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Biochemistry
A –1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA
Plant Pathology Department, Iowa State University, Ames, IA 50011
Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved June 14, 2002 (received for review April 12, 2002)
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Abstract |
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Abbreviations: BYDV, Barley yellow dwarf virus; ADSL, adjacent downstream stem loop; LDFE, long-distance frameshift element; WGE, wheat germ extract; UTR, untranslated region; RdRp, RNA-dependent RNA polymerase
The mechanism by which the mRNA interacts with the ribosome to bring about a frameshift remains a mystery. Somehow, the tRNAs in the ribosomal A and P sites simultaneously slip back one base on the mRNA before (5) or after (3) peptide bond formation, and translation resumes in the new (–1) reading frame. This requires pausing of the ribosome induced by the adjacent pseudoknot in the mRNA (10, 11). A long-standing enigma has been the observations that only certain pseudoknots facilitate frameshifting, whereas other very similar structures of comparable stability do not cause frameshifting (12–14), even though they induce pausing (15). Other elements, such as downstream stop codons and the RNA sequences surrounding the shifty site, can influence the effectiveness of these signals (16). Mutations in ribosomal proteins (17), translation factors (18), and nonsense-mediated decay factors (19) can alter frameshift efficiency. Their precise roles are unknown.
The true contributions of the above cis and trans acting components to
frameshifting in the context of the virus in its host can be difficult
to assess, as most studies rely on reporter plasmids using small
portions of viral sequence. Yet, viral gene expression and replication,
can be controlled by long-distance interactions on viral RNAs (20, 21).
In poliovirus (22) and bacteriophage Q
(23) RNAs, replicase binds
far upstream of the 3' end, shutting off translation, and is delivered
to the 3' end, where replication initiates, by protein–RNA
interactions in poliovirus (20) or long-distance base pairing in Q
RNA (24). These long-distance interactions allow these RNA viruses to
switch between replication and translation, events that are
incompatible on the same RNA. How other viruses regulate this switch is
poorly understood. Barley yellow dwarf virus (BYDV) uses
multiple long-distance interactions to regulate cap-independent
translation initiation (25), ribosomal frameshifting (26), and leaky
termination (27). The cap-independent translation element (3' TE) in
the 3' untranslated region (UTR) must form base pairs with a
stem loop in the 5' UTR to facilitate translation of this naturally
uncapped mRNA (25). The effects of such interactions would be missed in
studies that examine the translation signals out of their natural
sequence context.
ORF 2, which codes for the RNA-dependent RNA polymerase (RdRp) of BYDV, is expressed via –1 frameshift from the overlapping ORF 1 (Fig. 1A). Frameshifting takes place at the shifty site, G GGU UUU, yielding the 99-kDa fusion product (P1-2) of the 39-kDa ORF 1 product (P1) and 60-kDa ORF 2 product (28, 29). Beginning 6 nt downstream of the shifty heptanucleotide, a large bulged stem-loop structure is predicted (29). An additional sequence located 4 kilobases downstream in the 3' UTR of BYDV genomic RNA is necessary for frameshifting in wheat germ extract (WGE) (26). This includes a 50-base essential "core" element (nt 5,050–5,100) and an adjacent "enhancer" region (BYDV nt 5,101–5,283) that, when deleted, causes a 50% decrease in frameshifting (26). Here, we use a dual luciferase reporter system (30) to map the distant downstream element at higher resolution in vivo and in vitro. We find that an intramolecular association of this element with the bulge of the stem loop at the frameshift site via base pairing is required. We propose a role for this remarkable new type of frameshift signal, along with the long-distance cap-independent translation base pairing, in an elegant mechanism for regulating translation and replication.
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Materials and Methods |
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The 3' UTR mutations were generated by using full-length BYDV genomic clone pPAV6 as template (28). Deletions were generated via the ExSite PCR-based site-directed mutagenesis kit (Stratagene). The forward and reverse primers contain an NcoI site at either the 3' or the 5' end of the deletion desired. The PCR reaction was cleaved with NcoI, and then the two ends were ligated. The region between BamHI4837 and SmaI5677 sites was sequenced before ligation into the dual luciferase vector by using these sites. Point mutations were introduced by using the Quick Change method (Stratagene). Forward and reverse primers containing the desired mutations were used on pPAV6 template. Mutations were sequenced and cloned into the dual luciferase vector as described above.
Insertion mutants were generated by amplifying the BYDV sequence using a forward primer with BamHI and a reverse primer with SmaI sites at their 5' termini to produce the region of interest from pPAV6. For the insertion mutants into the KpnI site, the forward and reverse primers both contained a KpnI site. These fragments were generated by using PCR, gel purified, and ligated into pPAV6 that was digested with the same enzymes.
In Vitro Transcription. Uncapped and capped BYDV-derived RNAs were transcribed by using the Megascript and mMessage mMachine kits (Ambion, Austin, TX), respectively, from pPAV6 or its derivatives cleaved with SmaI (3' end of BYDV) or other indicated restriction enzymes. Dual luciferase vector transcripts were synthesized in the same manner but from plasmid cleaved with NheI, which retained the A60 tail. PstI-linearized DNA was filled in with Klenow polymerase fragment before transcription.
In Vitro Translation. In vitro translation was performed in WGE (Promega) as described previously (26). Bands were quantitated by using a STORM PhosphorImager and IMAGEQUANT software (Molecular Dynamics). To correct for the number of methionines in each product, the frameshift efficiency for the BYDV constructs was calculated as [(99-kDa counts/28 Met)/(99-kDa counts/28 Met + 30-kDa counts/10 Met)] x 100, and the dual luciferase constructs were [(100-kDa counts/22 Met)/(100-kDa counts/22 Met + 40-kDa counts/9 Met)] x 100.
Translation and Replication in Protoplasts. For luciferase assays, oat protoplasts were prepared from suspension cells, electroporated with 15 µg of RNA transcripts, and harvested after 4 to 6 h (25, 26). Cells were lysed in 100 µl of passive lysis buffer by shaking for 15 min at room temperature and spun to remove debris. Ten-microliter aliquots of supernatant were assayed by using the Stop and Glo dual luciferase kit (Promega). All mutants were tested in triplicate in the wild-type, in-frame, and out-of-frame vectors. The percent frameshifting was calculated by determining the fLUC/rLUC ratio for all constructs and then dividing this number by the ratio for the positive control (the same mutation cloned into the dual luciferase vector with the in-frame, GGGUUUCU shifty site). The results were compared with the negative control (dual luciferase vector with the nonshifting CGGCUUC sequence in the shifty site), which was around 20% of the wild-type frameshift signal. The percent frameshifting for the mutants was then reported as that compared with the wild-type control run on the same day. For RNA replication assays, protoplasts were electroporated with BYDV transcripts as above. After 24 h, total RNA was extracted and analyzed by Northern blot hybridization (26).
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Results |
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To determine whether it was simply proximity to the 3' end that reduced frameshifting, the 5,050–5,283 sequence was inserted in the unique KpnI4154 site in the expendable ORF 5 of the full-length BYDV transcript (Fig. 1B, PAV6K5050–5283). The transcript was truncated at the PstI5009 site (nt 5,009) leaving 850 nt of viral sequence (unnecessary for frameshifting; ref. 26) downstream of the ectopic 5,050–5,283 insertion in the KpnI4154 site. This truncated RNA frameshifted about as efficiently as wild type (Fig. 1B, lane 6). As a control, PstI truncation was shown to abolish frameshifting in the wild-type PAV6 RNA (Fig. 1B, lane 5). Therefore, the region between 5,050–5,283 contains the complete long-distance frameshift element (LDFE) necessary for frameshifting in vitro; however, it is less effective when located at the very 3' end.
Sequences Upstream and Downstream of the Shifty Site Are Necessary for Frameshifting in Vivo. For high resolution mapping of the frameshift signals in vivo and in vitro, we used a dual luciferase reporter that allows precise frameshift measurement. The dual luciferase reporter contains a Renilla luciferase (rLUC) gene in the zero frame, followed by a firefly luciferase (fLUC) ORF in the –1 frame, with the candidate frameshift sequence inserted between two reporters (30). The fLUC/rLUC ratio reveals the frameshift efficiency (Fig. 2A). We inserted the shifty site, the proposed adjacent downstream stem loop (ADSL), and substantial adjacent viral sequence upstream of the shifty site between the rLUC and fLUC ORFs (nt 1,044–1,250). Two additional regions of BYDV sequence, the 5' UTR (nt 1–152) and the 3' UTR (nt 4,908–5,677), were included so that the resulting transcript, DL-PAV206, had all of the UTR sequences necessary for cap- and poly(A)-independent translation (25). The missing viral RNA, bases 152–1,046 and 1,648–4,919, do not play a role in translation initiation or frameshifting in vitro (26, 32).
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A series of deletion mutants within the region spanning nt 5,016–5,426 was constructed in the dual luciferase vector to more precisely map the LDFE in vivo (Fig. 3). Deletion of nt 5,047–5,099 reduced frameshifting to background levels (about 0.2% frameshifting or 20% of that obtained with the full-length UTR). Deletions in nt 5,168–5,279 gave 30–50% of wild-type frameshifting. The sequence between nt 5,091–5,168 is not necessary for frameshifting. Thus, the essential core and enhancer sequences are two separate elements. Furthermore, in vivo, additional sequence was required beyond what is necessary for frameshift enhancer activity in vitro. Sequence between 5,319 and 5,355 was necessary for full frameshifting in vivo (Fig. 3), but no sequence downstream of nt 5,283 was necessary in vitro (26).
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Base Pairing Between the Adjacent Downstream Stem Loop and the Long-Distance Frameshift Element Is Essential for Viral RNA Replication. To test the role of the long-distance base pairing in the actual context of the replicating virus, we assayed the ability of full-length infectious BYDV RNA with altered frameshift elements to replicate in oat protoplasts. The frameshift is essential for BYDV replication because frameshifting is necessary for translation of the viral RdRp (26, 32). One constraint for mutating the ADSL RNA sequence is that the resulting alteration in the amino acid sequence of the RdRp could reduce its activity, independent of frameshift efficiency (32). To minimize this possibility, replication was tested only for the ADSL constructs with two-base mutations (PAV6 PT1230, PAV6 PT1230/PT5066). The PT1230 mutation introduces a relatively conservative change of serine to threonine in the RdRp, whereas the PT5066 mutation is in the UTR. These mutations had the same effects on frameshifting of full-length viral genomic RNA as on the reporter constructs in vitro (Fig. 4C). In protoplasts, the PAV6-derived transcripts with either of the two-base mutations did not replicate, as revealed by Northern blot analysis of viral RNA accumulation at 24 hpi (Fig. 4D, PAV PT1230 and PAV PT5066). In contrast, the double mutant, which restores the base-pairing interaction, replicated at wild-type levels (Fig. 4D, PAV PT1230/PT5066). These results demonstrate that the long-distance base pairing is required in the natural viral RNA replication process, as predicted by its essential role for frameshifting.
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Discussion |
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The frameshift enhancement by the sequence upstream of the shifty site (nt 1,044–1,139) in the dual luciferase reporter system (Fig. 2A) was unexpected. However, the eight bases immediately upstream of the HIV-1 and HTLV-2 shifty sites are known to affect frameshifting (16). The stem loop predicted by MFOLD (33) immediately upstream of the shifty site is conserved in all members of the Luteovirus and Dianthovirus genera. These upstream stem loop(s) may slow the ribosome in advance of the shifty site to enhance frameshifting. Alternatively, they may serve as "insulators" to prevent improper folding of the shifty site or ADSL with upstream sequences.
Interestingly, a bulged stem loop very similar to the ADSL of BYDV RNA stimulates Red clover necrotic mosaic dianthovirus (RCNMV) RNA frameshifting, in the absence of its viral 3' UTR, in rabbit reticulocyte lysates (8) and in dual luciferase reporter transcripts in WGE (J.K.B., unpublished results). However, whether the RCNMV 3' UTR further stimulates frameshifting remains to be tested. By analogy, in HIV RNA, an ADSL was thought to be sufficient to stimulate frameshifting (35), but for full frameshifting, the ADSL must interact with nearby downstream sequence to form a novel pseudoknot with a base triplex in the stem (9). Potential for long-distance base pairing between a 3' UTR element and the bulged stem loop adjacent to the frameshift site is conserved in SbDV (Fig. 4A) and in genus Dianthovirus, including RCNMV (not shown). The predicted base pairing of the SbDV putative LDFE to the ADSL bulge is long (7 bp) and GC-rich, but the predicted distant "stem" in SbDV is so weak (G:U-rich) that it may not exist (Fig. 4A). Nevertheless, this phylogenetic conservation supports a functional role for the long-distance base pairing.
The Long-Distance Loop:Bulge Base Pairing Is a Previously Uncharacterized Frameshift Structure. Gene 10 of bacteriophage T7 provides another example of –1 frameshifting stimulated by an element beyond the immediate vicinity of the frameshift site. It requires a sequence located 200 bases downstream of the shifty site that is predicted to form a stem loop (36). However, it is the sequence in this region and not predicted secondary structure that stimulates frameshifting. Perhaps the structures most similar to that of BYDV are found in Rous sarcoma virus (37) and gill-associated virus (38) RNAs. As in BYDV RNA, these structures consist of a long, bulged ADSL and a downstream region base-paired to a bulge in the ADSL. Unlike BYDV, the downstream element is not a stem loop; it is only 12–42 nt downstream of the ADSL, and the bulge to which its base pairs is on the 5' rather than the 3' side of the ADSL. A example of a different kind of recoding that is facilitated by a sequence in the 3' UTR is the incorporation of the amino acid selenocysteine at UGA codons (39). However, that interaction uses specialized translation elongation factors (39) rather than long-distance base pairing. Thus, the bulge-loop:stem-loop interaction in BYDV RNA is a different class of long-distance recoding element.
How RNA structures stimulate ribosomes to change frame is unknown, but one of their key roles in frameshifting is to pause the ribosome over the shifty site (11, 15). The properties that determine whether a structure stimulates frameshifting are unclear. A comparison of the high-resolution structures determined for the MMTV (40), BWYV (41), and other frameshift pseudoknots reveals no tell-tale structural features (4). It has been proposed that frameshift efficiency is determined by the ability of the pseudoknot to resist the ribosomal helicase activity during translation elongation (4, 9, 14). Yusupova et al. (42) propose that three ribosomal proteins surrounding the mRNA entry site into the 80S complex bind helical regions on the mRNA and separate the strands as the mRNA enters the ribosome. Therefore, the authors speculated that frameshift-stimulatory structures preclude efficient interactions with these helix-unwinding proteins. The bulge-loop:stem-loop helix formed by the long-distance interaction in BYDV RNA may coaxially stack with (one of) the flanking helices in the ADSL, stabilizing the long-distance base pairing, and forming a stable, kinked, helical structure that proves awkward for the ribosome to unwind. This could facilitate frameshifting by the above mRNA–ribosome interaction.
A Potential Translation–Replication Switch. The location of both the cap-independent translation initiation element and the frameshift element far downstream of the translated genes provides advantages for the virus. Owing to their location in the UTR, they are unfettered by protein coding constraints. Second, this location would prevent unproductive translation of 3' truncated mRNA that is either partially degraded or not fully synthesized. Most interestingly, a requirement for base pairing of 3' UTR elements to upstream sequences for translation (25) may provide a novel mechanism to prevent collisions between ribosomes and the replicase. Translating ribosomes block replicase molecules moving in the opposite direction on the same template RNA (22, 43). Poliovirus avoids this event as its replicase first binds the viral 5' UTR to shut off translation and is then delivered to the 3' end via protein–protein interactions to initiate (–) strand synthesis (20). According to our model (Fig. 5), early in infection newly translated replicase binds the 3' end of viral RNA and proceeds through the 3' UTR toward the 5' end as it synthesizes (–) strand. Upon reaching the LDFE and the 3' TE, replicase disrupts both sets of long-distance base pairing, thus blocking frameshifting and translation initiation. These events would clear ORF 2 and then ORF 1 of ribosomes, allowing replicase to complete full-length (–) strand synthesis from a ribosome-free (+) strand. As replicase subsequently produces (+) strand genomic RNA copies from the (–) strand template, eventually (+) strands would outnumber replicase molecules and be free to form the long-distance base-pairing interactions necessary for translation initiation and frameshifting, and the cycle would repeat. This model provides an elegant means to allow simultaneous replication and translation while avoiding nonproductive collisions between replicase and ribosome. This model is consistent with our unexplained observation that capped BYDV transcripts are far less infectious than uncapped transcripts (44). Presence of a 5' cap would favor continuous translation, preventng the replicase from copying ribosome-laden viral RNA. This mechanism may serve instead of, or in addition to, subcellular vesicles that may separate the sites of translation and replication of some viruses, such as BMV (45). The fact that frameshifting would be disrupted slightly before initiation by the replicase hints that expression of the RdRp (frameshift product of ORFs 1 + 2) may be regulated differently from the product of ORF 1 alone.
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Acknowledgements |
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Footnotes |
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To whom reprint requests should be addressed. E-mail:
wamiller{at}iastate.edu.
This paper was submitted directly (Track II) to the PNAS office.
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