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Journal of Virology, July 2007, p. 7695-7701, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00282-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Stable 2-Kilobase Latency-Associated Transcript of Herpes Simplex Virus Type 1 Can Alter the Assembly of the 60S Ribosomal Subunit and Is Exported from Nucleus to Cytoplasm by a CRM1-Dependent Pathway

Doina Atanasiu and Nigel W. Fraser^*

Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, Pennsylvania 19104

Received 9 February 2007/ Accepted 30 April 2007

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During latency of herpes simplex virus type 1 in the neurons of the peripheral nervous system, the major transcript detected is the 2-kb latency-associated transcript (LAT) intron. During lytic infection, this intron has been shown to associate with ribosomes, suggesting a role in modifying the translational machinery of infected cells. In this study we show, using LAT-transfected cells, that the interaction of the intron with the 60S ribosomal subunit leads to irreversible changes in the sedimentation profile of this subunit in the nucleus. Furthermore, the 2-kb LAT intron is transported to the cytoplasm as part of the 60S ribosomal subunit, using a CRM1-dependent pathway.

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During herpes simplex virus type 1 (HSV-1) latency, viral transcription is restricted to a family of RNAs encoded within the repeat segments of the viral genome. These transcripts are known as the latency-associated transcripts (LATs). The most abundant LAT is a stable intron approximately 2 kb in length (6) with a half-life of about 24 h (23). This transcript partially overlaps the 3' end of the ICP0 gene and is transcribed from the DNA strand complementary to that which encodes ICP0 mRNA (21).

The LAT stable intron is not unique. Recently, another stable intron has been reported in human cytomegalovirus (13). The 5-kb stable intron of this virus is almost exclusively nuclear during the immediate-early phase and predominantly nuclear during the late phase of viral replication (13). In productively infected tissue culture cells, the HSV-1 2-kb stable intron is also found in both the cytoplasm and the nucleus (18). However, the 2-kb intron of HSV-1 is retained in the nucleus of latently infected neurons (6).

The cytoplasmic 2-kb LAT intron comigrates with ribosomal subunits at the position of 50S (18). Other studies have suggested that the 2-kb LAT intron is present in the cytoplasm and is bound to polysomal fractions in latently infected neurons (7, 8). Although the in vitro translation of the 2-kb LAT intron is very inefficient (20), the presence of the intron in the polysomal fraction opens up the possibility that the 2-kb LAT intron is translated at some point during the HSV-1 life cycle. Although translation products from the 2-kb LAT intron have been reported (25), translation would have to occur in a tightly regulated and possibly transient fashion in order to explain the difficulty in detecting the expression of such proteins (15).

Previous studies have shown that in productively infected cells, the nuclear and cytoplasmic distribution of the 2-kb LAT intron is more similar to that of 28S rRNA than to that of cellular or viral mRNA (1). Another interesting finding is that the 2-kb LAT intron interacts with ribosomal proteins associated with the 60S ribosomal subunit (1). These data suggest that the 2-kb LAT intron may play a structural role in the ribosome life cycle and thus affect the functioning of the translational machinery.

In this study we show, using cell fractionation and sucrose gradients, that the 2-kb LAT interacts specifically with the large (60S) ribosomal subunit in the nucleoli, which leads to a change in its sedimentation profile. Furthermore, the export of the 2-kb LAT intron is dependent on the CRM1 pathway, suggesting that it is exported from the nucleus to the cytoplasm as part of the 60S ribosomal subunit. A mechanism for the transport of intron from nuclear spliceosome to cytoplasm is proposed.

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Cell line plasmids and viruses. SY5Y cells were grown in RPMI medium supplemented with 10% fetal calf serum. The following plasmids were used for transfections: pcDNAPst/Mlu (29), which encompasses the 2-kb LAT coding region, exon 1, and part of exon 2, and pXcm, pBfa, pHpa, which contain deletions of the 2-kb LAT coding region at the respective sites and were described elsewhere (11). pSty (16) has a deletion in exon 1 and was used to isolate the intron probe. The plasmid maps are shown in Fig. 1. HSV-1 strain 17⁺ and HSV 17⁺GUSB have been described previously (24).

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FIG. 1. Schematic representation of HSV-1 genome and plasmids. (A) A linear map of the HSV-1 genome with unique long (UL) and unique short (US) regions flanked by inverted repeat (IR) elements. The region encoding the LATs is enlarged to show the transcripts that map to this area. (B) Schematic representation of the plasmid deletion mutants used in this study. The pcDNAPst/Mlu plasmid encodes a wild-type intron. Xcm, Hpa, Bfa, and Sty are intron deletion mutants of pcDNAPst/Mlu. (C) The 0.9-kb LAT intron probe. (D) Diagram of 17+GUSB virus showing modification of the LAT gene.

Transfections. SY5Y cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, 10 µg of DNA, 1 µg of GFP (green fluorescent protein), and 15 µl of Lipofectamine 2000 were used per 60-mm dish containing serum-free medium. Five hours posttransfection, cells were fed with RPMI medium supplemented with 10% fetal calf serum. Cells were harvested 48 h from the start of transfection.

Infections. Subconfluent cell monolayers were infected with HSV-1 strains 17⁺ and 17⁺GUSB (28) at a multiplicity of infection of 5 for 18 h. For transfection/infection experiments, at 30 h posttransfection, cells were infected with the required virus at a multiplicity of infection of 5.

Drug treatment. Transfected cells were kept in cell cultivation medium containing 50 µg/ml of cycloheximide (Sigma), 250 µM of puromycin (Sigma), or 25 ng/ml of leptomycin B (LMB; a kind gift from Minoru Yoshida) for 3 h at 37°C. In all cases, control cells were maintained in medium without drugs.

Isolation and analysis of cellular fractions. The procedure for the isolation of cytoplasm, outer nuclear membrane, nucleoplasm, and extract of nuclear pellet was described elsewhere (1). Briefly, 10⁷ cells were harvested in 1x phosphate-buffered saline (PBS). Cells were resuspended in 1 ml EBKL buffer (25 mM HEPES buffer [pH 7.6], 5 mM MgCl₂, 1.5 mM KCl, 2 mM DTT [dithiothreitol], 1 mM PMSF [phenylmethylsulfonyl fluoride], and 4 g aprotinin/ml), lysed with loose Dounce homogenizer (20 to 30 tight strokes), and spun down for 5 min at 2,000 rpm. The supernatant was the cytoplasmic extract. The nuclei were washed with EBMK buffer (25 mM HEPES [pH 7.6], 5 mM MgCl₂, 1.5 mM KCl, 75 mM NaCl, 175 mM sucrose, and 2 mM DTT) containing 0.5% NP-40. The supernatant was the outer nuclear membrane. The pellet was incubated in 1 ml EBKL buffer (0.1% NP-40) for 10 min and then lysed with KCl to a final concentration of 0.2 M. The lysed nuclei were DNase treated for 15 min at 37°C. The supernatant of this step was the nucleoplasm. The pellet containing chromatin and nucleolar material was sonicated (three times for 10 s at 20 W each time) in EBMK (0.5% NP-40) followed by centrifugation at 10,000 rpm. The resulting supernatant was the extract of nuclear pellet. Equivalent A₂₆₀ concentrations were dot blotted onto nylon membranes (PerkinElmer Life Sciences Inc.) using a Bio-Rad dot blot apparatus. The membranes were UV cross-linked (auto setting; Stratalinker) and hybridized overnight with heat-denatured ³²P-labeled DNA probes. The blots were washed twice as described in the Northern procedure.

Isolation of cytoplasmic and nuclear extracts and sucrose gradients. After transfection or infection, cells were washed twice with PBS and harvested in reticulocyte standard buffer (RSB; 10 mM Tris [pH 7.4], 10 mM NaCl, and 10 mM MgCl₂). Cells were lysed by the addition of 1% NP-40 (Fluka) and centrifuged for 10 min at 3,000 rpm in a Beckman centrifuge through a sucrose cushion (0.33 M in RSB). The nuclei were resuspended in nuclei lysis buffer (20 mM HEPES [pH 7.9], 25% glycerol, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 1 mM PMSF, and 1 mM DTT) and lysed by mechanical disruption using a Dounce homogenizer. Both nuclear and cytoplasmic extracts were loaded on top of 5 to 50% linear sucrose gradients and spun at 38,000 rpm for 2 h in an SW41 rotor (Beckman). For each gradient, the absorbance at 254 nm was determined, and 0.5-ml fractions were collected using a GradiFrac apparatus (Pharmacia). Two-hundred-microliter aliquots of each fraction were mixed with 120 µl 37% formaldehyde, 80 µl of 20x SSC (3 M NaCl and 0.3 M sodium citrate; 1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and 400 µl formamide. After 15 min of incubation at 55°C, the samples were blotted in duplicate onto a nylon membrane (PerkinElmer Life Sciences) using a dot blot apparatus (Bio-Rad). Dot blots were cross-linked using a Stratagene UV cross-linker and hybridized as described for Northern blots. These dot blots were probed with 2-kb LAT intron, 28S rRNA, or 18S rRNA.

Preparation of ³²P-labeled probes. The intron probe was a 1-kb BstEII-BstEII fragment generated from pSty (16). The 0.9-kb 28S rDNA probe was produced by digesting pGEM-28S (1) with BamHI and BglII. The 18S rDNA probe was generated from pHrB plasmid (27) by XbaI and HindIII restriction digestion. The DNA probes were generated with a ³²P label using the RadPrime labeling system (Invitrogen) according to the manufacturer's instructions.

Data analysis. Dot blots were exposed to phosphor screens (Molecular Dynamics) and were developed using a Storm Phosphoimager (Molecular Dynamics). The data were analyzed using ImageQuant software (Mac version).

Metabolic cell labeling. Samples were labeled in vitro using [³⁵S]methionine-cysteine (Amersham). Briefly, at 46 h posttransfection, cells were starved in methionine-free media (Gibco) for 1 h and then labeled with 50 µCi of [³⁵S]Met-Cys per ml of media for 4 h at 37°C. Cells were washed twice with 1x PBS, harvested, and lysed on ice with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate). A cocktail of protease inhibitors (Complete cocktail; Roche) was present at all times. The protein concentration of lysates was estimated with a Bio-Rad protein assay kit.

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The 2-kb LAT intron induces a size change in cytoplasmic 60S ribosomal subunits. It has been shown that the HSV-1 2-kb LAT intron, which is expressed late in tissue culture infection, associates with ribosomal subunits (18). The affinity of this association is similar to that of rRNA, suggesting a structural rather than a messenger role for the LAT intron (1). To study this hypothesis, we have examined the sedimentation profile of the ribosomal subunits in SY5Y cells transfected with the pcDNAPst/Mlu plasmid—a plasmid that expresses the 2-kb intron from a truncated LAT transcript (29).

Cells were transfected with the pcDNA3 (mock) or the pcDNAPst/Mlu (intron-expressing) plasmid. Cytoplasmic extracts were prepared and loaded onto 5 to 50% sucrose gradients, as described in Materials and Methods. Aliquots from each fraction were blotted onto nylon membranes, and the 2-kb LAT (Fig. 2A) and 28S rRNAs (Fig. 2B through D) were detected with ³²P-labeled probes specific for each RNA, as described in Materials and Methods. Radioactivity was quantitated by phosphorimaging, and the data were expressed as a percentage of the total counts in a gradient. In mock-transfected cells, we found a normal distribution of 28S rRNA, with two visible peaks, one for 60S and one for 80S (Fig. 2C). However, when cells were transfected with a plasmid encoding the 2-kb LAT intron, aside from the expected LAT (Fig. 2A) (18) and ribosomal peaks (Fig. 2B), we found a lighter sedimenting particle, which we called the "pre-60S-size" particle (Fig. 2B), because it was detected with a 28S but not an 18S rRNA probe (Fig. 3). Analysis of cell extracts prepared from cells transfected with intron deletion mutants revealed a distribution of the 28S rRNA similar to that found in mock-transfected cells (Fig. 2D). This suggests that only full-length intron is able to induce a sedimentation change of the large ribosomal subunit, probably by some conformational requirements.

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FIG. 2. The 2-kb LAT induces a size change of the 60S ribosomal subunit in the cytoplasm. Sucrose gradient sedimentation was used to show the changes in ribosome caused by the LAT intron. SY5Y cells were transfected with plasmid DNA. Forty-eight hours posttransfection, cells were harvested and cytoplasmic extracts were loaded onto 5 to 50% sucrose gradients. Aliquots from each fraction were dot blotted onto nylon membranes and probed with 32P-labeled 28S (B, C, D) or intron (A) DNA probes. The following plasmids were transfected: (A) pcDNAPst/Mlu, (B) pcDNAPst/Mlu, (C) pcDNA3 (mock), and (D) intron deletion mutants (Xcm, Hpa, and Bfa). The labels "pre-60S-like," 60S, and 80S represent the relative sizes of the peaks identified with a 28S rRNA probe.

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FIG. 3. The presence of the "pre-60S-size" particle does not affect cellular translation. Typical 18S rRNA distribution in SY5Y cells transfected with the pcDNA3 (mock) or the pcDNAPst/Mlu plasmid.

Does the 2-kb LAT intron affect ribosome assembly or translation? It is known that in the absence of sufficient numbers of functional 60S subunits to pair with, 40S ribosomal subunits will accumulate in cells (2). Thus, we looked for the accumulation of 40S subunits following the expression of 2-kb LAT in transfected cells. Based on GFP expression from a cotransfected plasmid, it was estimated that approximately 40 to 60% of the cells were transfected, and from data generated from Fig. 2B (averaged over four independent experiments), that only approximately 12.5% of the 60S ribosome subunits were altered. However, the change induced in the 60S subunit by the 2-kb LAT intron did not prevent the assembly of ribosomes (Fig. 2B, C, D), suggesting the altered subunit can form a functional 80S ribosome. Furthermore, we did not see any 40S subunit accumulation (18S rRNA) in the cells transfected with the LAT-expressing plasmid compared to those infected with the control plasmid (Fig. 3).

In order to determine whether LAT-altered ribosomes affected translational levels in cells, we performed protein-labeling experiments. Mock- or 2-kb-intron-transfected cells were metabolically labeled 44 h posttransfection with 50 µCi [³⁵S]Met-Cys for 4 h. Cells were lysed, total cellular proteins were precipitated using trichloroacetic acid, and the levels of incorporation were measured as described in Materials and Methods. The incorporation levels for mock and 2-kb intron samples were similar (10⁷ cpm ± 3,000). These data suggest that the modified ribosomal particle either does not disrupt translation or is present at such low levels in the actively translating polysome population that the change in translation is below the level of detection.

The effect of the 2-kb intron on the 60S subribosomal particle in transfected cells cannot be corrected by virus superinfection. Productive infection of tissue culture cells did not show any change in the ribosome sedimentation (reference 18 and data not shown). This suggests that there may be other viral genes which can prevent formation of or correct the "pre-60S-size" particle formed in 2-kb LAT intron-transfected cells. Thus, the modified ribosomes may exist for only a short window in the HSV infection cycle (for example, during early reactivation). Other possibilities exist, for example, that the "pre-60S-size" particle is the result of the overproduction of intron in transfected cells. However, on hybridization with LAT and rRNA probes, labeled to approximately the same specific activity, it was noted that the rRNA probes always gave much stronger signals, suggesting that the copy numbers of LAT intron are much lower than those of rRNA in the transfected cells.

Using a 17⁺-based virus that has a deletion of approximately 1 kb at the two BstEII sites downstream of the LAT promoter and the insertion of a human ß-glucuronidase gene at this deletion, we examined the potential for the coinfection of this virus to correct the LAT intron effect on the 60S ribosome subunit. This deletion removes the putative splice donor site and 5' sequences from the stable 2-kb LAT intron (28). Thus, the virus cannot express the 2-kb LAT intron.

SY5Y cells were transfected with the pcDNAPst/Mlu plasmid for 32 h and then infected for 16 h with the mutant virus as described in Materials and Methods. Cytoplasmic extracts were isolated and separated through sucrose gradients as previously described. Using a ³²P-labeled 28S rDNA probe, we showed that the "pre-60S-size" particle that forms during transfection was not corrected by the LAT mutant virus (Fig. 4). Thus, we concluded that the modification to the 60S ribosomal subunit fractionation is irreversible. Because it cannot be corrected by other genes in the virus once it has occurred, we hypothesize that during normal infection, genes in the virus prevent the 2-kb LAT intron from modifying the ribosome rather than reversing the change after it has occurred.

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FIG. 4. The effect of the intron on the pre-60S-size particle is not corrected by the virus. 28S rRNA distribution in SY5Y cells transfected (circles) or transfected and infected with a LAT 17+GUSB mutant virus (squares).

The "pre-60S-size" particle is formed in the nuclei. The 2-kb LAT intron is spliced from a large precursor transcript in the nuclei. During the productive infection of cells, the intron can be found in both the cytoplasm and the nuclei (18). Thus, it is of interest to determine whether the intron associates with the 60S ribosome in the nuclei or the cytoplasm. Does the 2-kb intron attach to the maturing ribosome in the nuclei, and are both exported together, or does this interaction occur in the cytoplasm, altering a fully matured subribosomal particle? To address this question, we blocked protein biosynthesis with cycloheximide and measured the accumulation of "pre-60S-size" ribosomal particles.

SY5Y cells were transfected with either the pcDNA3 (mock) or pcDNAPst/Mlu (2-kb LAT-expressing) plasmid. Forty-five hours posttransfection, cells were treated for 3 h with 50 µg/ml cycloheximide. Cytoplasmic and nuclear extracts were separated through 5 to 50% sucrose gradients and probed with a 28S rDNA probe as described in Materials and Methods. The "pre-60S-size" particle could be identified in cytoplasmic extracts from 2-kb LAT-transfected cells (Fig. 5A), and it accumulated in the nuclei of cycloheximide-treated cells (Fig. 5B). These data support the hypothesis that the "pre-60S-size" particle forms in the nuclei and that export of the 2-kb intron requires ongoing protein synthesis.

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FIG. 5. The 2-kb LAT interacts with the 60S particle in the nuclei. Diagrams show typical 28S sedimentation in cytoplasmic (A) and nuclear (B) extracts. Prior to harvesting, cells were treated for 2 h with 50 µg/ml cycloheximide. A "pre-60S-size" particle can be seen in the nuclei of drug-treated cells (B; squares) and in the cytoplasm of untreated transfected cells (A; circles).

The 2-kb LAT intron is exported out of the nucleus via the LMB-sensitive CRM1 pathway. Before mRNA can leave the nucleus, appropriate processing events such as capping, splicing, and polyadenylation must occur. After proper nuclear processing, polyadenylated mRNAs are exported to the cytoplasm via the TAP pathway and translated, and their poly(A) tails are shortened by cytoplasmic nucleases (30). Because the 2-kb LAT intron is neither capped nor polyadenylated (20), it is less probable that the cell uses the TAP pathway to export it from the nucleus. As the 2-kb LAT has been shown to interact with ribosomes (1, 18), and ribosomes are known to be exported by the CRM1 pathway (9, 24), we hypothesized that this interaction is more likely to be responsible for the export of the intron from nuclei to the cytoplasm.

To test whether CRM1 plays a role in the export of the 2-kb LAT intron, we treated SY5Y-transfected cells with LMB—an inhibitor of the CRM1 pathway (12). If CRM1 is involved in the export of the 2-kb LAT intron, then treatment with LMB should affect the localization of the intron in the cell and lead to a nuclear accumulation. SY5Y cells were transfected with pcDNAPst/Mlu or pcDNA3 (mock). Forty-five hours posttransfection, cells were treated with 25 ng/ml LMB for 3 h. Cellular fractions were then isolated and tested for the presence of 2-kb LAT intron (Fig. 6B) and 28S rRNA (Fig. 6A) using ³²P-labeled DNA probes. The nuclear accumulation of 28S rRNA is entirely expected (Fig. 6A), as the 60S subunit is known to be exported by the CRM1 pathway (9). The 2-kb LAT intron distribution was similar to that of the 28S rRNA, with an accumulation of the intron into the nucleoli upon exposure to LMB (Fig. 6B). These data support the requirement of the CRM1 pathway for intron export.

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FIG. 6. The 2-kb LAT intron is exported from the nuclei via the CRM1 pathway. SY5Y cells were transfected with the pcDNA3 (mock) or the pcDNAPst/Mlu plasmid. Forty-five hours posttransfection, cells were treated with 25 ng/ml LMB. Subcellular compartments were isolated and the presence of 28S rRNA (A) and 2-kb LAT intron (B) was measured relative to untransfected cells. Percentage counts are normalized to the untransfected levels (100%) and clearly show nuclear accumulation of both 28S rRNA and 2-kb intron.

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Ribosomes from eukaryotic cells are complex ribonucleoprotein particles that are assembled in the cytoplasm from a small (40S) and a large (60S) subunit. Their biogenesis is a complicated process that includes the transcription of rRNA by RNA polymerase I as a large 45S pre-rRNA precursor (28S, 18S, and 5.8S rRNA), and by polymerase III from another gene to give 5S rRNA. During these processing reactions, many nonribosomal proteins as part of snoRNA particles associate with the pre-rRNAs in order to facilitate modification and processing, and about 80 ribosomal proteins are assembled onto rRNA (17).

In this study, we show that the 2-kb latency-associated transcript of HSV-1 interacts with the large ribosomal subunit and modifies its sedimentation profile (Fig. 2B). We call this modified particle "pre-60S size" because it sediments lighter than the 60S particle and contains 28S rRNA but not 18S rRNA. We hypothesize that the intron interacts with the forming 60S particle in the nucleoli (Fig. 5B) and may interfere with the nuclear processing of 28S rRNA, leading to changes in the protein composition of the particle.

In Saccharomyces cerevisiae Nog2p, a nuclear-nucleolar GTP-binding protein involved in rRNA maturation associates with particles highly enriched in 27S and 7S pre-rRNA that sediment in the 60S region of the gradient but not with 40S subunits, 80S ribosomes, or polysomes (19). It is possible that the LAT intron acts in a similar fashion.

In yeast, most pre-60S particles are nuclear-nucleolar localized and have not been detected in the cytoplasm (19). However, in LAT-expressing cells, the LAT-pre60S particle is transported to the cytoplasm.

Although we have not conclusively shown whether the modified particle is assembled into a functional ribosome, the 2-kb LAT intron has been found to be associated with polyribosomes during tissue culture infection and during latent infection of animals (7), suggesting that the LAT pre-60S-size particle is assembled into a functional ribosome.

Our translational studies have shown that the incorporation of [³⁵S]Met-Cys occurred at similar levels in both mock- and 2-kb LAT-transfected cells. Furthermore, no accumulation of 40S subunits was detected (Fig. 3), supporting the formation of 80S ribosomes. However, we could not detect modified forms of 80S subribosomal particles in cytoplasmic gradients. Had we found this particle, it would indicate incorporation of modified 60S particles into 80S ribosomes. The lack of findings may indicate that the change in size of the 80S particle is too small to detect on our sucrose isokinetic gradients.

While we could detect LAT pre-60S-size particles in cells transfected with full-length intron (Fig. 2B), we could not detect modified ribosomal particles with the LAT deletion mutants tested (Fig. 2D). These findings suggest the need for full-length 2-kb LAT intron in order to affect the change.

A growing body of evidence suggests that the intron plays a structural role in the ribosome and not as an mRNA (1, 15, 18). For example, the intron behaves like an rRNA in terms of affinity with ribosomal proteins (1). Furthermore, attempts to definitively show translation from the 2-kb intron have had little success in our hands (15).

Taken together, our data suggest a model for the processing of the LAT intron and export from the nuclei to the cytoplasm. After synthesis by RNA polymerase II in the nucleoplasm, the LAT gene is likely processed by the spliceosome complex to remove intronic sequences. The resulting stable intron (the 2-kb LAT) with a nonconsensus branch point (10, 25) reaches the nucleoli (1), the main compartment for eukaryotic ribosome biogenesis. It may be that the stable intron is transported from the spliceosome in the nucleoplasm to the nucleoli in a fashion similar to snoRNAs, which are cleaved from introns and transported to the nucleoli to modify rRNA (3, 14). While at the nucleoli, the intron might bind to snoRNPs (small nucleolar ribonucleoproteins) involved in the processing of rRNAs (26) or directly to the "pre-60S-size" subunit, and in this way appears to interfere with the later stages of maturation of this ribosome subunit. The subunits appear to diffuse slowly to the membrane (18) where the intron, associated with the newly formed particle, is exported from the nuclei via the CRM1 pathway (Fig. 6A). It has previously been shown that mutation of a nucleolar protein (Bop1) results in aberrant processing of the 60S ribosomal subunit; thus, it is possible that the LAT intron has a similar effect by interacting with a nucleolar protein (22).

In E. coli, SrmB and CsdA are cold shock RNA helicases involved in the early steps of 60S assembly and are required for growth at a low temperature (4, 5). A proposed role for these proteins is to derepress the synthesis of heat shock proteins by facilitating their translation during cold shock (4, 10). Similarly, we have shown that the presence of the 2-kb LAT intron in transfected cells results in an accumulation of Hsp70 during cold shock, which results in a faster recovery from cold shock (3). While the efficiency of transfection was estimated at 40 to 60%, it is estimated that only a small number of ribosomes are modified (15%). This may be due to the long half-life of the ribosomal subunits relative to the length of time posttransfection that the experiments were performed. This population of modified ribosomes would be used during reactivation to ensure the presence of an Hsp70 pool that would help in the translation of viral proteins, as well as host survival following the stress of reactivation.

In conclusion, we show that the presence of the 2-kb LAT intron can affect the sedimentation of the large ribosomal subunit. The modified ribosomal subunit containing the 2-kb LAT intron forms in the nuclei and is exported to the cytoplasm via the CRM1 pathway. Thus, the HSV-1 2-kb LAT intron is exported from the nuclei to the cytoplasm via the CRM1 pathway.

 
We thank Jay Gardner and Jared Gartner for their technical assistance. We thank Minoru Yoshida (Chemical Genetics Laboratory, Japan) for his generous gift of LMB.

This work was supported by National Institutes of Health grant NS 33768.

 
* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104. Phone: (215) 898-3847. Fax: (215) 898-3849. E-mail: nfraser{at}mail.med.upenn.edu

Published ahead of print on 9 May 2007.

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Journal of Virology, July 2007, p. 7695-7701, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00282-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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