JOURNAL OF VIROLOGY, Apr. 2009, p. 3637–3646 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.02201-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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Bunyamwera Orthobunyavirus S-Segment Untranslated Regions Mediate Poly(A) Tail-Independent Translation䌤 Gjon Blakqori, Ingeborg van Knippenberg, and Richard M. Elliott* Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom Received 17 October 2008/Accepted 27 January 2009
The mRNAs of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, possess a 5ⴕ cap structure but lack a 3ⴕ poly(A) tail, a common feature of eukaryotic mRNAs that greatly enhances translation efficiency. Viral mRNAs also contain untranslated regions (UTRs) that flank the coding sequence. Using model virus-like mRNAs that harbor the Renilla luciferase reporter gene, we found that the 3ⴕ UTR of the BUNV small-segment mRNA mediated efficient translation in the absence of a poly(A) tail. Viral UTRs did not increase RNA stability, and polyadenylation did not significantly enhance reporter activity. Translation of virus-like mRNAs in transfected cells was unaffected by knockdown of poly(A)-binding protein (PABP) but was markedly reduced by depletion of eukaryotic initiation factor 4G, suggesting a PABP-independent process for translation initiation. In BUNV-infected cells, translation of polyadenylated but not virus-like mRNAs was inhibited. Furthermore, we demonstrate that the viral nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm early in infection, followed by nuclear retention of PABP. Our results suggest that BUNV corrupts PABP function in order to inhibit translation of polyadenylated cellular mRNAs while its own mRNAs are translated in a PABP-independent process. liovirus proteases 2A and 3C cleave eIF4G and PABP, respectively, thereby effectively shutting off host cell protein synthesis (28, 30). PABP is also the target of the calicivirus 3C-like protease (29). The rotavirus NSP3 protein inhibits translation of polyadenylated mRNAs by disrupting the interaction of PABP with eIF4G through competition for the same binding site (37, 40). The Bunyaviridae family contains viruses with tripartite, negative-sense or ambisense RNA genomes, and is divided into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the family as a whole. The genome segments are termed small (S), medium (M), and large (L), according to their relative size. Each segment consists of a coding region that is flanked by 3⬘ and 5⬘ UTRs. The BUNV S segment encodes the nucleocapsid protein N and a nonstructural protein (NSs), the M segment encodes the two glycoproteins Gn and Gc as well as a second nonstructural protein NSm, and the L segment encodes the RNA-dependent RNA polymerase. The UTRs are multifunctional: they serve as promoters for transcription and replication, provide a signal for encapsidation by the N protein to form ribonucleoprotein particles (RNPs) and are also implicated in the packaging of RNPs into virions (14, 17, 36, 47). The mRNAs of bunyaviruses are distinct from their cellular counterparts in that although they contain a 5⬘ cap structure, acquired from host cell mRNAs by means of cap snatching, they do not have a poly(A) tail (13, 38, 39). Despite this difference, viral mRNAs are efficiently translated. In cells infected with orthobunyaviruses and phleboviruses, marked shutoff of host protein synthesis is observed. Shutoff has been mainly attributed to the transcription-inhibiting action of the NSs protein, though recombinant NSs-deficient orthobunyaviruses still cause some level of inhibition (5, 6, 43). What is less
The majority of cellular mRNAs possess a 5⬘ 7-methylguanosine cap and a 3⬘ polyadenosine [3⬘ poly(A)] tail. Efficient translation is achieved by the binding of various scaffolding proteins to these structures in order to mediate a closed-loop formation that allows the rapid reemployment of released ribosomal subunits. Major proteins involved in this process are the cap-binding eukaryotic initiation factor 4E (eIF4E), the poly(A)-binding protein (PABP), and eukaryotic initiation factor 4G (eIF4G), which bridges the interaction between eIF4E and PABP (22). Several viruses with a positive- or double-stranded RNA genome, however, have evolved distinct mechanisms for translation initiation. Members of the Picornaviridae and Flaviviridae families encode cap-less mRNAs containing 5⬘ internal ribosomal entry site sequences that depend on only a subset of the factors required for cap-dependent initiation (26). The mRNAs of caliciviruses are also noncapped but have a covalently linked viral protein (VPg) at their 5⬘ end that mediates the interaction with the cap-binding factor eIF4E (19, 23). An alternative or sometimes complementary strategy is the functional replacement of the poly(A) tail by a highly structured 3⬘ untranslated region (3⬘ UTR). Translation-enhancing activity has been demonstrated for the 3⬘ UTRs of flaviviruses, such as dengue virus, rotavirus, and many plant-infecting positive-strand RNA viruses (12, 15, 25, 45). In rotavirus-infected cells, the virus-encoded nonstructural NSP3 protein links the 3⬘ UTRs of viral mRNAs to eIF4G for efficient translation (45). Alternative strategies are often observed in viruses that have measures to interfere with the normal translation process. Po* Corresponding author. Mailing address: Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom. Phone: 44 1334 463396. Fax: 44 1334 462595. E-mail:
[email protected]. 䌤 Published ahead of print on 4 February 2009. 3637
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understood is how the efficient translation of poly(A) tail-less bunyavirus mRNAs is achieved. Here we investigated whether the viral UTRs influence translation efficiency by using model virus-like mRNAs that mimic the BUNV S-segment mRNA. MATERIALS AND METHODS Cells and viruses. HeLa cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and BHK-21 cells were kept in Glasgow minimum essential medium (GMEM) containing 10% newborn calf serum and 5% tryptose phosphate broth. Wild-type (wt) BUNV and rBUNV delNSs2, a recombinant virus that does not express NSs (21), were grown as described previously (46). Plasmid constructs. All plasmids were constructed using standard molecular cloning methods (3) and were confirmed by nucleotide sequencing. BUNV S-segment and globin gene untranslated regions were synthesized by PCR amplification of two partly overlapping primers. The primers were designed based on GenBank entries with accession numbers 28302128 (human beta-globin gene) and 9630660 (BUNV S-genome segment). The UTRs were inserted appropriately on either side of the Renilla luciferase gene into the NheI (5⬘ position) or XbaI (3⬘ position) restriction site of the expression vector phRL-CMV (Promega). Reverse primers for 3⬘ UTRs introduced a downstream SmaI restriction site. All primer sequences can be retrieved from the authors upon request. Plasmid phRL-CMVrev contains the Renilla luciferase open reading frame (ORF) in negative sense and was created by reversing the direction of the ORF in plasmid phRL-CMV. To this end, phRL-CMV was digested with restriction enzymes NheI and XbaI in order to excise the Renilla ORF. Both fragments, the vector backbone and Renilla ORF, were agarose gel purified, and the 5⬘ overhangs were filled in with T4 DNA polymerase (Promega). Finally, the two fragments were religated using the rapid dephosphorylation and ligation kit (Roche) and transformed into competent Escherichia coli. The resulting transformants were screened for negative orientation of the Renilla ORF. Plasmid pcDNA-PABP expressing myc-tagged PABP was kindly provided by Nicole Dalla Venezia (11). In vitro transcription. Plasmid phRL-CMV and derivatives containing BUNV or human beta-globin UTRs were doubly digested with either a combination of HindIII/XbaI (phRL-CMV and 5⬘ UTR-containing plasmids) or HindIII/SmaI (3⬘ UTR-containing plasmids) to generate fragments that start with the T7 RNA polymerase promoter and terminate after the Renilla luciferase ORF or inserted 3⬘ UTR, respectively. Possible RNase contamination was removed by adding 10 g proteinase K to the reaction mixture and incubating the mixture at 37°C for 1 h. One microgram of purified DNA was used in the mMessage Machine in vitro transcription kit (Ambion) for the synthesis of 5⬘ capped, poly(A) tail-less mRNAs. The template DNA was later degraded by the addition of 2 U Turbo DNase according to the manufacturer’s protocol. Where desired, a poly(A) tail was added to the RNA transcripts using the Poly(A)-Polymerase kit (Ambion). Transcripts were purified with RNeasy quick spin columns (Qiagen). Luciferase reporter assays. Subconfluent layers of BHK-21 cells, or HeLa cells with either PABP or eIF4G silenced, were transfected with 0.5 g in vitrotranscribed RNA using 2 l Lipofectamine 2000 (Invitrogen) in 200 l OptiMEM (Invitrogen). The cells were lysed 12 to 48 h posttransfection, as described in Results, and assayed for reporter activity using the Renilla luciferase reporter assay kit (Promega). Northern blotting. Approximately 8 ⫻ 105 BHK cells per cavity were seeded in six-well plates and immediately transfected with 1.5 g of the indicated mRNAs in 500 l OptiMEM containing 4.5 l Lipofectamine 2000. The transfection mix was replaced 4 hours later with GMEM supplemented with 10% newborn calf serum and 8% tryptose phosphate broth, and total RNA was harvested at 4, 8, 12, and 24 h posttransfection using the RNeasy kit (Qiagen). The harvested RNAs (2 g) were denatured in loading buffer containing 60% formamide (Sigma), separated by electrophoresis in a 1.2% Tris-acetate-EDTA (TAE) agarose gel and blotted onto a positively charged nylon membrane (Roche) (see reference 32 for details). Membranes were hybridized with digoxigenin-labeled probes complementary to either Renilla luciferase (transcribed using plasmid phRL-CMVrev as template) or actin mRNAs and developed as described in the manufacturer’s protocol (Roche). Posttranscriptional gene silencing. Approximately 2 ⫻ 105 HeLa cells were seeded in 24-well plates and grown overnight. For the knockdown of PABP, cells were transfected with 60 pmol of either short interfering RNA (siRNA) duplex siPABP#1 or control mismatch siRNA siPABP#2 INV (48) using 3 l Lipofectamine 2000 in 100 l OptiMEM. For the knockdown of eIF4G, cells were transfected with 1 g of either a plasmid that encodes the eIF4G-specific siRNA
J. VIROL. si31 or one that encodes the mismatch control siRNA si31M (9) kindly provided by Simon Morley, University of Sussex, United Kingdom, using 3 l of Lipofectamine 2000 in 100 l OptiMEM. The cells were trypsinized 24 h posttransfection, seeded into 12-well plates, and used for luciferase reporter assays 24 h later. Western blotting. Knockdown of PABP and eIF4G was confirmed by Western blotting. HeLa cells with either PABP or eIF4G silenced were lysed 62 h posttransfection in 100 l RIP buffer (50 mM Tris-HCl [pH 7.4], 5 mM EDTA, 300 mM NaCl, 1% Triton X-100) containing 25 units/ml Benzonase (Novagen) and Complete protease inhibitor cocktail (Roche). Lysates were separated on a 4 to 12% gradient Novex sodium dodecyl sulfate (SDS)-polyacrylamide gel (Invitrogen) and transferred onto a polyvinylidene difluoride membrane (Promega). Proteins were detected using antibodies against BUNV N protein, PABP (H-300; Santa Cruz), eIF4G (9) (kindly provided by Simon Morley), actin (I-19; Santa Cruz), or ␣-tubulin (clone B512; Sigma). Immunoprecipitation and mass spectrometry. BHK-21 cells were mock infected or infected with BUNV at a multiplicity of infection of 4 PFU per cell and incubated at 37°C. The cells were washed three times with phosphate-buffered saline (PBS) and resuspended in 0.5 ml per 106 cells of lysis buffer (40 mM HEPES [pH 7.9], 500 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM dithiothreitol) supplemented with Complete protease inhibitors (Roche) and 25 units/ml Benzonase (Novagen). Where indicated, lysates were treated with 20 g/ml RNase A (Ambion) at 37°C for 20 min. The lysate was incubated on ice with 65 l/ml Pansorbin (Calbiochem) to remove nonspecific binding factors and cleared by centrifugation at 10,000 ⫻ g for 30 min. The lysate (1.5 ml) was added to 20 l of a 50% slurry of anti-PAPB or anti-BUNV N antibody bound to protein G-Sepharose beads and incubated in a tumbler for 4 h at 4°C. The beads were washed once in lysis buffer, three times in lysis buffer lacking Triton X-100, and once in PBS. The beads were resuspended in 30 l sample buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 200 mM dithiothreitol, 0.2% bromophenol blue), and boiled for 3 min, and 15 l was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. For identification of proteins by mass spectrometry, the gel was stained with Coomassie blue, bands of interest were excised from the stained gel, and subjected to in-gel digestion with trypsin. The resultant peptides were analyzed by nanocapillary liquid chromatography coupled to electrospray ionization and tandem mass spectrometry. The tandem mass spectrometry data file generated was analyzed using the Mascot 2.1 search engine (Matrix Science, London, United Kingdom) against the MSDB database (February 2007). Immunofluorescence. BHK-21 cells were seeded on glass coverslips in six-well plates. The cells were left overnight and then infected with 1 PFU per cell of either wt BUNV or rBUNVdelNSs2 or left uninfected (mock infected). At selected time points, cells were fixed in PBS containing 4% paraformaldehyde and subsequently permeabilized with PBS containing 0.5% Triton X-100. The cells were incubated with primary antibodies against BUNV N and PABP (clone 10E10; Sigma) followed by incubation with fluorescence-labeled secondary antibodies (Sigma). Coverslips were mounted on microscope slides in 10 l Mowiol, and samples were analyzed by confocal fluorescence microscopy (Zeiss LSM 5 Exciter).
RESULTS Bunyamwera virus S-segment UTRs mediate efficient translation of nonpolyadenylated mRNAs. The BUNV genomic S segment is 961 nucleotides (nt) long, with UTRs of 84 and 174 nt flanking the coding region. The S-segment mRNA contains a short (12- to 17-nt) capped, host cell primer sequence and the complete genomic 3⬘ UTR at its 5⬘ end (84 nt), whereas the 3⬘ end of the mRNA is truncated at a signal 100 to 110 nt from the 5⬘ end of the template RNA, and is not polyadenylated (27). In order to clarify a possible role of the UTRs in translation, we inserted BUNV S nt 1 to 84 and 788 to 861 into the plasmid phRL-CMV to flank the luciferase reporter gene, thereby mimicking the viral S-segment mRNA. The resulting plasmid phRL-BUN served as the template to produce a capped, nonpolyadenylated virus-like mRNA in vitro, which was termed BUN-REN. Similarly, mRNAs were synthesized that either contained the UTRs of the human beta-globin mRNA (GLO-REN) or solely encoded the Renilla
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FIG. 1. Translation efficiency of nonpolyadenylated mRNAs. (A) Luciferase activity in BHK-21 cells. Cells were transfected with 0.5 g in vitro-transcribed, 5⬘ capped but nonpolyadenylated mRNAs, and 18 h later, the cells were lysed and assayed for luciferase activity. The black circles represent 5⬘ cap structures. (B) Stability of reporter signals. BHK-21 cells were transfected as described above for panel A, and cells were lysed at 12-h intervals over the course of 48 h and assayed for luciferase activity. The error bars show the standard deviations from the means for three replicate experiments. (C) Stability of transfected mRNAs. Northern blot analysis of total RNA (2 g/lane) extracted from transfected BHK-21 cells at the indicated time points (in hours after transfection). Renilla luciferase and actin mRNAs were detected with the respective digoxigeninlabeled probes. u, untransfected control.
luciferase sequence (CRL-REN) as illustrated in Fig. 1A. BHK-21 cells were transfected with the described mRNAs, lysed 18 h later, and assayed for Renilla luciferase activity, which reflects the translational capacity of the mRNAs. As expected, the capped but nonpolyadenylated Renilla luciferase mRNA (CRL-REN) was only poorly translated (Fig. 1A). The inclusion of the beta-globin UTRs (GLO-REN) resulted in a 10-fold increase in luciferase activity. Surprisingly, RNA con-
taining BUNV-derived UTRs mediated ⬎100-fold-stronger reporter signal (BUN-REN) than CRL-REN did. The observed increase of translational activity of nonpolyadenylated mRNAs conferred by the BUNV UTRs could reflect improved translation initiation, prolonged RNA stability, or a combination of the two. Time course experiments were conducted to test whether the BUNV UTRs had any effect on the longevity of the luciferase signal. As shown in Fig. 1B, the
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FIG. 2. Influence of polyadenylation on translation. (A) Agarose gel electrophoresis of in vitro-transcribed, nonpolyadenylated mRNA (⫺) and polyadenylated mRNA (⫹). m, size markers in bases. (B) Luciferase activity in transfected cells. BHK-21 cells were transfected with 0.5 g of either nonpolyadenylated or polyadenylated (AAAA) transcripts as indicated and lysed 18 h later, and Renilla luciferase activity was determined. The error bars show the standard deviations from the means for three replicate experiments.
reporter activities of all three types of nonpolyadenylated mRNAs decayed with similar rates, suggesting that BUNV UTRs do not confer enhanced RNA stability. This was further investigated by direct analysis of RNA stability by Northern blotting (Fig. 1C). There was a decrease in hybridization signal over time with all three transfected mRNA transcripts, and the BUNV-like mRNA appeared to be the least stable. Over the same time period, actin mRNA transcript levels appeared not to change. Taken together, these data suggest that the BUNV UTR sequences enhanced translation rather than merely conferring increased stability on the transfected RNA. Most cellular mRNAs contain a 3⬘ poly(A) tail up to several hundred nucleotides long that greatly enhances translation efficiency by supporting a closed-loop formation as well as increasing mRNA stability. We added poly(A) tails in vitro to all three mRNA species described above to determine their influence on translation; as seen in Fig. 2A, the electrophoretic migration of polyadenylated RNAs was significantly slower than that of unpolyadenylated RNAs, and we estimate that 300 to 500 A residues were added. Polyadenylation dramatically increased the translational capacity of CRL-REN and GLOREN, resulting in reporter activity that was 50- to 100-fold higher (Fig. 2B). By contrast, the activity of BUN-REN mRNA was only moderately enhanced, showing a sixfold increase of luciferase activity. Polyadenylation of the mRNAs increased the stability of all three types of mRNA equally when analyzed
by Northern blotting (data not shown). These results suggest that the BUNV S-segment UTRs contain a translation-enhancing element (TEE) that mediates efficient protein synthesis in the absence of a poly(A) tail. Translation-enhancing activity resides within the 3ⴕ UTR. In order to determine whether the observed translational activity of the BUNV UTRs localized to the 5⬘ or 3⬘ UTR or was the result of a synergistic effect of both UTRs, single-UTRcontaining reporter constructs were produced, and their translational activity was measured in transfected BHK-21 cells. While the absence of the 5⬘ UTR (BUN-REN3⬘861) was only slightly detrimental to reporter activity, the lack of the 3⬘ UTR (BUN-REN5⬘) almost completely abrogated luciferase expression (Fig. 3A). We used the RNA-folding algorithm Mfold (49) to examine the secondary structure of the 3⬘ UTR of the BUNV S-segment mRNA. Three stem-loop structures were predicted in the 3⬘ UTR, two smaller ones at either end that flank a larger structure (Fig. 3B). The last third of the UTR is highly conserved among many orthobunyavirus S segments, as is the presence of a stem-loop structure, which has been suggested to have a role in translation (4). Indeed, we found that deletion of the last 20 nucleotides, which completely removes the stemloop, led to a greater than 90% loss of reporter activity (BUNREN3⬘841), demonstrating the importance of this region for translation enhancement. These findings indicate that the 3⬘ UTR alone is sufficient and necessary for efficient protein
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FIG. 3. Translational activity resides within the 3⬘UTR. (A) Effect of omitting 3⬘ or 5⬘ UTR sequences on translation efficiency. BHK-21 cells were transfected with 0.5 g of various capped, nonpolyadenylated mRNAs that either lack the complete 3⬘UTR (BUN-REN5⬘), the complete 5⬘UTR (BUN-REN3⬘861), or the 20 terminal nucleotides of the 3⬘UTR (BUN-REN3⬘841). The CRL-REN and BUN-REN RNAs were included as controls. Cells were lysed 18 h after transfection and assayed for Renilla luciferase activity. The black circles represent 5⬘ cap structures. The error bars show the standard deviations from the means for three replicate experiments. (B) Predicted secondary structure of the BUNV S-segment mRNA 3⬘UTR. The black lines at the bottom of the figure indicate the regions present in BUN-REN3⬘861 and BUN-REN3⬘841.
synthesis and that at least one structural element within this sequence contributes to the overall activity of this newly discovered TEE. Translation of BUNV-like mRNAs is independent of the poly(A)-binding protein. The interaction of PABP with the poly(A) tail of polyadenylated mRNAs is important for establishing a closed-loop formation, but this requirement is not absolute. For example, the rotavirus 3⬘ UTR binds to the viral NSP3 protein which mediates the interaction with eIF4G in order to circularize viral mRNAs, making PABP redundant (45). Therefore, we assessed the requirement of PABP and eIF4G for the translation of conventional and BUNV-like mRNAs in cells that were limited in expression of these proteins by posttranscriptional gene silencing. To this aim, HeLa cells were transfected with a short interfering RNA that specifically blocks the expression of PABP, while control cells
were transfected with a nonsilencing mismatch siRNA (48). The targeted downregulation of PABP was confirmed by Western blotting (Fig. 4B). As shown in Fig. 4A, depletion of PABP was detrimental only to the translation of polyadenylated mRNAs, leading to a 50% loss of reporter activity, but not for the nonpolyadenylated mRNA BUN-REN, which remained largely unaffected. However, if cells were silenced for the expression of eIF4G, the translation of all mRNA species was inhibited (Fig. 4). It should be noted that the results are expressed as relative rather than absolute luciferase activity, and as the activity of CRL-REN is extremely low (Fig. 1A), the assay is too insensitive to reliably measure the effects of eIF4G depletion. Thus, the results indicate that the translation of BUNV-like mRNAs seems to be independent of PABP. Bunyamwera virus inhibits the translation of polyadenylated mRNAs. We compared the performance of conventional
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FIG. 4. Translational requirement for PABP and eIF4G. (A) Effect of depletion of PABP or eIF4G.For the knockdown of PABP, HeLa cells were transfected with 60 pmol of either a PABP-specific siRNA (RNA interference [RNAi] PABP) or a nonsilencing mismatch siRNA (control). For the knockdown of eIF4G, cells were transfected with 1 g of a plasmid that expresses the eIF4G-specific siRNA si31. Control cells were transfected with a plasmid encoding the mismatch siRNA si31M. After 48 h, the cells were transfected with 0.5 g of the indicated mRNA. The cells were lysed 14 h later and assayed for Renilla luciferase activity. The average of two experiments is shown. (B) Western blot analysis of PABP and eIF4G expression. HeLa cells silenced as described in Materials and Methods were lysed 62 h after transfection, and proteins were separated by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. The blots were probed for either PABP or eIF4G. The blots were also probed for actin and tubulin as loading controls.
and virus-like mRNAs in BUNV-infected cells in order to test for a possible impact of virus infection on translation efficiency. BHK-21 cells were infected with wt BUNV or rBUNdelNSs2, a recombinant virus that lacks the NSs protein (21), and then transfected with the different mRNAs (capped, nonpolyadeny-
lated, and capped, polyadenylated mRNAs) 9 h later. After a further 18 h, luciferase activity was measured. The translational activity of polyadenylated mRNAs was greatly reduced in cells which were infected with wt BUNV, while the activity of nonpolyadenylated RNAs (which of course is very low any-
FIG. 5. Impact of virus infection on translation. BHK-21 cells were infected with either wt BUNV or rBUNVdelNSs2 at 5 PFU per cell or left uninfected (mock infected). After 9 h, the cells were transfected with 0.5 g of the indicated mRNA, lysed 18 h later, and assayed for luciferase activity.
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way; Fig. 1A) was much more robust (Fig. 5). Expression of the viral NSs protein, which inhibits host cell transcription (43), was not required for this effect, as translation of poly(A)containing mRNAs was also inhibited in rBUNdelNSs2-infected cells. These results suggest that BUNV suppresses the poly(A) tail-dependent translation of host cell mRNAs. On the other hand, virus infection did not enhance the translation of virus-like mRNAs, as similar levels of translation were seen in uninfected or virus-infected cells. Poly(A)-binding protein is not degraded in Bunyamwera virus-infected cells. Our findings that translation of virus-like mRNAs was PABP independent and that infection diminished the activity of polyadenylated mRNAs pointed to PABP as a possible target for BUNV-mediated inhibition of host cell protein synthesis. Cleavage of PABP by virus-encoded proteases has been demonstrated for enteroviruses, caliciviruses, and retroviruses (2, 29, 30). We therefore assessed PABP stability in BHK-21 cells infected with BUNV expressing or lacking NSs. However, neither at early (8 h) nor at late (24 h) time points after infection was a decrease of PABP apparent (Fig. 6A). Bunyamwera virus N protein binds poly(A)-binding protein. As part of a survey to catalogue cellular factors that interact with BUNV proteins, we have applied a proteomics approach to look for proteins that are coimmunoprecipitated by anti-N antibodies. Intriguingly, one of the proteins identified by mass spectrometry as an interacting partner of the nucleocapsid protein was PABP. This interaction was confirmed by specific coimmunoprecipitation studies using anti-PABP antibodies on both in vitro and in vivo overexpressed proteins (not shown) as well as in lysates of infected cells (Fig. 6B). Immunoprecipitation analysis after infection with wild-type BUNV or rBUNdelNSs2 showed N protein coprecipitated with PABP and vice versa. Treatment with RNase A prior to immunoprecipitation reduced but did not fully abolish the interaction, indicating that it may be partially RNA mediated. Bunyamwera virus infection triggers PABP nuclear accumulation. We performed immunofluorescence analyses on BUNVinfected BHK cells to further investigate the interaction of N with PABP. Poly(A)-binding protein is involved in the nuclear export of mRNAs and is known to shuttle between the cytoplasm and nucleus, though it has a predominantly cytoplasmic localization (1). Indeed, we observed that PABP resides almost exclusively in the cytoplasm of noninfected control cells (Fig. 7A). In wt BUNV-infected cells, cytoplasmic colocalization of N and PABP was observed at early time points (Fig. 7B), while at later times a dramatic nuclear redistribution of PABP was observed, though most of the N protein remained in the cytoplasm (Fig. 7C and D). The nuclear accumulation of PABP was faster and more pronounced in cells infected with wt BUNV than in cells infected with rBUNVdelNSs2 (Fig. 7E to G), indicating that the NSs protein contributes to this effect. However, nuclear redistribution of PABP was also observed in cells that were infected with the NSs-deficient virus, although only after a prolonged phase of cytoplasmic colocalization of N and PABP. These data indicate that the BUNV nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm and that BUNV infection leads to nuclear localization of PABP, thereby possibly interfering with poly(A) tail-dependent translation.
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FIG. 6. Interaction between BUNV and PABP. (A) PABP stability in infected cells. BHK-21 cells were infected with either wt BUNV or rBUNVdelNSs2 at 5 PFU per cell or mock infected as indicated. Cells were lysed 10 and 24 h postinfection (p.i.) and analyzed for expression of PABP, BUNV N, and tubulin by Western blotting. (B) Coimmunoprecipitation of N protein with cellular PABP. BHK-21 cells were infected with wt BUNV (wt) or rBUNdelNSs2 (del) or mock infected (m), and at 12 h postinfection, cell lysates were treated with RNase A (⫹) or not treated with RNase A (⫺), followed by immunoprecipitation (IP) with anti-PABP or anti-N protein antibodies. Precipitated proteins were analyzed by SDS-PAGE, followed by Western blotting (WB) and immunodetection with anti-PABP and anti-N antibodies as indicated.
DISCUSSION Numerous positive- and double-stranded RNA viruses transcribe nonpolyadenylated mRNAs that feature translation-enhancing 3⬘ UTRs, often combined with the ability to inhibit the translation of polyadenylated host cell mRNAs. BUNV possesses a tripartite negative-strand RNA genome, encodes mRNAs that lack a poly(A) tail, and exerts a pronounced shutoff of host cell gene expression (16). However, the mechanism that allows viral mRNAs to be efficiently translated in infected cells is poorly understood. Frugulhetti and Rebello (18) reported that in Marituba orthobunyavirus-infected L-A9 cells, there was an increase in intracellular Na⫹ ion and decrease in K⫹ ion concentration that favored translation of viral mRNAs, whereas cellular mRNA translation was inhibited. In this study we provide evidence that the 3⬘ UTR of the BUNV S-segment mRNA constitutes a translation-enhancing element that mediates efficient translation in the absence of a poly(A) tail and that
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FIG. 7. Nuclear retention of PABP. BHK-21 cells were infected with wt BUNV or rBUNVdelNSs2 at 1 PFU per cell or mock infected. At 4, 8, and 22 h postinfection, the cells were fixed and analyzed by indirect immunofluorescence using antibodies to BUNV N (green) and PABP (red).
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BUNV interferes with the translation of polyadenylated mRNAs by manipulating the subcellular localization of PABP. For the plant-infecting tomato spotted wilt virus, the prototype of the Tospovirus genus within the Bunyaviridae family, a TEE was also predicted in the S-segment UTR based on RNA structure (44). Translation enhancement of the model RNA by the BUNV S-segment 3⬘ UTR was found to be at the level of translation per se rather than increased stability of the mRNA (Fig. 1). Similar characteristics have been reported for the 3⬘-located TEE of dengue virus (7). The observed faster degradation of the transfected BUNV-like model mRNA (Fig. 1C) is in line with the reported instability of the La Crosse orthobunyavirus (LACV) S-segment mRNA in infected mammalian cells (41). The complex stem-loop-containing secondary structure of the BUNV S-segment TEE is reminiscent of translation-enhancing 3⬘ UTRs of many animal- and plant-infecting positive-strand RNA viruses (7, 25). The translation-enhancing activity of these TEEs often depends on a cooperative functioning of several structural elements (7, 42). Complete removal of a predicted stem-loop at the 3⬘ end of the BUNV S-segment TEE severely reduced translational activity. Previously, Barr et al. (4) found that the region encompassing this stem-loop is highly conserved among various orthobunyavirus S segments, and since mutations in this region did not affect the efficiency of transcriptional termination by a downstream termination sequence, the authors speculated that this structure might be important for translation. This hypothesis is now supported by our data. In contrast, efficient translation of rotavirus mRNAs is mediated by the terminal four-nucleotide sequence GACC that is present in all genomic segments (8). No analogous sequence could be identified in the BUNV genome, again pointing toward a structural basis for translation enhancement. A detailed structural-functional analysis of the BUNV S-segment 3⬘ UTR will assist in revealing the roles of the identified structural elements in the translation-enhancing capacity of this newly discovered TEE. Translation of rotavirus mRNAs is thought to require binding of the viral NSP3 protein to the 3⬘ UTR in order to mediate interaction with eIF4G, although this has been challenged recently (34, 40). The BUNV S-segment UTRs enhanced translation in uninfected cells, and no stimulation was observed in BUNV-infected cells, strongly suggesting that no viral protein is necessary for the translation-enhancing activity. In contrast, translation of virus-like mRNAs relied heavily on the presence of eIF4G but was independent of PABP, implying that the 3⬘ UTR either binds directly to eIF4G or to some yet-to-be identified cellular factor that interacts with eIF4G. Expression of cellular proteins is markedly decreased upon infection with many bunyaviruses. For the orthobunyaviruses BUNV and LACV, as well as the phlebovirus Rift Valley fever virus, shutoff is primarily mediated by the NSs protein which inhibits transcription of mRNAs by cellular RNA polymerase II (RNAPII) (5, 6, 43). However, the NSs proteins of San Angelo orthobunyavirus and BUNV can also inhibit translation in Xenopus oocyte extracts and rabbit reticulocyte lysate systems, respectively (10, 21). Here we demonstrate that BUNV specifically inhibits translation of polyadenylated mRNAs, most likely by causing nuclear retention of PABP, thereby preventing it from assisting in the closed-loop formation. Nuclear redistribution of PABP occurred faster and
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was more pronounced in cells that were infected with NSsexpressing wt BUNV, implying that NSs plays some role in this process. Interestingly, nuclear accumulation of PABP can also be achieved by treatment of cells with chemical inhibitors of transcription like 5,6-dichlororibofuranosylbenzimidazole (1). Transcription inhibition presumably confines PABP to the nucleus through a lack of newly synthesized polyadenylated mRNAs with which the protein interacts for their conjoined translocation to the cytoplasm (31). Thus, the NSs-mediated nuclear retention of PABP is perhaps a secondary effect of its transcription-inhibiting effect on RNAPII. Our findings that virus lacking NSs was still able to inhibit translation of polyadenylated mRNAs and that this virus could also induce nuclear accumulation of PABP suggest that other viral proteins besides NSs take part in the downregulation of translation. Recombinant BUNV and LACV that lack NSs are still able to impose a moderate shutoff (5, 6). A recent study by Hart et al. (21) demonstrated that the individually expressed L and N proteins also exert some level of inhibition of reporter gene expression, although to a lesser extent than that by NSs. The L polymerase cleaves 5⬘ cap structures from host cell mRNAs, which it uses to prime transcription of viral mRNAs (38). This leads to the mRNA instability that was suggested to contribute to host cell shutoff (41). We found that the BUNV N protein binds to and colocalizes with PABP in the cytoplasm, particularly in cells infected with rBUNVdelNSs2, hinting that N might participate in translation inhibition. Recently, Ilkow et al. (24) demonstrated that PABP is sequestered by the capsid protein of rubella virus, which results in a block in translation. By contrast, the NSP3 protein of rotavirus indirectly influences the nuclear accumulation of PABP (20, 35). Whether BUNV N induces nuclear targeting of PABP is currently unclear, as is the importance of the interaction of these proteins for the BUNV life cycle. It is conceivable that binding of PABP by N assists in transcription, e.g., by mediating access to cellular mRNAs which are required for cap snatching by the viral polymerase, as has recently been discovered for the hantavirus N protein (33). In conclusion, the host cell shutoff observed in BUNV-infected cells is apparently the cumulative effect of a transcriptional block of RNAPII mediated by NSs, the inhibition of translation by the actions of NSs, N, and L proteins, as well as competition for free ribosomes by abundant viral mRNAs. Efforts to dissect the roles of individual BUNV proteins in nuclear retention of PABP and downregulation of cellular protein translation are currently under way. In this paper we have characterized further the poly(A) tail-independent translation by Bunyamwera virus. Although the underlying molecular mechanisms might prove to be different, our results suggest that certain positive- and negativestrand RNA viruses share a common protein expression strategy that is characterized by the establishment of a powerful host cell shutoff that is circumvented by viral mRNAs which adopt alternative ways for efficient translation. ACKNOWLEDGMENTS We thank Nicole Dalla Venezia, Simon Morley, Virginia Pain, and Xhiahong Shi for providing reagents and cell lines that were pivotal to this study. We thank Catherine Botting for mass spectrometry and Lucy Hill for technical assistance. This work was funded by Wellcome Trust grants to R.M.E.
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