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• W2057982258 abstract "Article22 November 2007free access Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights Baodong Wu Baodong Wu Department of Biology, York University, Toronto, Ontario, Canada Search for more papers by this author K Andrew White Corresponding Author K Andrew White Department of Biology, York University, Toronto, Ontario, Canada Search for more papers by this author Baodong Wu Baodong Wu Department of Biology, York University, Toronto, Ontario, Canada Search for more papers by this author K Andrew White Corresponding Author K Andrew White Department of Biology, York University, Toronto, Ontario, Canada Search for more papers by this author Author Information Baodong Wu1 and K Andrew White 1 1Department of Biology, York University, Toronto, Ontario, Canada *Corresponding author. Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. Tel.: +1 416 736 5243; Fax: +1 416 736 5698; E-mail: [email protected] The EMBO Journal (2007)26:5120-5130https://doi.org/10.1038/sj.emboj.7601931 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Eukaryotic positive-strand RNA viruses represent a distinct class of infectious agents that includes important pathogens of both plants and animals (Buck, 1996). Those that possess polycistronic genomes often transcribe subgenomic (sg) mRNAs during infections (Miller and Koev, 2000). These smaller virus-derived messages encode open reading frames (ORFs) that are translationally silent in the full-length viral genome and permit temporal and quantitative control of viral gene expression. The virally encoded RNA-dependent RNA polymerases (RdRps) responsible for replicating positive-strand RNA virus genomes—via minus-strand RNA intermediates—are also the same catalytic subunits that transcribe sg mRNAs (Miller and Koev, 2000). To date, three distinct mechanistic models for sg mRNA transcription have been identified. The internal initiation (II) model involves initiation of transcription at a sg mRNA promoter located internally within the full-length minus-strand of a viral genome (Miller et al, 1985), whereas the discontinuous template synthesis (DTS) model entails the use of a discontinuously synthesized minus-strand template for sg mRNA transcription (Sawicki and Sawicki, 1998; van Marle et al, 1999a; Pasternak et al, 2001; Sola et al, 2005). In the premature termination (PT) model, the RdRp terminates prematurely while synthesizing a full-length minus-strand copy of the viral RNA genome and the 3′-truncated minus-strand RNA generated serves as a template for sg mRNA transcription (Sit et al, 1998; White, 2002). This PT mechanism is believed to be utilized by a variety of viruses, including those that infect mammals (e.g., toroviruses; van Vliet et al, 2002; Smits et al, 2005), aquatic invertebrates (e.g., roniviruses; Cowley et al, 2002), fish (e.g., betanodaviruses; Iwamoto et al, 2005), plants (e.g., dianthoviruses; Sit et al, 1998 and closteroviruses; Gowda et al, 2001), and insects (e.g., nodaviruses; Lindenbach et al, 2002). The PT mechanism has been studied most extensively in Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) (White and Nagy, 2004). The positive-strand RNA genome of TBSV encodes five proteins (Hearne et al, 1990). The 5′ proximal p33 and its read-through product p92 (the RdRp) are translated directly from the viral genome, whereas more 3′-proximally positioned proteins are translated from two sg mRNAs that are transcribed during infections (Figure 1A; White and Nagy, 2004). Of these five viral proteins, only the p92 RdRp and its auxiliary factor p33 are required for genome replication and sg mRNA transcription (White and Nagy, 2004). 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. Download figure Download PowerPoint Studies of TBSV have identified numerous RNA elements in the viral genome that are involved in the PT mechanism, including distant RNA–RNA interactions (Zhang et al, 1999; Choi et al, 2001; Choi and White, 2002; Lin and White, 2004). Long-range interactions occur in the positive strand and involve RNA sequences located immediately 5′ to the transcriptional initiation sites for sg mRNA1 and sg mRNA2, termed receptor sequence-1 (RS1) and RS2, respectively (Figure 1B). These two RS elements base pair with corresponding activator sequence-1 (AS1) and AS2, located ∼1000 and ∼2100 nt upstream, respectively (Figure 1B). The RNA structures formed by the AS/RS interactions are essential for the RdRp termination events that occur during minus-strand synthesis, and lead to production of 3′-truncated templates (Choi and White, 2002; Lin and White, 2004). Interestingly, the long-range nature of these interactions is not essential, because an AS/RS element can be functionally replaced by a stable RNA hairpin positioned just upstream from its cognate transcription initiation site (Lin and White, 2004). Termination efficiency at these RNA structures is also influenced by the distance between the base paired element and the site of initiation (i.e., the length of the so-called spacer element) (Figure 1B; Lin et al, 2007). Subsequent utilization of 3′-truncated minus-strand templates for transcription relies on linear sg mRNA promoters located at their 3′-ends (Figure 1B). These promoters are structurally and functionally similar to that used by the virus to produce full-length positive-strand genomes (Lin and White, 2004). Thus, mechanistically, the main feature distinguishing TBSV sg mRNA transcription from genome replication is the manner by which the corresponding minus-strand templates are generated (i.e., termination internally versus termination at the 5′ end of the template). Currently, no protein factors have been identified for any of the viruses that utilize a PT mechanism. For TBSV, previous mutational analysis of the RS1 element suggested a possible role for this sequence in modulating sg mRNA2 levels in vivo (Choi and White, 2002). However, because the RS1 sequence also encodes the C-terminus of the p92 RdRp (Figures 1A and 2A), it was unclear whether the defect was RNA- or protein-based. In this study, we systematically investigated the function of the C-terminus of the TBSV RdRp and demonstrate that viral genome replication can be efficiently uncoupled from sg mRNA transcription at the protein level. Our analyses also revealed the steps in the PT mechanism requiring the C-terminal activity and alluded to a plausible evolutionary scheme by which the virus could gain or enhance its transcriptional activity. Moreover, an important role for global RNA virus genome structure in modulating RdRp evolution was uncovered. Specific and general implications of these findings are discussed. 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. Download figure Download PowerPoint Results The C-terminus of the RdRp is involved in sg mRNA transcription An infectious clone of a previously constructed modified TBSV genome, AS1m1, was used for in vivo analysis of RdRp activity (Choi and White, 2002). In AS1m1, the upstream AS1 element contains translationally silent substitutions that prevent it from base pairing with its RS1 partner sequence (Figure 2A, inset). These changes uncouple the RdRp-coding function of RS1 from its RNA structure-related function, thus allowing for unfettered mutational analysis of the C-terminus of p92 within the viral genome. However, since sg mRNA1 is inactivated in AS1m1, the effect of RdRp modifications on sg mRNA transcription was monitored by assessing the accumulation levels of sg mRNA2. Plant protoplasts were transfected with in vitro-transcribed wt (T100) and modified TBSV genomes, and the levels of viral RNA accumulation were determined by northern blot analysis following a 24-h incubation. C-terminal RdRp deletion analysis—that involved introduction of tandem termination codons at appropriate positions in the 3′ region of the p92 ORF—revealed that removal of the ultimate Ala residue in Cd1 caused a relative ∼5-fold decrease in sg mRNA2 accumulation (Figure 2B). Additional deletions of up to four residues in Cd2 through Cd4 resulted in ∼10-fold reductions in sg mRNA2 levels, whereas removal of one additional residue in Cd5 led to a ∼20-fold drop. This progressive decrease in sg mRNA2 was also observed at earlier time points and at lower incubation temperatures (data not shown). Interestingly, when six or more C-terminal residues were deleted (Cd6 through Cd10), no viral RNAs accumulated, signifying a lethal defect eliminating all forms of RNA synthesis (Figure 2B). For the viable mutants, western blot analysis of p92 revealed very low but relatively comparable levels of accumulation (Figure 2C), suggesting similar levels of protein stability. The importance of the five C-terminal residues for sg mRNA transcription was investigated further by substituting each with three different amino acids of similar or contrasting chemical properties (Figure 2D). The most notable defects were observed at the third and fifth positions from the end, where substitutions of Ala or Asp, respectively, with (Asp, Lys) or (Asn, Lys) caused sg mRNA2 levels to drop by ∼80–90%. For the other modifications tested, none inhibited sg mRNA2 to levels below ∼46% (Figure 2D). Overall, the C-terminal substitution analysis revealed that (i) amino-acid identities are somewhat flexible, (ii) severe defects in sg mRNA transcription can also be induced through amino-acid substitutions and (iii) the Ser residue is not a phosphoregulator of transcriptional activity. Mutant viral genomes are transcriptionally defective at the protein level The results above pointed to a defective RdRp; however, the possibility that the long-distance RNA–RNA interactions essential for sg mRNA2 transcription may have been adversely affected by the sequence modifications needed to be critically addressed. To this end, sg mRNA2 transcription was uncoupled from its two essential long-range interactions, DE/CE and AS2/RS2 (Figure 1A; Zhang et al, 1999; Lin and White, 2004). This was accomplished by deleting CE and replacing RS2 with a local RNA hairpin positioned just upstream from the sg mRNA2 transcription initiation site (Figure 3A). When the C-terminally truncated RdRp mutants Cd1H through Cd5H were tested along with the AS1H control (encoding wt RdRp), the results were similar to those observed earlier (Figure 2B), except that the defects in transcription were less prominent (Figure 3A). This reduced inhibition could be related to differences in the structure and/or stability of the local RNA hairpin compared with those of the wt long-distance AS2/RS2 interaction. Importantly, the general trend of the transcriptional defect was reproducible for sg mRNA2 in a context lacking its cognate essential long-distance RNA–RNA interactions. 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. Download figure Download PowerPoint To further substantiate the proposed RdRp-dependent defect, two additional approaches were pursued. In the first, five substitutions were introduced simultaneously at degenerate codon positions in the C-terminal region of the RdRp (Figure 3B). When tested, the Psg1-S5 genome transcribed sg mRNA2 efficiently (Figure 3B), thereby excluding this RNA sequence from being directly involved in modulating sg mRNA2 production. In the final approach, it was reasoned that if the RdRp was responsible for the reductions of sg mRNA2, then it should be possible to confer the defect in trans to another transcriptionally active viral RNA replicon. To test this hypothesis, small non-coding TBSV replicons (White, 1996) containing RNA hairpin-type transcriptional cassettes (Lin and White, 2004) were cotransfected with viral genomes containing a wt or C-terminally truncated RdRp (Figure 3C). Replicon-derived sg RNAs (sg Reps) were generated from HL65 and HL128 (containing the more stable RNA hairpins) when they were cotransfected with AS1m1, which provided wt RdRp in trans (Figure 3C). In contrast, when the same replicons were complemented with Cd4, a viral genome producing an RdRp truncated by four C-terminal residues, there was notably reduced levels of sg Rep accumulation (Figure 3C). The ability to confer the defect in trans further supports a protein-based activity. Thus, all three approaches used to assess the origin of the transcriptional defect point to the RdRp as the causal factor. The C-terminus of the RdRp facilitates the termination step of the PT transcriptional mechanism We next turned our attention to determining what step(s) in the PT mechanism the RdRp C-terminus was facilitating. An early event in this process is the attenuation of the RdRp that leads to its termination and the production of minus-stand intermediates (Figure 1B). In C-terminally truncated mutants, Cd1 through Cd5, relative levels of sg mRNA minus-strand accumulation were reduced to ∼30–50% that of the AS1m1 control (Figure 4A). Overall, the accumulation of these minus-strand sg mRNA templates was lowered by ∼2- to 3-fold; however, their corresponding plus-strand sg mRNAs were decreased by ∼5- to 20-fold (Figure 2B). This implied that the reduction in minus-strand templates had an amplified detrimental effect on plus-strand transcription, or that additional factors were contributing to the outcome. 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. Download figure Download PowerPoint One possibility considered was that the 3′-termini of the minus strands generated were aberrant (i.e., contained extra or deleted residues relative to the 3′ end of the promoter sequence)—a feature known to adversely affect tombusvirus promoter activity (Panavas et al, 2002; Lin and White, 2004). Analysis of the 3′-termini of minus-strand templates from AS1m1 or Cd4 showed that the latter had approximately twice as many templates with 3′ ends that were extended past the initiating nucleotide (Figure 4B). Some of the Cd4-derived templates also contained non-templated residues. The higher proportion of aberrant 3′-termini generated by the C-terminally truncated Cd4 would act to decrease overall promoter activity and contribute to the reduced levels of sg mRNA. Primer extension analysis of the 5′ ends of sg mRNA2s from infections with Cd4 (and other C-terminally truncated mutants) revealed only authentic 5′ termini, indicating no defects in terms of the accuracy of initiation of transcription (data not shown). Influence of base paired and linear components of the RNA attenuation signal on sg mRNA transcription The RdRp attenuation signal includes both base paired and linear RNA elements, and its activity is affected by both the thermodynamic stability of the base paired structure and the length of the associated linear spacer element (Lin and White, 2004; Lin et al, 2007). Accordingly, these two components were examined with respect to the RdRp C-terminus. First, the transcriptional efficiency mediated by different sized RNA hairpins was assessed to determine how the C-terminal RdRp mutants would respond to structures with increasing thermodynamic stability (Figure 5A). This analysis was carried out using viral genomes that expressed an RdRp truncated by four residues (i.e., Cd4 based). Consistent with our other results (Figure 3A and C), the genomes expressing the mutant RdRp (Cd4-2AS-H1 through -H4) did not utilize the RNA hairpin signals as efficiently as their counterparts expressing the wt RdRp (2AS-H1 through -H4; Figure 5B). This relationship, depicted graphically in Figure 5C, indicates a general lower sensitivity of the mutant RdRp for detecting the base paired component of the RNA attenuation signal, particularly for H1 through H3 (note difference in slopes). In contrast, the corresponding relative accumulation profiles were comparable when spacer elements ranging in lengths from 0 to 6 nt were tested (Figure 6). Thus, a mutant RdRp lacking four C-terminal residues has spacer length requirements that are similar to those of the wt RdRp. 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint Sg mRNA promoter activity is adversely affected by C-terminally truncated RdRp We next sought to determine if the C-terminal truncations were also affecting the ability of the RdRp to utilize the wt sg mRNA2 promoter. In TBSV, sg mRNA promoters and the genome plus-strand promoter have similar linear structures and sequence identities (Figure 7A) and are interchangeable (Lin and White, 2004). Thus, to address the potential effects of the C-terminal truncations on sg mRNA promoter activity in a context independent of the sg mRNA-specific transcription attenuation components, we repositioned the sg mRNA2 promoter to the end of a small viral replicon (Rep-HL47) and compared the level of replication with that of a wt replicon containing the genomic promoter (Figure 7A). The mutant RdRps were provided in trans—through cotransfection with mutant viral genomes Cd1 through Cd5—and effects of the C-terminal deletions on the activities of the two promoters were monitored via replicon accumulation. Results from these cotransfection studies revealed that with increasing C-terminal deletions, accumulation levels of Rep-HL47 were more negatively affected than those of the wt replicon (Figure 7B). This general trend for the defects suggests that the C-terminal truncations preferentially affect the ability of the mutant RdRps to utilize the wt sg mRNA2 promoter (Figure 7C). Thus, even if these mutant RdRps were to generate wt promoters (i.e., minus-strand templates with 3′ termini corresponding to the transcription initiation site), they would be slightly compromised (∼10 to ∼35%) in their ability to utilize them. 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). Download figure Download PowerPoint RdRp coding and functional RNA elements involved in sg mRNA transcription overlap An interesting aspect of the TBSV RdRp is that the RNA sequence encoding its transcription-specific C-terminus is also an RNA regulatory element involved in directing sg mRNA1 transcription (Choi and White, 2002). In particular, the RS1 element shares its sequence with the codons that specify the last three residues of the RdRp (refer to Figure 2A). This is a fascinating example of precisely overlapping sub-elements where both components mediate the same viral process, that is, sg mRNA transcription. This tight coupling must, in turn, place unique evolutionary constraints on these elements. Despite such limitations, it is interesting to note that two tombusviruses, Cucumber Bulgarian virus (CBV) and Maize necrotic streak virus (MNeSV), contain modifications in this region that maintain both RNA- and protein-based functions. In these two viral genomes, the predicted AS1/RS1 interactions contain a GC base pair (which maintains the AS1/RS1 interaction) in place of the corresponding AU base pair in TBSV (compare structures of A/R-5GC and T100, respectively, in Figure 8A). In CBV and MNeSV, the nucleotide substitutions in their AS1 and RS1 elements result in the maintenance of Leu residues internally and Val-to-Ala substitutions at the C-termini of their RdRps, respectively. To assess the effects of these modifications in the context of TBSV, we constructed a mutant genome that contained the double substitution of the AU-to-GC base pair, as well as the single substitution mutants A-to-G (at AS1) or U-to-C (at RS1; Figure 8A). As anticipated, sg mRNA1 levels were preferentially affected, showing relative accumulation levels of ∼92% (for the UG wobble pair; mutant AS-5G), ∼10% (for the AC mismatch; RS-5C) and ∼113% (for the compensatory GC pair; A/R-5GC), as compared with that for the wt T100 (Figure 8B). Thus, although coding and RNA-based regulatory activities are tightly coupled in a common sequence, the AS1 and RS1 elements possess the capacity to evolve while maintaining both of these functions. Further relevance of this finding to RdRp evolution is presented in the following section. 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. Not" @default.
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• W2057982258 title "Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights" @default.
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