of the levels in the KOS1.1 sample). We have observed significant variation in this value between experiments: as noted below, in eight trials, the levels of VP16 mRNA observed at 12 h after infection with d27-1 ranged from 25 to 116% of wild-type levels, with a mean of 68%. We suggest a possible explanation for this variation in the next section.
The translational yield of VP16 mRNA is greatly decreased in cells infected with viruses lacking ICP27. In order to determine whether and how ICP27 modulates VP16 gene expression, we undertook a series of experiments that carefully compared the levels of VP16 protein and mRNA in cells infected with HSV strains containing or lacking ICP27. First, we monitored VP16 protein levels by Western blot analysis over a 12-h time course in cells infected with the wild-type virus or the ICP27-null mutant, d27-1 (Fig. 2A). Note that the Western blotting conditions we used were empirically determined to yield quantitative data, in which the VP16 protein levels were in a linear range compared to a standard curve. VP16 protein was detectable at 6 h postinfection in wild-type-infected cells and accumulated to high levels by 12 h. In d27-1-infected cells, however, VP16 protein accumulation was greatly decreased, yielding less than 3% of wild-type levels by 12 h postinfection. Clearly, therefore, ICP27 is required at some stage for VP16 gene expression. Next, VP16 mRNA levels were determined by Northern blot analysis over a duplicate 12-h time course on the same day (Fig. 2B). In wild-type-infected cells, VP16 RNA began to accumulate at 4 h postinfection and levels rose rapidly thereafter, reaching a plateau at 8 to 10 h. Accumulation of VP16 RNA in d27-1-infected cells was delayed, and RNA levels were reduced, relative to that for the wild-type infection at all time points analyzed. However, as the infection proceeded, VP16 mRNA continued to accumulate in d27-1-infected cells rather than reaching a plateau, and hence at 12 h postinfection the levels were approximately 50% of those for the wild-type infection, compared to only 10% at 6 h postinfection. Although significant, these reductions in VP16 mRNA levels were not sufficient to explain the striking decrease in VP16 protein accumulation (compare the graphs in Fig. 2A and B). That is, at each time analyzed, VP16 protein levels were decreased far more than the corresponding mRNA levels.
We have conducted eight independent trials in which VP16 mRNA and protein levels were compared at 12 h postinfection, and we found that VP16 mRNA levels in d27-1-infected cells were either modestly reduced from, or very similar to, wild-type levels (range, 25 to 116% of wild-type levels; mean, 68%). We consider it likely that much of this variation stems from differences in the rate of progression of the lytic cycle between experiments, predicted to yield variable mutant/wild-type mRNA ratios (see Fig. 2B). In contrast, VP16 protein levels were reduced to less than 8% of the quantities in KOS1.1-infected cells (range, 1.4 to 7.8% of wild-type levels; mean, 4.7%). Taken together, these data suggest that ICP27 strongly stimulates efficient accumulation of VP16 protein and argue that it does so primarily by acting downstream of the production of cytoplasmic mRNA.
We wanted to confirm that the defect in VP16 protein accumulation was due to the lack of ICP27 and not to another peculiarity of the d27-1 virus. Thus, we quantified VP16 RNA and protein levels at 12 h postinfection in cells infected with either the wild-type virus KOS1.1, the d27-1 mutant (using several independently prepared stocks), d27-lacZ1 (a precursor of the d27-1 virus), or, most importantly, a completely independent ICP27-null virus, 5dl1.2. Table 1 shows the relative VP16 RNA-to-protein ratios normalized to the ratios in KOS1.1-infected cells. In all cases, the RNA-to-protein ratio was significantly higher in cells infected with viruses lacking ICP27 (9- to 81-fold). These data demonstrate that compared to that with the wild-type virus, there is a large defect in the accumulation of VP16 protein in cells infected with viruses lacking ICP27 that cannot be accounted for by a drop in mRNA levels, and this effect is not unique to the d27-1 virus.
The decrease in VP16 protein levels in the absence of ICP27 could reflect a decrease in the translation rate of VP16 mRNA or, alternatively, an enhanced turnover rate of VP16 protein, or a combination of both effects. To determine if the stability of VP16 protein was decreased in d27-1-infected cells, Vero cells were infected with KOS1.1 or d27-1 for 12 h to allow VP16 protein to accumulate, and the cells were then treated with cycloheximide to arrest further protein synthesis. Cells were lysed at 2, 4, and 6 h post-cycloheximide treatment, and VP16 protein levels were analyzed by Western blotting (Fig. 3). The results showed no significant decrease in VP16 levels following cycloheximide treatment in either KOS1.1- or d27-1-infected cells, indicating that VP16 protein stability is unaltered in the absence of ICP27. Thus, the defect in VP16 protein accumulation in cells infected with viruses lacking ICP27 likely is due to decreased synthesis. We therefore conclude that the translational yield of VP16 mRNA, defined as the amount of VP16 protein translated per mRNA molecule (49, 50), is severely impaired in the absence of ICP27.
Polyribosomal distribution of VP16 mRNA. To further analyze the effects of ICP27 on the translation of VP16 mRNA, we examined the distribution of the VP16 transcript on polyribosomes in HSV-infected cells. In this experiment, cells were infected with KOS1.1 or d27-1, and postmitochondrial supernatants, prepared at 12 h postinfection, were fractionated by velocity sedimentation through sucrose density gradients. RNA was isolated from 20 equal fractions collected from the top of the gradient, and these fractions were subjected to Northern blot analysis to determine the distribution of VP16 mRNA across the gradient. The UV absorbance profiles across each gradient are shown in Fig. 4A. The two profiles are very similar, although the d27-1 profile shows a higher peak in fractions 6 to 8, corresponding to the ribosomal subunits, and slightly less absorbance in the fractions at the bottom of the gradient, where the large polyribosomes sediment (fractions 11 to 20). Figure 4B shows the RNA extracted from each fraction of the gradients, separated on an agarose gel and stained with ethidium bromide. Note that the first four fractions contain only 5 or 18S RNA and thus lack intact ribosomes. Therefore, any mRNAs that sediment in these fractions are not undergoing translation. Figure 4C shows the distribution of VP16 mRNA in these gradients. The KOS1.1 Northern blot showed considerable smears of radioactivity above and below the band corresponding to VP16 mRNA. This is most likely due to the read-through transcription of both strands of the viral genome that occurs at late times postinfection (87). This smear is largely absent in d27-1-infected cells, presumably due to the impairment of DNA replication. The distribution of KOS1.1 VP16 mRNA on the sucrose gradient showed substantial amounts associated with large polyribosomes (40% in fractions 17 to 20) and the remainder of the RNA spread more or less evenly throughout the upper portion of the gradient (Fig. 4D). To verify that the VP16 mRNA was indeed associated with active translation complexes, infected cells were treated with puromycin, which causes nascent peptide release and dissociation of polysomes. This treatment induced a shift in VP16 RNA from the bottom of the gradient (fractions 17 to 20) to fractions higher up in the gradient (fractions 9 to 15) (data not shown). Similarly, disruption of polysomes in the postmitochondrial supernatant by EDTA caused all of the VP16 mRNA to fractionate at the top of the gradient with ribosomal subunits (data not shown). Thus, VP16 RNA is associated with actively translating polyribosomes in cells infected with KOS1.1.
The ribosomal distribution of VP16 mRNA in cells infected with d27-1 differed dramatically (Fig. 4C and D). More than 50% of the mRNA was recovered in fractions 3 to 6, with the majority associated with the 40S ribosomal subunit in fraction 4 (see the ethidium bromide-stained gel in Fig. 4B). Only a minor fraction of the VP16 mRNA was associated with polyribosomes in d27-1-infected cells. These results, taken together with the RNA/protein ratios (Table 1), demonstrate that ICP27 is required for efficient translation of VP16 mRNA in HSV-infected cells, and they provide strong evidence that translation is blocked at the level of initiation in the absence of ICP27.
Infection with the ICP27 mutant virus does not alter the phosphorylation of the translation factor eIF2. Translational arrest mediated by PKR-induced Ser51 phosphorylation of the translation initiation factor eIF2 is widely observed in virus-infected cells (reviewed in reference 15). Phosphorylation of the eIF2 subunit is reversed in HSV-1 infection by the action of the 134.5 gene product, which binds to protein phosphatase 1 and promotes the dephosphorylation of eIF2 (21). In view of the effects of ICP27 on the translation of VP16 mRNA, we asked whether Ser51 phosphorylation of eIF2 was induced in cells infected with the d27-1 virus. The contents of total eIF2 and phospho-Ser51-eIF2 were determined by Western blotting (Fig. 5). The results showed that HSV infection with either KOS1.1 or d27-1 did not increase the extent of Ser51 phosphorylation of eIF2 above that present in mock-infected cells. As a positive control, uninfected cells were treated with thapsigargin, a compound that depletes calcium levels in the endoplasmic reticulum, thereby inducing Ser51 phosphorylation of eIF2 by the PERK/PEK kinase (60). As shown in Fig. 5, thapsigargin treatment readily induced Ser51 phosphorylation of eIF2. These data argue that the reduced translational efficiency of VP16 mRNA in the absence of ICP27 is not due to increased phosphorylation of eIF2.
Polyribosomal distribution of cellular RNAs in the absence of ICP27. The observed translational defect of VP16 RNA in cells infected with viruses lacking ICP27 is striking, because it is well known that ICP27 knockout viruses fail to shut off cellular protein synthesis. This seems to imply that there is no particular global arrest of protein synthesis in infected cells lacking ICP27. To verify this, we analyzed the polyribosomal distribution of two cellular RNAs, GAPDH and ?-actin, in uninfected cells or cells infected with d27-1. The wild-type virus KOS1.1 was not analyzed, because virtually no cellular mRNA remains at 12 h postinfection. The UV absorbance profiles of the sucrose gradients (Fig. 6A) and the ethidium bromide-stained gels of total RNA extracted from each fraction (Fig. 6B) show a considerable loss of polyribosomes in d27-1-infected cells compared to uninfected cells. This most likely reflects the activity of the virion host shutoff protein, vhs, which has previously been documented to induce the disruption of polyribosomes (12). Nonetheless, the distribution of the GAPDH and ?-actin mRNAs in the d27-1 sucrose gradients was very similar to that in uninfected cells (Fig. 6C and D). Approximately two-thirds of the total GAPDH (Fig. 6C) or ?-actin (Fig. 6D) mRNA in mock-infected cells sedimented with large polyribosomes in fractions 18 to 20, demonstrating that the mRNAs were undergoing active translation. The abundance of the cellular mRNAs in d27-1-infected cells was reduced to approximately 40% of uninfected levels, presumably due to vhs-induced loss of cytoplasmic mRNAs. However, the polyribosomal distribution of GAPDH and ?-actin transcripts was virtually unaltered, with approximately 70% of the transcripts in fractions 18 to 20. Collectively, these data demonstrate that these two cellular transcripts are translated efficiently in HSV-infected cells lacking ICP27. Thus, the requirement for ICP27 for efficient translation in HSV-infected cells is not a global phenomenon. These results have implications for the role of ICP27 in host shutoff (see Discussion).
Comparison of polyribosomal distributions of VP16, TK, and ICP8 mRNAs. ICP27 is known to influence the expression of many HSV genes, both positively and negatively, and it was of interest to assess which other viral mRNAs, if any, have reduced translational efficiencies in cells infected with viruses lacking ICP27. We therefore compared the polyribosomal distributions of VP16, TK, and ICP8 mRNAs in KOS1.1- and d27-1 infected cells at both 6 and 12 h postinfection. To obtain higher resolution at the bottom of the sucrose gradients, we used conditions under which the polysomes sedimented higher up in the gradient, with a peak in fractions 15 to 17. RNA was also recovered from the pellet for analysis. Figure 7A shows the distribution of VP16 RNA under these conditions. In KOS1.1-infected cells, a significant percentage of the VP16 RNA was recovered in the pellet (10 to 20%; fraction 21), particularly at 6 h postinfection. This suggests that a proportion of VP16 mRNA is associated with extremely large polysomes, indicating very active translation. It is interesting that the polysome peak in fractions 15 to 17 is significantly larger at 6 than at 12 h postinfection, with a concomitant shift to polysomes with a smaller average size, suggesting that translation efficiency is declining at later times of infection. In contrast, the amount of VP16 mRNA recovered in the pellet in the d27-1 samples was reduced two- to fourfold, and the great majority at both 6 and 12 h was associated with individual ribosome subunits and small polysomes in fractions 3 to 6. We conclude that the VP16 transcript requires ICP27 for efficient translation throughout infection.
The distribution profiles of the TK transcript are shown in Fig. 7B. At 6 h postinfection, the distribution profiles of the TK transcript in cells infected with KOS1.1 and cells infected with d27-1 were virtually identical: approximately 40% was associated with large polyribosomes (fractions 16 to 18), and 10 to 15% was associated with the pellet (fraction 21). This suggests that ICP27 is not required for efficient translation of the TK transcript at this early time postinfection. Lending support to this conclusion, we found that the TK RNA/protein ratios at 4 and 6 h postinfection were very similar in KOS1.1- and d27-1-infected cells (less than a twofold difference [data not shown]). The TK polysomal distribution profiles at 12 h postinfection were quite different, with most of the RNA in d27-1-infected cells sedimenting at the top of the gradient (55% in fractions 3 to 6) and a minor proportion in the polysomal fractions (15% in fractions 16 to 18). The TK RNA abundance in wild-type-infected cells was down-regulated to 20% of the levels in d27-1-infected cells by 12 h postinfection (data not shown). The remaining transcript was diffusely distributed through the upper half of the sucrose gradient, and a small fraction (7%) was associated with polysomes (Fig. 7B). We conclude that the TK transcript is actively translated early in infection in an ICP27-independent manner but that the translational efficiency decreases as infection proceeds.
Finally, the polysomal distribution profiles of ICP8 mRNA are shown in Fig. 7C. At 6 h postinfection, approximately 40% of the transcript sedimented in polysomal fractions in both KOS1.1- and d27-1-infected cells, suggesting that the translational efficiency of ICP8 is not affected by ICP27. In accord with this conclusion, it has been documented that expression of ICP8 does not require ICP27 (83). It was somewhat surprising that 40 to 50% of the mRNA sedimented in fractions 6 to 10 in both KOS1.1- and d27-1-infected cells. Since ICP8 is a large transcript of 4 kb, these fractions may represent small polyribosomes. The data seem to suggest that ICP8 mRNA is not assembled efficiently into large polyribosomes. It is not clear why this is the case, but the result may indicate that translation of ICP8 mRNA is subject to ICP27-independent differential regulation during infection. Interestingly, the proportion of total RNA in these fractions was even greater at 12 h (65%), which again suggests a progressive decline in translational efficiency as infection proceeds. Taken together, these data strongly suggest that neither the TK nor the ICP8 transcript exhibits the striking dependence on ICP27 for efficient translation that we observe for VP16 mRNA. However, the efficiencies of translation of all three mRNAs seem to decline at late times, and this effect is independent of ICP27. This finding is consistent with the progressive decrease in translational efficiency of HSV-1 mRNAs previously documented by Laurent et al. (31).
DISCUSSION
We report here a previously unrecognized function of ICP27: translational regulation of viral gene expression. As reviewed in the introduction, ICP27 modulates many of the posttranscriptional events that are required for gene expression, including polyadenylation, pre-mRNA splicing, and nuclear RNA export. More recently, ICP27 has been shown to increase the expression of two late genes by increasing their rates of transcription. Now, translation joins the list of the many cellular pathways influenced by ICP27.
We have analyzed the effects of ICP27 on nuclear/cytoplasmic RNA distribution, mRNA abundance, protein accumulation, and mRNA polyribosomal distribution of the essential HSV-1 transactivator VP16. We provide strong evidence that ICP27 is not required for cytoplasmic accumulation of the VP16 transcript (Fig. 1). This conclusion is in complete agreement with the results of Pearson et al. (54), who observed that the nuclear/cytoplasmic distributions of several HSV-1 mRNAs (gB, gC, VP16, UL42, UL26.5, and the short UL24 transcript) were not affected by the presence or absence of ICP27. In fact, the only transcripts in which cytoplasmic localization was found to be defective in the absence of ICP27 were the long UL24 transcripts. These observations led Pearson et al. to suggest that ICP27-independent modes of HSV-1 RNA export must exist. In this context, it is noteworthy that REF, the ICP27 binding partner thought to promote ICP27-dependent RNA export, is dispensable for RNA nuclear export in Drosphila melanogaster and Caenorhabditis elegans (17, 37).
The main effects of ICP27 on VP16 gene expression were found at the levels of mRNA abundance and translational efficiency. VP16 RNA levels were somewhat reduced in the absence of ICP27 at all times of infection (see Fig. 2). However, VP16 protein levels were reduced to a far greater extent. The most striking and novel finding we report here is that ICP27 greatly enhances the translational efficiency of the cytoplasmic mRNA, resulting in VP16 protein yields that are 9- to 80-fold greater per RNA molecule than those in the absence of ICP27. The increased accumulation of VP16 protein was not due to changes in its turnover rate but rather correlated with a dramatic mobilization of VP16 mRNA from 40S ribosomal subunits into actively translating polysomes. These effects appear to be mRNA specific, since two cellular RNAs and two early HSV RNAs did not show the same dependence on ICP27 for translational stimulation. Pearson et al. recently reported a large defect in the accumulation of UL24 protein in the absence of ICP27, compared to a minor reduction in short UL24 transcript levels, leading the authors to speculate that the translation of these transcripts is impaired in the absence of ICP27 (54). Thus, it is likely that the requirement of ICP27 for efficient translation is not unique to the VP16 message. It is possible that identification of additional mRNA targets of such regulation might be facilitated by examining infections conducted under more-restrictive conditions, such as low-multiplicity infection of primary cells. In this context, it will be important to learn if the requirement is manifest during infection of other cell types.
Our experiments have not addressed whether the effects of ICP27 on the translational enhancement of VP16 mRNA are direct or indirect. ICP27 stimulates the expression of some of the E genes that participate in DNA replication, and as a consequence the ability of ICP27 mutant viruses to replicate DNA is defective. The expression of many L genes is compromised in d27-1 infection because of the impairment of DNA replication, and furthermore, several late genes strictly require ICP27 directly for their expression. Thus, it is not clear if the requirement for ICP27 for efficient VP16 translation is direct or rather reflects reduced levels of one or more other viral proteins. Several considerations make it tempting to speculate that ICP27 is acting directly. These are outlined below.
Our findings that ICP27 increases the abundance and translational efficiency of VP16 mRNA, but not its nuclear export, are reminiscent of several recent studies that have analyzed how the process of splicing enhances gene expression (38, 49, 50, 88). It has been noted for many years that the presence of an intron in an mRNA can dramatically boost the levels of gene expression. It is now recognized that the process of splicing does not merely serve to remove unwanted sequences from RNA but also enhances other vital mRNA-processing events such as polyadenylation and nuclear export. Many of these effects may be attributable to the EJC, a protein "mark" that is deposited 20 to 24 nucleotides upstream of exon-exon junctions during the process of splicing (26, 32, 33, 65, 81, 90; reviewed in reference 61). At least seven proteins comprise the EJC, some of which (such as REF) are thought to promote nuclear export of the spliced RNA by recruiting the TAP/NXF1 nuclear export factor. Furthermore, several EJC proteins remain bound to the RNA in the cytoplasm, and at least one of these, Y14, remains associated with mRNA until it engages the ribosome and is not removed until the mRNA has undergone translation (8, 27, 28). It has been suggested that this is the means by which the splicing history of an RNA is communicated to the translation machinery in the process of nonsense-mediated decay, a degradation pathway for RNAs that bear premature termination codons (8, 29, 32, 40; reviewed in reference 39). The laboratories of Moore and Cullen have recently reported that splicing-dependent enhancement of gene expression occurs primarily at the levels of mRNA abundance and increased translational efficiency rather than nuclear RNA export, and these effects are due to the action of the EJC proteins RNPS1, Y14, and Magoh (38, 49, 50, 88). Factors involved in nonsense-mediated decay were also found to stimulate translation when tethered to an intronless reporter mRNA (49). The similarities between our data and these reports are intriguing. In particular, we note that splicing was found to enhance the translational utilization of ?-globin mRNA, a highly intron-dependent gene, more than 30-fold (38, 88). This is very similar to the magnitude of the ICP27-mediated translational enhancement of the VP16 transcript. These considerations may give some clues as to how ICP27 stimulates the translation of VP16 mRNA. Being intronless, the VP16 transcript could not acquire EJC proteins via splicing. Perhaps ICP27 binds to VP16 mRNA and recruits the translation-stimulatory EJC proteins in a splicing-independent manner, possibly through its documented interaction with REF. Indeed, the VP16 transcript is one of several HSV-1 mRNAs that have been shown to contain a binding site for ICP27 protein (78). However, the ICP8 mRNA also carries an ICP27 binding site (78), yet we saw no alterations in the polyribosomal distribution profiles of ICP8 mRNA in the presence or absence of ICP27. Thus, it is not clear to what extent, if any, the translational effects of ICP27 are mediated through direct binding to RNA.
Other links between the EJC, splicing, and translation have recently been uncovered. A protein termed eIF4AIII, which is highly similar to translation initiation factors eIFAI and eIFAII, has been shown to be an integral part of the EJC (2, 13, 53, 76). eIF4AIII is a nucleocytoplasmic shuttling protein, interacts with Y14 and Magoh, and has been shown to inhibit translation in vitro by binding eIF4G (35). It is interesting to speculate that ICP27 could modulate the function of this protein in HSV-infected cells and thereby influence the translational efficiencies of either viral or cellular transcripts. A further example that illustrates how nuclear events can regulate translation is the recent finding that shuttling SR proteins, which play important roles in constitutive and alternative splicing, associate with polyribosomes and stimulate translation when tethered to a reporter RNA (73). This observation is intriguing in view of the many known interactions between ICP27 and the splicing machinery: (i) ICP27 colocalizes with and redistributes splicing factors (55, 70, 71); (ii) ICP27 binds the SAP145 splicing factor (1); and (iii) extracts of infected cells containing ICP27 show defective splicing in vitro (1, 20, 36). Recently, Sciabica et al. showed that ICP27 interacts with and alters the phosphorylation of some SR proteins (74). Thus, it is conceivable that ICP27 might modulate translation via alterations in the activity of shuttling SR proteins.
Our data suggest that VP16 mRNA encounters a barrier to translational initiation in infected cells in the absence of ICP27. Since the translational inhibition is mRNA specific (?-actin, GAPDH, TK, and ICP8 transcripts are exempt), it seems possible that VP16 mRNA bears specific cis-acting sequences that hinder translation, and that ICP27 acts to overcome this negative effect. Such cis-acting sequences might be structural features that act as a translational impediment or might correspond to binding sites for trans-acting factors that repress translation. In the latter scenario, ICP27 could act to antagonize the function of the factors that bind the repression element. One example of this type of regulation is the control of translational silencing of the cellular 15-lipoxygenase (LOX) and human papillomavirus type 16 L2 mRNAs (6, 51, 52). These RNAs are silenced at the level of translational initiation in undifferentiated erythroid and epithelial cells, respectively, by binding of cellular hnRNP proteins K and E1/E2 to negative elements located in their 3' untranslated regions (6, 51). This repression system is inactivated during differentiation, in part through phosphorylation of hnRNP K by src-family kinases, leading to translational activation (52). Perhaps a similar type of silencing reversal is the mechanism by which ICP27 promotes the translation of VP16 mRNA. Indeed, ICP27 has been shown to bind hnRNP K (85). Furthermore, the block to the translational initiation of the LOX and L2 mRNAs occurs after recruitment of the 40S ribosomal subunit to the mRNAs but prior to joining of the 60S subunit (51). Our data are consistent with a similar block to VP16 translation in cells infected with d27-1, because most of the mRNA sediments with the 40S subunit.
Another mechanism by which ICP27 might act to regulate the translation of VP16 mRNA is correct localization of the RNA to a subcellular compartment that is highly active in translation. A precedent for this idea comes from the HIV Rev protein, which promotes the cytoplasmic accumulation of unspliced and partially spliced HIV RNAs that possess a Rev binding site. Rev interacts with a cellular protein, termed hRIP, and it has recently been shown that this interaction is necessary for releasing the HIV RNAs from the nuclear periphery into the cytoplasm (68). While this study did not address whether the movement of the HIV RNAs from the nuclear periphery to the cytoplasm correlated with an enhanced translational yield, it raises the possibility that VP16 mRNA could be similarly mislocalized in the absence of ICP27, resulting in sequestration away from the translational apparatus.
Our data have not addressed whether the translation of VP16 mRNA is impaired in uninfected cells or whether, instead, HSV infection induces a translational barrier that is overcome by the expression of ICP27. The best-documented virus-induced translational repression system is triggered by PKR-mediated phosphorylation of the translation initiation factor eIF2 (14, 15). This translational inhibition is counteracted in HSV infection by the action of the 134.5 gene product, which binds to protein phosphatase 1 and redirects it to dephosphorylate eIF2 (21). Because 134.5 is encoded by a late gene, it seemed possible that its expression would be reduced during infection with d27-1, leading to increased accumulation of phosphorylated eIF2. However, we did not detect an increase in phospho-Ser51 eIF2 levels in cells infected with d27-1 (Fig. 5). Thus, it is very unlikely that phosphorylation of eIF2 underlies the ICP27-dependent translational regulation that we have uncovered. Consistent with this conclusion, the phenotype of 134.5 mutants is characterized by global translational arrest (5), while the effects of deleting ICP27 are mRNA specific. In addition, Vero cells are fully permissive for 134.5 mutants (5, 59).
We find that the cellular mRNAs for GAPDH and ?-actin are associated with polyribosomes in cells infected with the ICP27-null mutant, a distribution similar to that in uninfected cells. This observation is interesting because it has been recognized for several years that translational control plays an important role in the down-regulation of cellular gene expression during HSV-1 infection. Simonin et al. (77) and Greco et al. (18) reported that while the levels of the cellular mRNAs encoding actin and ribosomal proteins decrease in parallel in HSV-infected cells, the translation rates of these RNAs are differentially controlled, such that the synthesis of ribosomal proteins persists while actin synthesis is strongly inhibited. These authors also reported that the actin mRNA is progressively shifted from polyribosomes to an association with the 40S ribosomal subunit, indicating that translation is inhibited at a step prior to binding of the 60S subunit. Our data suggest that this shift does not occur in cells infected with viruses lacking ICP27, implying that ICP27 or an ICP27-dependent gene product is responsible. These observations raise the possibility that ICP27 impairs the translation of cellular mRNAs, contributing to the ICP27-dependent shutoff of host protein synthesis.
Finally, it is clear that not all HSV mRNAs require ICP27 in order to be translated efficiently. The TK and ICP8 transcripts exhibited no obvious changes in their polyribosomal association in the presence and absence of ICP27. Preliminary data suggest that the same is true for the UL30 and gB mRNAs (K. S. Ellison, R. M. Maranchuk, and J. R. Smiley, unpublished data). Thus, it remains to be seen which other HSV transcripts, if any, are targets for this function of ICP27. Interestingly, our polysomal analysis of VP16, TK, and ICP8 RNAs suggests that the translation rates of all three decline at late times of infection, and this decline appears to be independent of ICP27. This finding is consistent with those of Laurent et al. (31), who showed that the rates of synthesis of several viral proteins from all three temporal classes decline progressively, even though the corresponding mRNAs are maintained at high levels. The transcripts encoding these proteins were shown to be partially displaced from polyribosomes to particles that cosediment with the 40S ribosomal subunit, exactly as we find for the VP16, TK, and ICP8 mRNAs. Thus, our results, in combination with those of Laurent et al., suggest that the shutoff of viral protein synthesis results from a mechanism that represses translation at the initiation step. Unlike the shutoff of cellular proteins, however, the down-regulation of viral protein synthesis apparently does not require ICP27.
In summary, we have uncovered a previously unknown role for ICP27 in the translational regulation of gene expression and have identified a natural viral target for this activity. Not surprisingly, many questions remain outstanding. First, are the effects of ICP27 on translation direct or, instead, mediated by another ICP27-induced viral protein? Second, what is the mechanism that restricts the translation of VP16 mRNA in the absence of ICP27, and is it induced by virus infection? Third, which other HSV mRNAs require ICP27 for efficient translation? Fourth, what domains of the ICP27 protein are required for this activity? The answers to these and other questions will generate many important insights into the complex interactions between HSV-1 and the host cell.
ACKNOWLEDGMENTS
We thank Holly Saffran for expert technical help, support, and comments on the manuscript and Sarah Richart for comments on the manuscript.
This research was supported by a grant from the Canadian Institutes of Health Research to J.R.S.
REFERENCES
Bryant, H. E., S. E. Wadd, A. I. Lamond, S. J. Silverstein, and J. B. Clements. 2001. Herpes simplex virus IE63 (ICP27) protein interacts with spliceosome-associated protein 145 and inhibits splicing prior to the first catalytic step. J. Virol. 75:4376-4385.
Chan, C. C., J. Dostie, M. D. Diem, W. Feng, M. Mann, J. Rappsilber, and G. Dreyfuss. 2004. eIF4A3 is a novel component of the exon junction complex. RNA 10:200-209.
Chen, I.-H. B., K. S. Sciabica, and R. M. Sandri-Goldin. 2002. ICP27 interacts with the RNA export factor Aly/REF to direct herpes simpex virus type 1 intronless mRNAs to the TAP export pathway. J. Virol. 76:12877-12889.
Cheung, P., K. S. Ellison, R. Verity, and J. R. Smiley. 2000. Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha-globin pre-mRNA in infected HeLa cells. J. Virol. 74:2913-2919.
Chou, J., J. J. Chen, M. Gross, and B. Roizman 1995. Association of a Mr 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 and premature shutoff of protein synthesis after infection with 134.5– mutants of herpes simplex virus 1. Proc. Natl. Acad. Sci. USA 92:10516-10520.
Collier, B., L. Goobar-Larsson, M. Sokolowski, and S. Schwartz. 1998. Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2. J. Biol. Chem. 273:22648-22656.
Cullen, B. R. 1998. Retroviruses as model systems for the study of nuclear RNA export pathways. Virology 249:203-210.
Dostie, J., and G. Dreyfuss. 2002. Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr. Biol. 12:1060-1067.
Elgadi, M. M., and J. R. Smiley. 1999. Picornavirus internal ribosome entry site elements target RNA cleavage events induced by the herpes simplex virus virion host shutoff protein. J. Virol. 73:9222-9231.
Ellison, K. S., S. A. Rice, R. Verity, and J. R. Smiley. 2000. Processing of -globin and ICP0 mRNA in cells infected with herpes simplex virus type 1 ICP27 mutants. J. Virol. 74:7307-7319.
Everett, R. D. 1986. The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2, and 3 can activate HSV-1 gene expression in trans. J. Gen. Virol. 67:2507-2513.
Fenwick, M. L., and R. D. Everett. 1990. Inactivation of the shutoff gene (UL41) of herpes simplex virus types 1 and 2. J. Gen. Virol. 71:2961-2967.
Ferraiuolo, M. A., C.-S. Lee, L. W. Ler, J. L. Hsu, M. Costa-Mattioli, M.-J. Luo, R. Reed, and N. Sonenberg. 2004. A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc. Natl. Acad. Sci. USA 101:4118-4123.
Gale, M., and M. G. Katze. 1998. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78:29-46.
Gale, M. J., S.-L. Tan, and M. G. Katze. 2000. Translational control of viral gene expression in eukaryotes. Microbiol. Mol. Biol. Rev. 64:239-280.
Gao, M., and D. M. Knipe. 1993. Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants. J. Virol. 67:876-885.
Gatfield, D., and E. Izaurralde. 2002. REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. J. Cell Biol. 159:579-588.
Greco, A., A.-M. Laurent, and J.-J. Madjar. 1997. Repression of beta-actin synthesis and persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1 infection are under translational control. Mol. Gen. Genet. 256:320-327.
Hardwicke, M. A., and R. M. Sandri-Goldin. 1994. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J. Virol. 68:4797-4810.
Hardy, W. R., and R. M. Sandri-Goldin. 1994. Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J. Virol. 68:7790-7799.
He, B., M. Gross, and B. Roizman. 1997. The 134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1 to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl. Acad. Sci. USA 94:843-848.
Hughes, R. G., and W. H. Munyon. 1975. Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation. J. Virol. 16:275-283.
Ingram, A., A. Phelan, J. Dunlop, and J. B. Clements. 1996. Immediate early protein ICP63 of herpes simplex virus type 1 binds RNA directly. J. Gen. Virol. 77:1847-1851.
Jean, S., K. M. LeVan, B. Song, M. Levine, and D. M. Knipe. 2001. Herpes simplex virus 1 ICP27 is required for transcription of two viral late (g2) genes in infected cells. Virology 283:273-284.
Jenkins, H. L., and C. A. Spencer. 2001. RNA polymerase II holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells. J. Virol. 75:9872-9884.
Kang, Y., and B. R. Cullen. 1999. The human TAP protein is a nuclear mRNA export factor that contains novel RNA binding and nucleocytoplasmic transport sequences. Genes Dev. 13:1126-1139.
Kataoka, N., J. Yong, V. N. Kim, F. Velazquez, R. A. Perkinson, F. Wang, and G. Dreyfuss. 2000. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell 6:673-682.
Kim, V. N., J. Jong, N. Kataoka, and G. Dreyfuss. 2001. The Y14 protein communicates to the cytoplasm the positions of exon-exon junctions. EMBO J. 20:2062-2068.
Kim, V. N., N. Kataoka, and G. Dreyfuss. 2001. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. Science 293:1832-1836.
Koffa, M. D., J. B. Clements, E. Izaurralde, S. Wadd, S. A. Wilson, I. W. Mattaj, and S. Kuersten. 2001. Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway. EMBO J. 20:5769-5778.
Laurent, A.-M., J.-J. Madjar, and A. Greco. 1998. Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the rate limiting step. J. Gen. Virol. 79:2765-2775.
Le Hir, H., D. Gatfield, E. Izaurralde, and M. J. Moore. 2001. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 20:4987-4997.
Le Hir, H., E. Izaurralde, L. E. Maquat, and M. J. Moore. 2000. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19:6860-6869.
Lengyel, J., C. Guy, V. Leong, S. Borge, and S. A. Rice. 2002. Mapping of functional regions in the amino-terminal portion of the herpes simplex virus ICP27 regulatory protein: importance of the leucine-rich nuclear export signal and RGG box RNA-binding domain. J. Virol. 76:11866-11879.
Li, Q., H. Imataka, S. Morino, G. W. J. Rogers, N. J. Richter-Cook, W. C. Merrick, and N. Sonenberg. 1999. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol. Cell. Biol. 19:7336-7346.
Lindberg, A., and J.-P. Kreivi. 2002. Splicing inhibition at the level of spliceosome assembly in the presence of herpes simplex virus protein ICP27. Virology 294:189-198.
Longman, D., I. L. Johnstone, and J. F. Caceres. 2003. The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans. RNA 9:881-891.
Lu, S., and B. R. Cullen. 2003. Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 9:618-630.
Lykke-Anderson, J. 2001. mRNA quality control: marking the message for life and death. Curr. Biol. 11:88-91.
Lykke-Anderson, J., M. D. Shu, and J. A. Steitz. 2001. Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293:1836-1839.
McCarthy, A. M., L. McMahan, and P. A. Schaffer. 1989. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J. Virol. 63:18-27.
McGregor, F., A. Phelan, J. Dunlop, and J. B. Clements. 1996. Regulation of herpes simplex virus poly(A) site usage and the action of immediate-early protein IE63 in the early-late switch. J. Virol. 70:1931-1940.
McLauchlan, J., P. C. Loney, R. M. Sandri-Goldin, and J. B. Clements. 1992. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. J. Virol. 66:6939-6945.
McLauchlan, J., S. Simpson, and J. B. Clements. 1989. Herpes simplex virus induces a processing factor that stimulates poly(A) site usage. Cell 59:1093-1105.
McMahan, L., and P. A. Schaffer. 1990. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to the C-terminal regions and are required to modulate viral gene expression very early in infection. J. Virol. 64:3471-3485.
Mears, W. E., V. Lam, and S. A. Rice. 1995. Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. J. Virol. 69:935-947.
Mears, W. E., and S. A. Rice. 1998. The herpes simplex virus immediate-early protein ICP27 shuttles between nucleus and cytoplasm. Virology 242:128-137.
Mears, W. E., and S. A. Rice. 1996. The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation. J. Virol. 70:7445-7453.
Nott, A., H. Le Hir, and M. J. Moore. 2004. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes Dev. 18:210-222.
Nott, A., S. H. Meislin, and M. J. Moore. 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA 9:607-617.
Ostareck, D. H., A. Ostareck-Lederer, I. N. Shatsky, and M. W. Hentze. 2001. Lipoxygenase mRNA silencing in erythroid differentiation: the 3' UTR regulatory complex controls 60S ribosomal subunit joining. Cell 104:281-290.
Ostareck-Lederer, A., D. H. Ostareck, C. Cans, G. Neubauer, K. Bomsztyk, G. Superti-Furga, and M. W. Hentze. 2002. c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs. Mol. Cell. Biol. 22:4535-4543.
Palacios, I. M., D. Gatfield, D. St Johnston, and E. Izaurralde. 2004. An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427:753-757.
Pearson, A., D. M. Knipe, and D. M. Coen. 2004. ICP27 selectively regulates the cytoplasmic localization of a subset of viral transcripts in herpes simplex virus type 1-infected cells. J. Virol. 78:23-32.
Phelan, A., M. Carmo-Fonseca, J. McLauchlan, A. I. Lamond, and J. B. Clements. 1993. A herpes simplex virus type-1 immediate early gene product IE63 regulates small ribonucleoprotein distribution. Proc. Natl. Acad. Sci. USA 90:9056-9060.
Phelan, A., and J. B. Clements. 1997. Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm. J. Gen. Virol. 78:3327-3331.
Phelan, A., J. Dunlop, and J. B. Clements. 1996. Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts. J. Virol. 70:5255-5265.
Pollard, V. W., and M. H. Malim. 1998. The HIV-1 Rev protein. Annu. Rev. Microbiol. 52:491-532.
Poon, A. P., and B. Roizman. 1997. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant 134.5 genes of herpes simplex virus 1. Virology 229:98-105.
Prostko, C. R., M. A. Brostrom, E. M. Malara, and C. O. Brostrom. 1992. Phosphorylation of eukaryotic initiation factor (eIF) 2 and inhibition of eIF-2B in GH3 pituitary cells by perturbants of early protein processing that induce GRP78. J. Biol. Chem. 267:16751-16754.
Reed, R., and B. Hurt. 2002. A conserved RNA export machinery coupled to pre-mRNA splicing. Cell 108:523-531.
Rice, S. A., and D. M. Knipe. 1988. Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27. J. Virol. 62:3814-3823.
Rice, S. A., and D. M. Knipe. 1990. Genetic evidence for two distinct transactivation functions of the herpes simplex virus protein ICP27. J. Virol. 64:1704-1715.
Rice, S. A., L. Su, and D. M. Knipe. 1989. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J. Virol. 63:3399-3407.
Rodrigues, J. P., M. Rode, D. Gatfield, B. J. Blencowe, M. Carmo-Fonseca, and E. Izaurralde. 2001. REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc. Natl. Acad. Sci. USA 98:1030-1035.
Roizman, B., and D. M. Knipe. 2001. Herpes simplex viruses and their replication, p. 2381-2398. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Strauss (ed.), Field's virology, 4th ed. Lippincott, Williams & Wilkins, Philadelphia, Pa.
Sacks, W. R., C. C. Greene, D. P. Ashman, and P. A. Schaffer. 1985. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J. Virol. 55:796-805.
Sanchez-Velar, N., E. B. Udofia, Z. Yu, and M. L. Zapp. 2004. hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region. Genes Dev. 18:23-34.
Sandri-Goldin, R. M. 1998. ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 12:868-879.
Sandri-Goldin, R. M., and M. K. Hibbard. 1996. The herpes simplex virus type 1 regulatory protein ICP27 coimmunoprecipitates with anti-Sm antiserum, and the C terminus appears to be required for this interaction. J. Virol. 70:108-118.
Sandri-Goldin, R. M., M. K. Hibbard, and M. A. Hardwicke. 1995. The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing. J. Virol. 69:6063-6076.
Sandri-Goldin, R. M., and G. E. Mendoza. 1992. A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Genes Dev. 6:848-863.
Sanford, J. R., N. K. Gray, K. Beckmann, and J. F. Caceres. 2004. A novel role for shuttling SR proteins in mRNA translation. Genes Dev. 18:755-768.
Sciabica, K. S., Q. J. Dai, and R. M. Sandri-Goldin. 2003. ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation. EMBO J. 22:1608-1619.
Sekulovich, R. E., K. Leary, and R. M. Sandri-Goldin. 1988. The herpes simplex virus type 1 protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. J. Virol. 62:4510-4522.
Shibuya, T., T. O. Tange, N. Sonenberg, and M. J. Moore. 2004. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11:346-351.
Simonin, D., J.-J. Diaz, T. Masse, and J.-J. Madjar. 1997. Persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1. J. Gen. Virol. 78:435-443.
Sokolowski, M., J. E. Scott, R. P. Heaney, A. H. Patel, and J. B. Clements. 2003. Identification of herpes simplex virus RNAs that interact specifically with regulatory protein ICP27 in vivo. J. Biol. Chem. 278:33540-33549.
Soliman, T. M., R. M. Sandri-Goldin, and S. J. Silverstein. 1997. Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins. J. Virol. 71:9188-9197.
Soliman, T. M., and S. J. Silverstein. 2000. Herpesvirus mRNAs are sorted for export via Crm1-dependent and -independent pathways. J. Virol. 74:2814-2825.
Stutz, F., A. Bachi, T. Doerks, I. C. Braun, B. Seraphin, M. Wilm, P. Bork, and E. Izaurralde. 2000. REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6:638-650.
Stutz, F., and M. Rosbash. 1998. Nuclear RNA export. Genes Dev. 12:3303-3319.
Uprichard, S. L., and D. M. Knipe. 1996. Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes. J. Virol. 70:1969-1980.
Varmuza, S. L., and J. R. Smiley. 1985. Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences. Cell 41:793-802.
Wadd, S., H. Bryant, O. Filhol, J. E. Scott, T.-Y. Hsieh, R. D. Everett, and J. B. Clements. 1999. The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2. J. Biol. Chem. 274:28991-28998.
Weinheimer, S. P., B. A. Boyd, S. K. Durham, J. L. Resnick, and D. R. O'Boyle II. 1992. Deletion of the VP16 open reading frame of herpes simplex virus type 1. J. Virol. 66:258-269.
Weinheimer, S. P., and S. L. McKnight. 1987. Transcriptional and post-transcriptional controls establish the cascade of herpes simplex virus protein synthesis. J. Mol. Biol. 195:819-833.
Wiegand, H. L., S. Lu, and B. R. Cullen. 2003. Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proc. Natl. Acad. Sci. USA 100:11327-11332.
Zhou, C., and D. M. Knipe. 2002. Association of herpes simplex virus type 1 ICP8 and ICP27 proteins with cellular RNA polymerase II holoenzyme. J. Virol. 76:5893-5904.
Zhou, Z., M.-J. Luo, K. Straesser, J. Katahira, E. Hurt, and R. Reed. 2000. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407:401-405.(Kimberly S. Ellison, Robe)