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CORD-19:13743b59fc40891e47e6574c9777201082685b84 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-108 Sentence denotes Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights
T2 110-118 Sentence denotes Abstract
T3 119-288 Sentence denotes 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.
T4 289-457 Sentence denotes 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.
T5 458-635 Sentence denotes 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.
T6 636-820 Sentence denotes 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).
T7 821-1106 Sentence denotes 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.
T8 1107-1342 Sentence denotes 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.
T9 1344-1506 Sentence denotes Eukaryotic positive-strand RNA viruses represent a distinct class of infectious agents that includes important pathogens of both plants and animals (Buck, 1996) .
T10 1507-1630 Sentence denotes Those that possess polycistronic genomes often transcribe subgenomic (sg) mRNAs during infections (Miller and Koev, 2000) .
T11 1631-1836 Sentence denotes 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.
T12 1837-2079 Sentence denotes 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) .
T13 2080-2170 Sentence denotes To date, three distinct mechanistic models for sg mRNA transcription have been identified.
T14 2171-2609 Sentence denotes 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) .
T15 2610-2887 Sentence denotes 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 0 -truncated minus-strand RNA generated serves as a template for sg mRNA transcription (Sit et al, 1998; White, 2002) .
T16 2888-3319 Sentence denotes 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) .
T17 3320-3471 Sentence denotes The PT mechanism has been studied most extensively in Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) (White and Nagy, 2004) .
T18 3472-3555 Sentence denotes The positive-strand RNA genome of TBSV encodes five proteins (Hearne et al, 1990) .
T19 3556-3826 Sentence denotes The 5 0 proximal p33 and its read-through product p92 (the RdRp) are translated directly from the viral genome, whereas more 3 0 -proximally positioned proteins are translated from two sg mRNAs that are transcribed during infections ( Figure 1A ; White and Nagy, 2004) .
T20 3827-3990 Sentence denotes 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) .
T21 3991-4225 Sentence denotes 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) .
T22 4226-4465 Sentence denotes Long-range interactions occur in the positive strand and involve RNA sequences located immediately 5 0 to the transcriptional initiation sites for sg mRNA1 and sg mRNA2, termed receptor sequence-1 (RS1) and RS2, respectively ( Figure 1B ).
T23 4466-4617 Sentence denotes These two RS elements base pair with corresponding activator sequence-1 (AS1) and AS2, located B1000 and B2100 nt upstream, respectively ( Figure 1B) .
T24 4618-4854 Sentence denotes 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 0 -truncated templates (Choi and White, 2002; Lin and White, 2004) .
T25 4855-5105 Sentence denotes 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) .
T26 5106-5330 Sentence denotes 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) .
T27 5331-5489 Sentence denotes Subsequent utilization of 3 0 -truncated minus-strand templates for transcription relies on linear sg mRNA promoters located at their 3 0 -ends ( Figure 1B ).
T28 5490-5644 Sentence denotes These promoters are structurally and functionally similar to that used by the virus to produce full-length positive-strand genomes (Lin and White, 2004) .
T29 5645-5910 Sentence denotes 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 0 end of the template).
T30 5911-6013 Sentence denotes Currently, no protein factors have been identified for any of the viruses that utilize a PT mechanism.
T31 6014-6178 Sentence denotes 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) .
T32 6179-6339 Sentence denotes 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.
T33 6340-6557 Sentence denotes 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.
T34 6558-6766 Sentence denotes 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.
T35 6767-6876 Sentence denotes Moreover, an important role for global RNA virus genome structure in modulating RdRp evolution was uncovered.
T36 6877-6943 Sentence denotes Specific and general implications of these findings are discussed.
T37 6944-7211 Sentence denotes 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 is translated directly from the viral genome by readthrough of the p33 stop codon (UAG).
T38 7212-7312 Sentence denotes Proteins encoded downstream are translated from two sg mRNAs that are transcribed during infections.
T39 7313-7453 Sentence denotes The relative positions (arrowheads) of interacting RNA elements that are involved in sg mRNA transcription are shown above the viral genome.
T40 7454-7660 Sentence denotes 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.
T41 7661-7749 Sentence denotes Relevant sequences of the TBSV genome are shown with corresponding coordinates provided.
T42 7750-7916 Sentence denotes 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.
T43 7917-8009 Sentence denotes Stem-loop structures containing AS1 and AS2 are connected by an 11-nt-long sequence (boxed).
T44 8010-8169 Sentence denotes 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).
T45 8170-8277 Sentence denotes A PT mechanism for sg mRNA2 transcription is depicted at the bottom with relevant terminal sequences shown.
T46 8278-8413 Sentence denotes The minus-strand generated by the termination event contains the sg mRNA promoter (shaded) and templates the transcription of sg mRNA2.
T47 8414-8452 Sentence denotes RdRp activity (Choi and White, 2002) .
T48 8453-8618 Sentence denotes In AS1m1, the upstream AS1 element contains translationally silent substitutions that prevent it from base pairing with its RS1 partner sequence (Figure 2A , inset).
T49 8619-8813 Sentence denotes 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.
T50 8814-8984 Sentence denotes 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.
T51 8985-9194 Sentence denotes 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.
T52 9195-9474 Sentence denotes C-terminal RdRp deletion analysis-that involved introduction of tandem termination codons at appropriate positions in the 3 0 region of the p92 ORF-revealed that removal of the ultimate Ala residue in Cd1 caused a relative B5-fold decrease in sg mRNA2 accumulation ( Figure 2B ).
T53 9475-9662 Sentence denotes Additional deletions of up to four residues in Cd2 through Cd4 resulted in B10-fold reductions in sg mRNA2 levels, whereas removal of one additional residue in Cd5 led to a B20-fold drop.
T54 9663-9796 Sentence denotes This progressive decrease in sg mRNA2 was also observed at earlier time points and at lower incubation temperatures (data not shown).
T55 9797-9989 Sentence denotes 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 ).
T56 9990-10172 Sentence denotes 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.
T57 10173-10385 Sentence denotes 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 ).
T58 10386-10593 Sentence denotes 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 B80-90%.
T59 10594-10689 Sentence denotes For the other modifications tested, none inhibited sg mRNA2 to levels below B46% ( Figure 2D ).
T60 10690-10976 Sentence denotes 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 aminoacid substitutions and (iii) the Ser residue is not a phosphoregulator of transcriptional activity.
T61 10977-11227 Sentence denotes 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.
T62 11228-11399 Sentence denotes 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) .
T63 11400-11567 Sentence denotes 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 ).
T64 11568-11832 Sentence denotes 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 ).
T65 11833-12158 Sentence denotes This reduced inhibition could be related to differences in the structure and/ or stability of the local RNA hairpin compared with those of 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.
T66 12159-12233 Sentence denotes Amino acids in the C-terminus of p92 are presented under the RNA sequence.
T67 12234-12374 Sentence denotes The nucleotide substitutions in mutant AS1m1 are indicated by shading (see inset). (B) Deletion analysis of C-terminal residues of the RdRp.
T68 12375-12638 Sentence denotes 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.
T69 12639-12726 Sentence denotes The identities of the viral genomes used in the transfections are indicated at the top.
T70 12727-12814 Sentence denotes The positions of the viral genome (g) and sg mRNAs (sg1 and sg2) are shown to the left.
T71 12815-13300 Sentence denotes 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.
T72 13301-13437 Sentence denotes Viral proteins were detected with antiserum generated against a peptide corresponding to a region in p33 (which is also present in p92).
T73 13438-13637 Sentence denotes 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.
T74 13638-13766 Sentence denotes The wt residues are presented at the top of the northern blot, with the corresponding substitutions indicated immediately below.
T75 13767-13868 Sentence denotes The left and right panels in panel D are from the same experiment but were analyzed on separate gels.
T76 13869-14026 Sentence denotes The associated values were generated relative to the same internal control, AS1m1, that was included in both blots. the wt long-distance AS2/RS2 interaction.
T77 14027-14195 Sentence denotes Importantly, the general trend of the transcriptional defect was reproducible for sg mRNA2 in a context lacking its cognate essential longdistance RNA-RNA interactions.
T78 14196-14295 Sentence denotes To further substantiate the proposed RdRp-dependent defect, two additional approaches were pursued.
T79 14296-14441 Sentence denotes In the first, five substitutions were introduced simultaneously at degenerate codon positions in the C-terminal region of the RdRp ( Figure 3B ).
T80 14442-14621 Sentence denotes 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.
T81 14622-14838 Sentence denotes 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.
T82 14839-15085 Sentence denotes 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 ).
T83 15086-15285 Sentence denotes 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 ).
T84 15286-15493 Sentence denotes 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 ).
T85 15494-15578 Sentence denotes The ability to confer the defect in trans further supports a protein-based activity.
T86 15579-15701 Sentence denotes Thus, all three approaches used to assess the origin of the transcriptional defect point to the RdRp as the causal factor.
T87 15702-15912 Sentence denotes 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.
T88 15913-16067 Sentence denotes 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 ).
T89 16068-16237 Sentence denotes In C-terminally truncated mutants, Cd1 through Cd5, relative levels of sg mRNA minus-strand accumulation were reduced to B30-50% that of the AS1m1 control ( Figure 4A ).
T90 16238-16427 Sentence denotes Overall, the accumulation of these minus-strand sg mRNA templates was lowered by B2to 3-fold; however, their corresponding plus-strand sg mRNAs were decreased by B5to 20-fold ( Figure 2B ).
T91 16428-16588 Sentence denotes This implied that the reduction in minus-strand templates had an Free energy changes (DG, at 221) for formation of each RNA structure are indicated in kcal/mol.
T92 16589-16669 Sentence denotes The sg RNA that is transcribed from these replicons (sg Rep) is indicated below.
T93 16670-16933 Sentence denotes 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.
T94 16934-17157 Sentence denotes The bands located between the genome (g) and sg mRNA2 (sg2) likely represent multimers of the replicon. amplified detrimental effect on plus-strand transcription, or that additional factors were contributing to the outcome.
T95 17158-17459 Sentence denotes One possibility considered was that the 3 0 -termini of the minus strands generated were aberrant (i.e., contained extra or deleted residues relative to the 3 0 end of the promoter sequence)-a feature known to adversely affect tombusvirus promoter activity (Panavas et al, 2002; Lin and White, 2004) .
T96 17460-17676 Sentence denotes Analysis of the 3 0 -termini of minus-strand templates from AS1m1 or Cd4 showed that the latter had approximately twice as many templates with 3 0 ends that were extended past the initiating nucleotide ( Figure 4B ).
T97 17677-17749 Sentence denotes Some of the Cd4-derived templates also contained non-templated residues.
T98 17750-17937 Sentence denotes The higher proportion of aberrant 3 0 -termini generated by the C-terminally truncated Cd4 would act to decrease overall promoter activity and contribute to the reduced levels of sg mRNA.
T99 17938-18193 Sentence denotes Primer extension analysis of the 5 0 ends of sg mRNA2s from infections with Cd4 (and other C-terminally truncated mutants) revealed only authentic 5 0 termini, indicating no defects in terms of the accuracy of initiation of transcription (data not shown).
T100 18194-18462 Sentence denotes 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) .
T101 18463-18547 Sentence denotes Accordingly, these two components were examined with respect to the RdRp C-terminus.
T102 18548-18767 Sentence denotes 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 ).
T103 18768-18886 Sentence denotes This analysis was carried out using viral genomes that expressed an RdRp truncated by four residues (i.e., Cd4 based).
T104 18887-19138 Sentence denotes 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 ).
T105 19139-19381 Sentence denotes 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).
T106 19382-19540 Sentence denotes In contrast, the corresponding relative accumulation profiles were comparable when spacer elements ranging in lengths from 0 to 6 nt were tested ( Figure 6 ).
T107 19541-19666 Sentence denotes Thus, a mutant RdRp lacking four C-terminal residues has spacer length requirements that are similar to those of the wt RdRp.
T108 19667-19805 Sentence denotes 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.
T109 19806-19985 Sentence denotes 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) .
T110 19986-20368 Sentence denotes 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 ).
T111 20369-20594 Sentence denotes 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.
T112 20595-20795 Sentence denotes 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 ).
T113 20796-20976 Sentence denotes 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 ).
T114 20977-21222 Sentence denotes Thus, even if these mutant RdRps were to generate wt promoters (i.e., minus-strand templates with 3 0 termini corresponding to the transcription initiation site), they would be slightly compromised (B10 to B35%) in their ability to utilize them.
T115 21224-21438 Sentence denotes 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) .
T116 21439-21577 Sentence denotes In particular, the RS1 element shares its sequence with the codons that specify the last three residues of the RdRp (refer to Figure 2A ).
T117 21578-21731 Sentence denotes This is a fascinating example of precisely overlapping sub-elements where both components mediate the same viral process, that is, sg mRNA transcription.
T118 21732-21823 Sentence denotes This tight coupling must, in turn, place unique evolutionary constraints on these elements.
T119 21824-22060 Sentence denotes 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.
T120 22061-22310 Sentence denotes 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 ).
T121 22311-22516 Sentence denotes 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.
T122 22517-22773 Sentence denotes 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 ).
T123 22774-23058 Sentence denotes As anticipated, sg mRNA1 levels were preferentially affected, showing relative accumulation levels of B92% (for the UG wobble pair; mutant AS-5G), B10% (for the AC mismatch; RS-5C) and B113% (for the compensatory GC pair; A/R-5GC), as compared with that for the wt T100 ( Figure 8B ).
T124 23059-23257 Sentence denotes 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.
T125 23258-23348 Sentence denotes Further relevance of this finding to RdRp evolution is presented in the following section.
T126 23349-23455 Sentence denotes Our results have identified the C-terminus of the TBSV RdRp as a key determinant of sg mRNA transcription.
T127 23456-23702 Sentence denotes Deletion of up to five C-terminal residues caused major reductions in sg 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.
T128 23703-23970 Sentence denotes Free energy changes (DG, at 221) 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 Cterminally truncated by four residues (Cd4-2AS-H1 through -H4).
T129 23971-24219 Sentence denotes 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 (DG).
T130 24220-24365 Sentence denotes 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.
T131 24366-24409 Sentence denotes Vertical bars indicate standard deviations.
T132 24410-24758 Sentence denotes 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.
T133 24759-24969 Sentence denotes 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.
T134 24970-25146 Sentence denotes 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.
T135 25147-25285 Sentence denotes Vertical bars indicate standard deviations. mRNA accumulation, with comparatively minor effects on viral genome replication ( Figure 9A ).
T136 25286-25519 Sentence denotes However, removal of one additional residue completely abolished TBSV genome replication, indicating a tight, but separable, physical coupling of crucial RNA replication and transcription functions within the C-terminus ( Figure 9A ).
T137 25520-25668 Sentence denotes The uncoupling of replication from transcription has been reported in plus-strand RNA viruses that utilize transcriptional mechanisms other than PT.
T138 25669-25956 Sentence denotes For example, in the alphavirus Sindbis virus (SINV)-that employs an II transcriptional mechanism-mutation of centrally located Arg residues in the nsP4 RdRp prevented it from binding to its sg mRNA promoter and specifically abolished sg mRNA transcription in vitro Stollar, 2004, 2007) .
T139 25957-26096 Sentence denotes However, this uncoupling was not reproducible in vivo, because the same mutations proved to be lethal to the virus (Li and Stollar, 2004) .
T140 26097-26401 Sentence denotes In contrast, mutations in either SINV nsP3 (unknown function) or in nsP2 (a protease, in addition to other functions) of Semliki Forest virus (also an alphavirus) caused significant defects in sg mRNA transcription in vivo, while maintaining virus viability (LaStarza et al, 1994; Suopanki et al, 1998) .
T141 26402-26748 Sentence denotes The uncoupling of sg mRNA transcription in vivo has also been achieved in an arterivirus, Equine arteritis virus, where mutation of two different proteins that contain zinc-binding domains, nsp1 and nsp10 (neither is the RdRp), caused specific defects in the DTS mechanism of sg mRNA transcription (van Marle et al, 1999a, b; Tijms et al, 2001) .
T142 26749-27011 Sentence denotes These examples illustrate that the II and DTS transcriptional processes in these viruses are complex and involve multiple viral proteins; however, with the exception of SINV nsP4, the specific roles played by these assorted viral factors remain to be determined.
T143 27012-27107 Sentence denotes Before this study, nothing was known about the protein components involved in the PT mechanism.
T144 27108-27185 Sentence denotes Accordingly, the results from this study are significant in several respects.
T145 27186-27331 Sentence denotes The TBSV RdRp represents the first protein factor to be identified as a key regulator of the PT mechanism of sg mRNA transcription-for any virus.
T146 27332-27508 Sentence denotes Also noteworthy is that this is the first demonstration, for a plant virus, that genome replication can be efficiently uncoupled-at the protein levelfrom sg mRNA transcription.
T147 27509-27537 Sentence denotes Moreover, this is the only .
T148 27538-27732 Sentence denotes 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.
T149 27733-27776 Sentence denotes Vertical bars indicate standard deviations.
T150 27777-27979 Sentence denotes The differences between Rep (wt) and HL47 accumulation levels were statistically significant for Cd4 (Po0.01) and Cd5 (Po0.05). example of a viral RdRp specifically modulating sg mRNA synthesis in vivo.
T151 27980-28116 Sentence denotes These findings increase our general understanding of the PT mechanism and provide novel insights into fundamental viral RdRp activities.
T152 28117-28279 Sentence denotes The PT mechanism involves a defined series of steps and, through systematic analyses, we have identified those that are facilitated by the C-terminus of the RdRp.
T153 28280-28467 Sentence denotes As summarized in Figure 9B , our results indicate major involvement at the level of sg mRNA minus-strand production and a possible lesser role with subsequent use of the sg mRNA promoter.
T154 28468-28602 Sentence denotes Viral RdRps take on a general right-hand topology that includes fingers, palm and thumb domains (Ferrer-Orta et al, 2006; Figure 9B ).
T155 28603-28770 Sentence denotes Our results indicate that in TBSV, the extreme C-terminus mediates efficient RdRp detection of the base paired components of the RNA attenuation signals (Figure 9Bi) .
T156 28771-28955 Sentence denotes Accordingly, the important C-terminal residues must either allow the RdRp to communicate directly with the RNA structures or mediate an indirect interaction via an auxiliary factor(s).
T157 28956-29260 Sentence denotes Active tombusvirus replicase complexes purified from transfected yeast cells contain at least four different host proteins (in addition to p33 and p92) (Serva and Nagy, 2006) and can direct complementary strand synthesis in vitro when incubated with exogenous viral RNA templates (Serva and Nagy, 2006) .
T158 29261-29464 Sentence denotes However, this extract, as well as similar replicase extracts from plants, is thus far unable to recognize the RNA attenuation signals for sg mRNA transcription in vitro (PD Nagy, personal communication).
T159 29465-29600 Sentence denotes Therefore, either the extracts are missing a key factor(s) or the isolated complexes are perturbed in a way that inhibits this process.
T160 29601-29854 Sentence denotes If the RdRp interacts directly with the attenuation signal, the C-terminal residues could function to organize the RdRp into a conformation that induces it to pause when it encounters the attenuation signal-thus providing an opportunity for termination.
T161 29855-30093 Sentence denotes Results from the spacer analysis revealed similar length requirements for wt and mutant RdRps (Figure 6) , indicating that the C-terminal residues are not involved in defining the span of intervening sequence that is optimal for function.
T162 30094-30249 Sentence denotes Conversely, the mutant RdRp showed altered positions of minus-strand termination as well as enhanced incorporation of non-templated residues ( Figure 4B ).
T163 30250-30399 Sentence denotes The latter activity is a known property of viral RdRps (Nagy and Simon, 1997) and, for TBSV, C-terminal modifications appear to enhance this feature.
T164 30400-30494 Sentence denotes For the wt RdRp, B60% of its minus-strand 3 0 -ends corresponded to the initiating nucleotide.
T165 30495-30602 Sentence denotes In contrast, only B30% of the 3 0 -ends generated by the mutant RdRp mapped to this position ( Figure 4B ).
T166 30603-30887 Sentence denotes Since aberrant 3 0 -termini negatively affect promoter utilization in TBSV, the higher proportion of 3 0 -extensions generated by the mutant RdRp could largely account for the proportionally greater reduction in sg mRNA accumulation-versus corresponding minus-strands ( Figure 9Bii ).
T167 30888-31059 Sentence denotes In some cases, this imbalance may also be related to reduced ability of certain C-terminally truncated RdRps (e.g., Cd4) to utilize the sg mRNA promoter ( Figure 9Biii) .
T168 31060-31201 Sentence denotes Thus, the combined effect of several distinct defects likely account for the overall reductions in sg mRNA accumulation levels ( Figure 9B) .
T169 31202-31403 Sentence denotes Collectively, these studies have provided important new mechanistic insights into the role of the RdRp in mediating sg mRNA transcription under physiological conditions (i.e., in host cell infections).
T170 31404-31652 Sentence denotes Specifically, the RdRp was found to (i) mediate efficient generation of minus-strand sg mRNA templates, (ii) facilitate accurate termination of minusstrands, and (iii) possibly promote efficient utilization of the wt sg mRNA promoter ( Figure 9B ).
T171 31653-31854 Sentence denotes In a theoretical evolutionary model, it is logical to suppose that replication of a viral RNA genome (e.g., encoding a polyprotein) preceded the emergence of sg mRNA transcription (Tijms et al, 2001) .
T172 31855-31998 Sentence denotes Indeed, for the PT mechanism, this scenario is supported by our demonstration that replication can be effectively uncoupled from transcription.
T173 31999-32077 Sentence denotes How then was sg mRNA transcription introduced into a viral replication system?
T174 32078-32302 Sentence denotes The necessity of the C-terminus for transcription, but not for replication, suggests a potential pathway for gaining (or enhancing) sg mRNA transcription through the C-terminal extension of a more primitive tombusvirus RdRp.
T175 32303-32552 Sentence denotes Although other possibilities exist, this type of read-through genetic mechanism has been implicated in the evolution of cellular proteins derived from humans (Fiddes and Goodman, 1980) , Drosophila (Levine et al, 2006) and yeast (Namy et al, 2003) .
T176 32553-32685 Sentence denotes In our proposed viral scenario, the primitive RdRp would originally have limited or no ability to recognize RNA attenuation signals.
T177 32686-32939 Sentence denotes However, in gaining this function-via extension of its ORF-it would then be able to efficiently generate smaller minus-sense RNAs that could potentially be used as templates to synthesize a new class of smaller viral RNA (i.e., sg (m)RNAs; Figure 10A ).
T178 32940-33411 Sentence denotes This scheme is attractive because the RdRp would not need to gain new structural 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.
T179 33412-33513 Sentence denotes P92 is depicted with classical right hand RdRp topology (fingers, F; palm, P; and thumb, T, domains).
T180 33514-33669 Sentence denotes 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.
T181 33670-33930 Sentence denotes See text for details. features to allow it to bind to a distinct sg mRNA promoter (as is the case for SINV; Li and Stollar, 2004) , since it would utilize the same type of plus-strand promoter that functions for viral genome replication (Lin and White, 2004) .
T182 33931-34088 Sentence denotes In using a common type of plus-strand promoter for both processes, the RdRp adaptation necessary to gain the ability to transcribe sg mRNAs would be reduced.
T183 34089-34320 Sentence denotes In this regard, the biogenesis of the PT system represents a simple but ingenious mechanism for relocating dormant internally positioned sg mRNA promoters to the 3 0 termini of minus-strand templateswhere they become highly active.
T184 34321-34468 Sentence denotes Another intriguing feature of the RdRp C-terminus is that the sequence encoding it is also the RS1 transcriptional regulatory element (Figure 2A ).
T185 34469-34616 Sentence denotes This relationship is further complicated by the necessity for the RS1 element to base pair with its AS1 partner sequence located B1000 nt upstream.
T186 34617-34812 Sentence denotes This convoluted interdependence suggests that AS1, via its basepairing requirement with RS1, could influence the coding of the RdRp C-terminus-and thus the structure and function of this protein.
T187 34813-35070 Sentence denotes The coexistence and coevolution of localized functional RNA elements and their corresponding coding sequences in mRNAs has been explored previously both in silico (Konecny et al, 2000; Pedersen et al, 2004) and in natural populations (Parmley et al, 2007) .
T188 35071-35326 Sentence denotes However, the concept that an RNA sequence can act via a long-range tertiary RNA-RNA interaction to influence protein coding at a distal location has not been investigated and, as described below, our data support the occurrence of such an event in nature.
T189 35327-35524 Sentence denotes As mentioned earlier, two tombusviruses (CBV and MNeSV) contain a GC base pair in the AS1/RS1 interaction in place of the AU base pair in TBSV (also present in nine other tombusviruses; not shown).
T190 35525-35697 Sentence denotes Considering that 10 out of 14 sequenced tombusviruses contain the AU base pair, it is reasonable to believe that the GC variant arose from the former 'consensus' base pair.
T191 35698-35991 Sentence denotes This concept is also supported by the observation that the introduction of the GC base pair into a TBSV context induced slightly improved levels of sg mRNA1 accumulation (i.e., B113%; Figure 8 ), which would presumably confer enhanced fitness by providing more coat protein for virus assembly.
T192 35992-36334 Sentence denotes In terms of the order of appearance of the individual substitutions, the presence of GU wobble base pairs in two tombusviruses (Cucumber necrosis virus (CNV) and TBSV-statice isolate (TBSV-S); refer to Figure 8A ) suggests that the A-to-G substitution at the distal AS1 occurred first, followed by a compensating U-to-C at RS1 ( Figure 10B ).
T193 36335-36590 Sentence denotes This order is also supported experimentally in TBSV, where the GU base pair-containing intermediate was found to be somewhat compromised (B92%), but considerably more transcriptionally active than the AC mismatchcontaining intermediate (B10%) (Figure 8 ).
T194 36591-36850 Sentence denotes The greater functionality of the GU wobble base pair would aid it competitively and allow it to persist long enough-as suggested by its existence in natural tombusvirus populations of CNV and TBSV-S-for the compensatory change to occur (Rousset et al, 1991) .
T195 36851-37079 Sentence denotes Therefore, following this line of reasoning, the substitution at the distal AS1 element would initiate the cascade of events that lead to the modification of the C-terminus of the RdRp encoded B1000 nt downstream ( Figure 10B ).
T196 37080-37371 Sentence denotes Moreover, as mfold analysis predicts formation of the AS1/RS1 interaction in the context of a folded full-length TBSV RNA genome (Choi and White, 2002) , these data suggest a previously unappreciated role for the global structure of an RNA virus genome in modulating viral protein evolution.
T197 37372-37755 Sentence denotes The implications of this finding are also relevant to understanding evolutionary processes in other viruses such as Hepatitis C virus (Kim et al, 2003) , Dengue virus (Alvarez et al, 2005) , Flock house virus (Lindenbach et al, 2002) and Barley yellow dwarf virus (Miller and White, 2006) , all of which contain functional long-distance RNA-RNA interactions involving coding regions.
T198 37756-38019 Sentence denotes Furthermore, as there is computational (Meyer and Miklos, 2005) and experimental (Nackley et al, 2006) evidence for functional higher-order RNA structures in coding regions of cellular mRNAs, the relevance of this concept will likely extend beyond viral messages.
T199 38020-38490 Sentence denotes Constructs previously described that were used in this study include: T100, the wt TBSV genome construct (Hearne et al, 1990 ); AS1m1 and Psg1-S5, modified TBSV genomes with 3-nt substitutions in AS1 (Choi and White, 2002) or five silent mutations in RS1 (Zhang et al, 1999) , respectively; a TBSV replicon corresponding to a prototypical TBSV DI RNA (Wu et al, 2001 ) and HL47, a replicon containing a sg RNA promoter as its plus-strand promoter (Lin and White, 2004) .
T200 38491-38634 Sentence denotes Based on the above constructs, additional modifications were introduced and the relevant differences are presented in the accompanying figures.
T201 38635-38790 Sentence denotes All modifications were made using PCRbased mutagenesis and standard cloning techniques (Sambrook The 5 0 portion of the TBSV genome is shown schematically.
T202 38791-39001 Sentence denotes 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.
T203 39002-39151 Sentence denotes 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.
T204 39152-39293 Sentence denotes 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.
T205 39294-39411 Sentence denotes Nucleotide substitutions are indicated along with effects on the corresponding amino-acid sequences (in parentheses).
T206 39412-39448 Sentence denotes See text for details. et al, 1989) .
T207 39449-39590 Sentence denotes The PCR-derived regions introduced into the constructs were sequenced completely to ensure that only the intended modifications were present.
T208 39591-39711 Sentence denotes In vitro RNA transcripts of viral RNAs were generated using T7 RNA polymerase as described previously (Wu et al, 2001) .
T209 39712-39857 Sentence denotes Preparation and inoculation of cucumber protoplasts and extraction of total nucleic acids were carried out as outlined in Choi and White (2002) .
T210 39858-39992 Sentence denotes Briefly, isolated cucumber protoplasts (3 Â10 5 ) were transfected with RNA transcripts (3 mg for genomic RNA; 1 mg for replicon RNA).
T211 39993-40077 Sentence denotes The transfected protoplasts were incubated at 221C for 24 h under constant lighting.
T212 40078-40144 Sentence denotes Total nucleic acids were isolated and analyzed as described below.
T213 40145-40359 Sentence denotes Total nucleic acid preparations isolated from virus-inoculated protoplasts were subjected to northern blot analysis for detection of plus-and minus-strand viral RNAs as described previously (Choi and White, 2002) .
T214 40360-40564 Sentence denotes Nucleic acids were either treated with glyoxal and separated in 1.4% agarose gels, or denatured in formamidecontaining buffer and separated in 4.5% polyacrylamide-8 M urea gels ( Figures 3C and 7B only) .
T215 40565-40662 Sentence denotes Equal loading of lanes was confirmed before transfer via staining the gels with ethidium bromide.
T216 40663-40856 Sentence denotes Following electrophoretic transfer to nylon membranes, positivestrand viral RNAs were detected using 32 P-labeled DNA oligonucleotide probes and negative strands were detected by RNA riboprobe.
T217 40857-40944 Sentence denotes Relative isotopic levels were determined using PharosFx Plus Molecular Imager (BioRad).
T218 40945-41205 Sentence denotes Sequence analysis of the 3 0 termini of minus-strand sg mRNA2 was accomplished by ligation with a phosphorylated oligonucleotide with RNA ligase, reverse transcription-PCR amplification of the cDNA, and subsequent cloning and sequencing (Lin and White, 2004) .
T219 41206-41315 Sentence denotes RNA structures were predicted and free energy changes calculated using mfold version 2.3 Zuker et al, 1999) .
T220 41316-41460 Sentence denotes After a 24-h incubation period, protoplasts were vortexed in 50 ml 2 Â SDS-PAGE loading buffer for 30 s, followed by heating at 1001C for 5 min.
T221 41461-41501 Sentence denotes Proteins were separated on 12% SDS-PAGE.
T222 41502-41658 Sentence denotes After electrotransfer to a nitrocellulose membrane, p33 and p92 were detected using a polyclonal antibody against a peptide of p33 (McCartney et al, 2005) .
T223 41659-41837 Sentence denotes HRP-conjugated secondary antibody (Sigma, A0545) and Amersham ECL Plus western blotting detection reagents (GE Healthcare, RPN 2132) were used for further processing of the blot.