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. 2005 Jul;79(13):8303-15.
doi: 10.1128/JVI.79.13.8303-8315.2005.

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

Wei-Wei Chiu  1 , Richard M Kinney, Theo W Dreher
Affiliations

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

Wei-Wei Chiu et al. J Virol. 2005 Jul.

Abstract

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.

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Figures

FIG. 1.
FIG. 1.
Luciferase reporter constructs used for assaying the roles of flaviviral UTRs in regulating translational expression. (A) The 10.7-kb DEN2 genome is diagrammed with the 5′ cap (asterisk), 5′ UTR, the single long ORF, and the 3′ UTR that ends in a conserved stem-loop (SLA) and has no poly(A) tail highlighted. Distinctive features of the UTRs are indicated. Gray boxes indicate structural features that are conserved among mosquito-borne flaviviruses: 5′SL in the 5′ UTR (2), SLA and SLB at the 3′ end (3, 43), and the pseudo-repeated elements DB1 and DB2 further upstream in the 3′ UTR. DB1 and DB2, which resemble stalked dumbbells (36), include a conserved sequence (CS2, not shown). Black boxes indicate the conserved sequence elements, CS1 and cCS1 (16), that are complementary over 11 nucleotides. UVR represents the upstream variable region of the 3′ UTR that is not well conserved among flaviviruses. (B) LUC reporter mRNA constructs with DEN2, WNV, Sindbis virus (Sin), or rabbit α-globin UTRs. All pUC-based LUC reporter constructs have the upstream NotI site and T7 promoter (black arrowhead) and downstream KpnI, Acc65I/SacI, and EcoRI sites (not always shown). They also include an LUC coding region modified with an in-frame PstI site at the 5′ end (placed in front of the second natural codon, GAA, of the LUC ORF) and a HindIII site just upstream of the stop codon (created with silent mutations introduced into the LUC ORF). DLD RNA, which has DEN 5′ and 3′ UTRs, is made by transcription from the indicated template after linearization with SnaBI. The terminal sequences of DLD RNA differ from authentic DEN RNA by the substitution of a G for an A residue at the 5′ end and addition of UAC at the 3′ end. Translation starts with the first two authentic DEN codons and terminates with the authentic DEN UAG stop codon. DCLD RNA differs from DLD RNA by containing the first 36 codons of the DEN capsid coding region, which includes cCS1 and corresponds roughly to the capsid coding region present in flavivirus subgenomic replicons (22, 30, 35, 37). WLW and WCLW RNAs are analogous to DLD and DCLD RNAs, with all viral sequences from WNV. In these RNAs, a nonviral G residue is present at the 5′ end and an additional UAC at the 3′ end. Thirty-one codons of the capsid ORF are present in WCLW RNA. SinLSin RNA has the 59-nucleotide SIN 5′ UTR (with an additional G residue at the 5′ end to facilitate transcription), the first two codons of the SIN nonstructural polyprotein ORF, and the 318-nucleotide SIN 3′ UTR (GenBank accession no. J02362) with an XhoI site added to the 3′ end. A 72-nucleotide poly(A) tract that ends with an Acc65I site is present downstream of the XhoI site. GLG RNA contains 5′ and 3′ UTRs (36 nt and 87 nt, respectively) derived from rabbit α-globin mRNA (GenBank accession no. J00658). The first two codons of the α-globin coding region precede the two codons encoded by the PstI site of the modified LUC ORF. GCLG RNA is a derivative of GLG RNA that encodes the same capsid-LUC fusion protein as DCLD RNA. Variants with or without an A72 tail can be made by plasmid linearization with Acc65I or XhoI, respectively.
FIG. 2.
FIG. 2.
LUC variants with N-terminal fusions have similar specific activities. The indicated RNAs were translated in a rabbit reticulocyte lysate in the presence of [35S]methionine. (A) Translation products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%); the major product, LUC, is indicated by an arrowhead. These samples are shown to indicate the quality of the LUC product, not the translational efficiencies of the various RNAs. (B) Relative specific activities (relative light units per pmol) of LUC proteins made in reticulocyte lysates (averages from three replicates with each error bar representing one standard deviation). The N-terminal sequences of LUC proteins made by these RNAs are indicated (single-letter amino acid code); intervening capsid sequences are indicated by three periods.
FIG. 3.
FIG. 3.
Efficient LUC expression from 5′-capped flavivirus reporter mRNAs in Vero cells. Vero cells were electroporated with the indicated RNAs, and LUC extracts were prepared after various incubation times. LUC activities and protein levels in each extract were assayed to determine corrected LUC yields (relative light units [RLU] per mg protein). Representative time courses from single experiments (average of duplicates) are shown in the time courses of panels A and B. The tabulated results report LUC expression relative to that from DCLD RNA, with the (maximal) linear rate representing translational efficiency. The maximum LUC accumulation for each RNA is also reported, as is the LUC expression half-life, which reflects the combined RNA and LUC protein stability. The tabulated results reflect averages from at least two separate transfection experiments performed on different days, involving duplicate sets of independently made RNA transcripts in each case. Standard errors are indicated. The asterisk in front of each RNA name represents a 5′ m7GpppG cap.
FIG. 4.
FIG. 4.
Contributions of DEN 5′ and 3′ UTRs to translational expression in Vero cells. Data of LUC expression from the indicated capped (A) or uncapped (B) RNAs are as described in the legend to Fig. 3. DCLΔ and GCLΔ RNAs have 3′ UTRs comprised of only 15 vector-derived nucleotides (UAAAAUGGAUCUCGA). Note the different scales on the y axes in panels A and B. Expression data for capped (*) DCLGpA RNA are tabulated at the top of panel A, but the graphed time course is not shown, in order to avoid cluttering the graph. For clarity, standard errors of LUC expression have been omitted for the less-efficiently expressed RNAs in panels A and B; standard errors for these determinations averaged about 20% of the means. RLU, relative light units. ND, not determined. (C) The relative linear rates of LUC expression from the various RNAs are compared graphically. (D) Assay of the physical stabilities of selected 32P-labeled transcripts electroporated and incubated in Vero cells for the indicated times before extraction and analysis in the denatured state in a 1% agarose gel. The estimated half-lives are written at the bottom.
FIG. 5.
FIG. 5.
Complementarity between cCS1 and CS1 is not required for LUC expression from DCLD RNA. (A) The sequences of wild-type and variant cyclization sequences cCS1 (5′, indicated in gray lettering) and CS1 (3′, black lettering) are shown. The wild-type cCS1 and CS1 sequences, which form a perfect 11-bp duplex, are shown in uppercase letters. The three mutant RNAs were constructed by transposing these sequences to the other end of the genome. Single-base changes in mCS1 and mcCS1, indicated by underlined italics, were necessary to avoid the insertion of a UGA stop codon within the capsid ORF when CS1 was moved to the 5′ location. The most stable long-distance base-pairing schemes are shown, with the sequence in the 5′ region of the mRNA in the upper line. (B) Data of LUC expression from the indicated capped (*) RNAs are as described in the legend to Fig. 3. (C) The relative linear rates of LUC expression from the various RNAs are compared graphically. RLU, relative light units.
FIG. 6.
FIG. 6.
Several features from the DEN 3′ UTR contribute to efficient translation. (A) The sequences of conserved features of the DEN2 3′ UTR are shown in their proposed foldings. The native DEN stop codon UAG is shown on the left, and the additional nonviral nucleotides at the 3′ end of DCLD RNA are shown on the right. The NcoI restriction site used for producing the */NcoI variant of DCLD RNA is marked. The foldings of DB1 and DB2 are according to reference , while the foldings of SLA and SLB are according to references and . The numbered nucleotides (numbering from the 5′ end of the DEN genome) represent the deletion boundaries in the deletion derivatives of DCLD studied in panels B through D. (B to D). Data of LUC expression from the indicated capped (*) RNAs is as described in the legend to Fig. 3. The y axes are the same in each case. RLU, relative light units.
FIG. 7.
FIG. 7.
Translational efficiencies and RNA functional half-lives of 5′-capped DCLD variants with deletions in the 3′ UTR. Graphical representations of relative translation efficiencies (linear rates of LUC accumulation) (A) and LUC expression half-lives (B) are shown for the indicated RNAs. Because all of the RNAs encode the same form of LUC, LUC expression half-lives are proportional to RNA functional half-lives. Data are taken from Fig. 4 and 6. Error bars represent standard errors.
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