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. 2007 Dec 12;26(24):5120-30.
doi: 10.1038/sj.emboj.7601931. Epub 2007 Nov 22.

Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights

Baodong Wu  1 , K Andrew White
Affiliations

Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights

Baodong Wu et al. EMBO J. .

Abstract

Many eukaryotic positive-strand RNA viruses transcribe subgenomic (sg) mRNAs that are virus-derived messages that template the translation of a subset of viral proteins. Currently, the premature termination (PT) mechanism of sg mRNA transcription, a process thought to operate in a variety of viruses, is best understood in tombusviruses. The viral RNA elements involved in regulating this mechanism have been well characterized in several systems; however, no corresponding protein factors have been identified yet. Here we show that tombusvirus genome replication can be effectively uncoupled from sg mRNA transcription in vivo by C-terminal modifications in its RNA-dependent RNA polymerase (RdRp). Systematic analysis of the PT transcriptional pathway using viral genomes harboring mutant RdRps revealed that the C-terminus functions primarily at an early step in this mechanism by mediating both efficient and accurate production of minus-strand templates for sg mRNA transcription. Our results also suggest a simple evolutionary scheme by which the virus could gain or enhance its transcriptional activity, and define global folding of the viral RNA genome as a previously unappreciated determinant of RdRp evolution.

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Figures

Figure 1
Figure 1
TBSV genome and sg mRNA transcriptional RNA elements. (A) Schematic linear representation of the TBSV RNA genome and its coding organization. Boxes indicate encoded proteins. The p92 RdRp is translated directly from the viral genome by readthrough of the p33 stop codon (UAG). Proteins encoded downstream are translated from two sg mRNAs that are transcribed during infections. The relative positions (arrowheads) of interacting RNA elements that are involved in sg mRNA transcription are shown above the viral genome. Initiation sites for sg mRNA transcription are labeled sg1 and sg2 and bold arrows represent corresponding structures of the two sg mRNAs below. (B) RNA elements that regulate sg mRNA transcription in TBSV. Relevant sequences of the TBSV genome are shown with corresponding coordinates provided. The AS1/RS1 base pairing interaction is essential for sg mRNA 1 transcription, whereas the DE/CE and AS2/RS2 interactions are essential for transcription of sg mRNA2. Stem-loop structures containing AS1 and AS2 are connected by an 11-nt-long sequence (boxed). Initiation sites for the two sg mRNAs are indicated by small arrows and are separated from their respective AS/RS interactions by spacer elements (underlined). A PT mechanism for sg mRNA2 transcription is depicted at the bottom with relevant terminal sequences shown. The minus-strand generated by the termination event contains the sg mRNA promoter (shaded) and templates the transcription of sg mRNA2.
Figure 2
Figure 2
Mutational analysis of the C-terminus of the TBSV RdRp. (A) The AS1/RS1 interaction in the wt TBSV genome (i.e., T100) and corresponding coding region for the C-terminus of p92. Amino acids in the C-terminus of p92 are presented under the RNA sequence. The nucleotide substitutions in mutant AS1m1 are indicated by shading (see inset). (B) Deletion analysis of C-terminal residues of the RdRp. Mutant viral genomes containing deletions of 1–10 C-terminal amino acids (Cd1 through Cd10, respectively) were transfected into plant protoplasts and sg mRNA2 levels quantified by northern blot analysis with a virus-specific DNA probe following a 24 h incubation. The identities of the viral genomes used in the transfections are indicated at the top. The positions of the viral genome (g) and sg mRNAs (sg1 and sg2) are shown to the left. The values below, in this and in other similar experiments, correspond to means from three independent experiments (with standard deviations) and represent the ratios of sg mRNA2 levels to their corresponding genomic RNA levels, all normalized to that for the control (in this case, AS1m1), set at 100. (C) Western blot analysis of p92 (and p33) accumulation in transfected protoplasts for wt TBSV (T100), control viral genome AS1m1, and C-terminally truncated mutants Cd1 through Cd5. Viral proteins were detected with antiserum generated against a peptide corresponding to a region in p33 (which is also present in p92). Accordingly, both p33 and p92 are detected by this antiserum. (D) Transcriptional activity of viral genome mutants containing single substitutions in each of the five C-terminal residues in the RdRp. The wt residues are presented at the top of the northern blot, with the corresponding substitutions indicated immediately below. The left and right panels in panel D are from the same experiment but were analyzed on separate gels. The associated values were generated relative to the same internal control, AS1m1, that was included in both blots.
Figure 3
Figure 3
Context-independent activity of mutant RdRps. (A) A local RNA hairpin (left) was substituted for the long-range AS2/RS2 interaction in mutant viral genomes. The identities of the mutant viral genomes used in the transfections are indicated at the top of the Northern blot. The relative sg mRNA2 values shown below were calculated as described in the legend to Figure 2B. (B) The five translationally silent substitutions in the C-terminal coding region of p92 in viral genome mutant Psg1-S5 are indicated by shading (top). Northern blot analysis of Psg1-S5 and control AS1m1. (C) HL127, HL65, and HL128 are small viral replicons (Replicon) containing different sized hairpin-type transcriptional cassettes (left). Free energy changes (ΔG, at 22°) for formation of each RNA structure are indicated in kcal/mol. The sg RNA that is transcribed from these replicons (sg Rep) is indicated below. Each replicon was cotransfected into protoplasts along with helper viral genome AS1m1 (wt RdRp) or Cd4 (mutant RdRp C-terminally truncated by four residues), and relative sg Rep accumulation levels assessed by northern blotting following a 24-h incubation period. The bands located between the genome (g) and sg mRNA2 (sg2) likely represent multimers of the replicon.
Figure 4
Figure 4
Minus-strand RNA analysis of C-terminally truncated RdRp mutants. (A) Northern blot analysis of minus-strand accumulation levels in protoplasts was performed 24 h post-transfection using a positive-sense RNA probe complementary to genomic and sg mRNAs. The mutants examined are indicated at the top and the positions of minus-strand viral genome (g (−)) and sg mRNA2 (sg2 (−)) are shown on the left. (B) Sequence analysis of the 3′ termini of sg mRNA2 minus strands. 3′-RACE was used to determine the terminal sequences for AS1m1 (wt RdRp) and Cd4 (mutant RdRp). The sequences of 3′-terminal regions of sg mRNA2 minus strands are shown below corresponding positive-strand sequence. The numbers in parentheses indicate the number of times each sequence was observed. Non-templated residues are underlined.
Figure 5
Figure 5
Effect of hairpin stability on sg mRNA2 transcription by C-terminally truncated RdRp. (A) Depiction of RNA hairpin cassettes introduced into modified viral genomes. Free energy changes (ΔG, at 22°) for formation of each RNA structure are indicated in kcal/mol. (B) Hairpins were tested in genomic contexts that expressed either wt RdRp (2AS-H1 through -H4) or an RdRp C-terminally truncated by four residues (Cd4-2AS-H1 through -H4). Northern blot analysis of viral RNA accumulation 24 h post-transfection of protoplasts is presented. (C) Graphical representation of relative accumulation values for sg mRNA2 observed in panel B plotted against predicted RNA hairpin stability (ΔG). For relative sg mRNA2 accumulation levels presented, that for 2AS-H4 was set at 100 and the other values were normalized relative to this number. Vertical bars indicate standard deviations.
Figure 6
Figure 6
Effect of spacer length on transcription from C-terminally truncated RdRp mutants. (A) In viral genome mutants S0 through S6, spacer sequences of 0–6 uridylates (Us) were tested for transcriptional activity in protoplast transfections. (B) Northern blot analysis of sg mRNA2 accumulation 24 h post-transfection of protoplasts is presented. The Cd4H series blot was exposed approximately twice as long as the AS1H series blot. (C) Graphical representation of relative accumulation values for sg mRNA2 observed in panel B plotted against spacer length. For relative sg mRNA2 accumulation levels presented, those for AS1H-S2 and Cd4H-S2 were set at 100 and the other corresponding values were normalized relative to those numbers. Vertical bars indicate standard deviations.
Figure 7
Figure 7
Sg mRNA2 promoter activity with wt and C-terminally truncated RdRps. (A) Schematic representation of a replicon with complementary wt or sg mRNA2 5′-terminal minus-strand sequences (i.e., promoters) expanded above. Nucleotide differences in the two sequences are highlighted. (B) Northern blot analysis of replicon RNA accumulation levels when protoplasts were cotransfected with viral genomes expressing wt (AS1m1) or C-terminally truncated (Cd1 through Cd5) RdRps. (C) Graphical representation of relative accumulation values for replicons observed in panel B plotted against viral genomes expressing wt and mutant RdRps (provided in trans). For relative accumulation levels presented, that for the wt replicon cotransfected with AS1m1 (wt RdRp) was set at 100 and the other corresponding values were normalized relative to that number. Vertical bars indicate standard deviations. The differences between Rep (wt) and HL47 accumulation levels were statistically significant for Cd4 (P<0.01) and Cd5 (P<0.05).
Figure 8
Figure 8
Analysis of AS1/RS1 interaction in wt TBSV genome (T100). (A) AS1 and RS1 sequences from wt (T100) and mutant viral genomes (AS-5G, RS-5C and A/R-5GC) and their corresponding encoded p92 amino acids. Substituted nucleotides are shaded and corresponding amino-acid changes are boxed. Note, the sequences shown for mutants AS-5G and A/R-5GC are present naturally in (CNV and TBSV-S) and (CBV and MNeSV), respectively. (B) Northern blot analysis of mutant viral genomes isolated from protoplasts 24 h post transfection. Relative levels of sg mRNA1 (sg1) accumulation are indicated at the bottom.
Figure 9
Figure 9
Role of C-terminus of TBSV RdRp in viral RNA synthesis. (A) Summary schematic diagram depicting the role of the C-terminal residues of p92 in sg mRNA transcription and viral genome replication as determined by progressive terminal deletion analysis. (B) A PT model for TBSV sg mRNA transcription highlighting the role of the C-terminus of p92 in the different steps of the process. P92 is depicted with classical right hand RdRp topology (fingers, F; palm, P; and thumb, T, domains). The C-terminus (C) in the cartoon is circled and the arrows point to the steps (i, ii, and iii) in the PT mechanism that are facilitated by these residues. See text for details.
Figure 10
Figure 10
Models for RdRp evolution. (A) Gain or enhancement of sg mRNA transcription function via extension of the p92 RdRp ORF. The 5′ portion of the TBSV genome is shown schematically. The genome at the top encodes a primitive RdRp lacking notable transcriptional activity, whereas that below contains a C-terminally extended p92 ORF (hatched box) that confers enhanced transcriptional activity. See text for details. (B) Influence of a long-distance RNA–RNA interaction (i.e., global folding) on the structure of the C-terminus of the p92 RdRp. The genome at the top has gained an A-to-G nucleotide substitution in AS1, whereas that below contains a second compensating mutation in RS1. Nucleotide substitutions are indicated along with effects on the corresponding amino-acid sequences (in parentheses). See text for details.
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References

    1. Alvarez DE, Lodeiro MF, Luduena SJ, Pietrasanta LI, Gamarnik AV (2005) Long-range RNA–RNA interactions circularize the dengue virus genome. J Virol 79: 6631–6643 - PMC - PubMed
    1. Buck KW (1996) Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47: 159–251 - PMC - PubMed
    1. Choi IR, Ostrovsky M, Zhang G, White KA (2001) Regulatory activity of distal and core RNA elements in Tombusvirus subgenomic mRNA2 transcription. J Biol Chem 276: 41761–41768 - PubMed
    1. Choi IR, White KA (2002) An RNA activator of subgenomic mRNA1 transcription in tomato bushy stunt virus. J Biol Chem 277: 3760–3766 - PubMed
    1. Cowley JA, Dimmock CM, Walker PJ (2002) Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences. J Gen Virol 83: 927–935 - PubMed

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