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

Abstract

Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.

Fig 1.

Mapping of long-distance frameshift element in vitro. (A) BYDV genome organization. Frameshift elements are shown as gray boxes; numbering indicates base positions (nt) in BYDV genome. (B) Wheat germ translation products of viral transcripts. PAGE and quantification of frameshifting are described in Materials and Methods. Frameshift efficiencies (% fs) are shown under each lane. Translation of capped transcripts from wild-type SmaI-linearized pPAV6 (lane 1), and mutant containing only bases 5,050–5,283 downstream of the BamHI₄₈₃₇ site (lane 2). Lanes 4–6, translation of uncapped transcripts from SmaI or PstI-cut pPAV6 or from PstI-cut pPAV6K5050–5283 that contains bases 5,050–5,283 in the KpnI₄₁₅₄ site. Mobilities of the products of BYDV ORF1 (39 kDa), ORF 1 + 2 (99 kDa), respectively, and Brome mosaic virus (BMV) RNA translation products (lane 3) are marked.

Fig 2.

Frameshifting in the dual luciferase vector. (A) Map of vector showing predicted BYDV RNA secondary structure around the shifty site inserted between the two LUC ORFs. The BYDV 3′ UTR is inserted after fLUC. BYDV sequences are represented by black boxes. Frameshift rates (±SD) in oat protoplasts (in vivo) were calculated as described in Materials and Methods. The percentage of frameshifting is calculated by using the DL-PAV206 control value for that individual assay. All constructs were tested in triplicate at least twice except for DL-PAV-SL, which was tested once. (B) In vitro translation products of dual luciferase constructs with different shifty site sequences. The rLUC (40 kDa) and rLUC-fLUC (100 kDa) products are marked. DL-PSHSIL206 has a disrupted shifty site (see Materials and Methods). Both LUC ORFs are fused in the same frame in DL-PM4 206.

Fig 3.

Frameshifting in oat protoplasts of BYDV 3′ end deletion mutants. All deletion constructs are in the DL-PAV206 SmaI-linearized background. Results are graphed as the % of the frameshift efficiency (fs) of DL-PAV206 (fs = 1.10%; Fig. 2A), which contains the full-length 3′ UTR (nt 4,809–5,677). Each sample was tested in triplicate two independent times; error bars represent the SD.

Fig 4.

Frameshifting and replication of BYDV mutants that destroy and restore the long-distance base pairing. (A) Predicted secondary structures (MFOLD) of BYDV and the related SbDV RNAs showing the base-pairing interaction (boxed in BYDV structure) of the long-distance frameshift element (LDFE) with the bulge in the adjacent downstream stem loop (ADSL bulge). (B) Frameshift efficiencies of dual luciferase constructs with mutations in ADSL bulge and/or LDFE that disrupt and restore potential base pairing. Frameshift efficiencies are averages from three independent experiments. The nucleotide sequence for the two regions for each mutant is shown with altered bases in bold. (C) PAGE of wheat germ translation products of BYDV dual luciferase (100-kDa frameshift product) and of full-length viral (PAV6) constructs containing the same sets of two-base mutations in the ADSL and/or LDFE. (D) Northern blot hybridization of RNA from oat protoplasts inoculated with the same wild-type and mutant PAV6 transcripts used in C. BYDV genomic (gRNA) and subgenomic RNAs (sgRNA) produced during virus replication are marked. (Lower) Ethidium bromide staining of gel used for the above blot as a control for gel loading. This experiment was repeated three times with the same results.

Fig 5.

RNA traffic signal hypothesis for regulation of BYDV translation and replication. Black line indicates BYDV genomic RNA showing relevant secondary structural elements with key regions in color (not to scale). Black boxes indicate ORFs 1 and 2. For simplicity, other ORFs, nascent proteins, and (−) strand RNA are not shown. (A) Base pairing between the 3′ TE and 5′ UTR (magenta) (25) and between the LDFE and ADSL (gold) allow ribosomes (gray ovals) to translate ORF 1 and ORF 2. (B) As the viral RdRp molecules (blue spheres) accumulate, they initiate (−) strand synthesis at the 3′-terminal structure (46). They proceed upstream along the template and melt out the ADSL:LDFE base pairing. This blocks frameshifting (vertical bars), clearing ORF 2 of ribosomes, briefly allowing translation of ORF1 only. (C) Next, the RdRp reaches the 3′ TE and disrupts the 3′TE:5′ UTR base pairing, preventing translation initiation. This clears ORF1 and the entire RNA of ribosomes, allowing the RdRps to continue to the 5′ end. Subsequent rounds of replication would cause (+) strand RNA to accumulate in excess of RdRp molecules, returning the process to A as shown.