Epistemic_Statements  

CORD-19:13743b59fc40891e47e6574c9777201082685b84 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 458-635 Epistemic_statement 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.
T2 636-820 Epistemic_statement 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).
T3 1107-1342 Epistemic_statement 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.
T4 2888-3319 Epistemic_statement 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) .
T5 3320-3471 Epistemic_statement denotes The PT mechanism has been studied most extensively in Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) (White and Nagy, 2004) .
T6 4855-5105 Epistemic_statement 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) .
T7 6014-6178 Epistemic_statement 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) .
T8 6179-6339 Epistemic_statement 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.
T9 6340-6557 Epistemic_statement 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.
T10 6558-6766 Epistemic_statement 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.
T11 8010-8169 Epistemic_statement 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).
T12 8814-8984 Epistemic_statement 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.
T13 9797-9989 Epistemic_statement 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 ).
T14 9990-10172 Epistemic_statement 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.
T15 10690-10976 Epistemic_statement 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.
T16 10977-11227 Epistemic_statement 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.
T17 11833-12036 Epistemic_statement 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.
T18 12234-12316 Epistemic_statement denotes The nucleotide substitutions in mutant AS1m1 are indicated by shading (see inset).
T19 14622-14838 Epistemic_statement 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.
T20 14839-15085 Epistemic_statement 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 ).
T21 15286-15493 Epistemic_statement 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 ).
T22 15494-15578 Epistemic_statement denotes The ability to confer the defect in trans further supports a protein-based activity.
T23 15702-15912 Epistemic_statement 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.
T24 16238-16427 Epistemic_statement 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 ).
T25 16428-16588 Epistemic_statement 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.
T26 16934-17037 Epistemic_statement denotes The bands located between the genome (g) and sg mRNA2 (sg2) likely represent multimers of the replicon.
T27 17158-17459 Epistemic_statement 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) .
T28 17750-17937 Epistemic_statement 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.
T29 18194-18462 Epistemic_statement 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) .
T30 18548-18767 Epistemic_statement 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 ).
T31 19139-19381 Epistemic_statement 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).
T32 19667-19805 Epistemic_statement 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.
T33 19986-20368 Epistemic_statement 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 ).
T34 20796-20976 Epistemic_statement 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 ).
T35 21224-21438 Epistemic_statement 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) .
T36 21732-21823 Epistemic_statement denotes This tight coupling must, in turn, place unique evolutionary constraints on these elements.
T37 21824-22060 Epistemic_statement 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.
T38 23703-23798 Epistemic_statement denotes Free energy changes (DG, at 221) for formation of each RNA structure are indicated in kcal/mol.
T39 25286-25519 Epistemic_statement 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 ).
T40 25957-26096 Epistemic_statement denotes However, this uncoupling was not reproducible in vivo, because the same mutations proved to be lethal to the virus (Li and Stollar, 2004) .
T41 26749-27011 Epistemic_statement 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.
T42 27332-27508 Epistemic_statement 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.
T43 28280-28467 Epistemic_statement 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.
T44 28603-28770 Epistemic_statement 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) .
T45 28956-29260 Epistemic_statement 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) .
T46 29261-29464 Epistemic_statement 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).
T47 29601-29854 Epistemic_statement 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.
T48 29855-30093 Epistemic_statement 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.
T49 30250-30399 Epistemic_statement 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.
T50 30603-30887 Epistemic_statement 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 ).
T51 30888-31059 Epistemic_statement 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) .
T52 31060-31201 Epistemic_statement denotes Thus, the combined effect of several distinct defects likely account for the overall reductions in sg mRNA accumulation levels ( Figure 9B) .
T53 31855-31998 Epistemic_statement denotes Indeed, for the PT mechanism, this scenario is supported by our demonstration that replication can be effectively uncoupled from transcription.
T54 31999-32077 Epistemic_statement denotes How then was sg mRNA transcription introduced into a viral replication system?
T55 32078-32302 Epistemic_statement 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.
T56 32303-32552 Epistemic_statement 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) .
T57 32553-32685 Epistemic_statement denotes In our proposed viral scenario, the primitive RdRp would originally have limited or no ability to recognize RNA attenuation signals.
T58 32686-32939 Epistemic_statement 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 ).
T59 32940-33085 Epistemic_statement 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.
T60 33692-33930 Epistemic_statement denotes 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) .
T61 33931-34088 Epistemic_statement 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.
T62 34469-34616 Epistemic_statement 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.
T63 34617-34812 Epistemic_statement 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.
T64 34813-35070 Epistemic_statement 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) .
T65 35071-35326 Epistemic_statement 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.
T66 35525-35697 Epistemic_statement 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.
T67 35698-35991 Epistemic_statement 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.
T68 35992-36334 Epistemic_statement 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 ).
T69 36591-36850 Epistemic_statement 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) .
T70 36851-37079 Epistemic_statement 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 ).
T71 37080-37371 Epistemic_statement 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.
T72 39294-39411 Epistemic_statement denotes Nucleotide substitutions are indicated along with effects on the corresponding amino-acid sequences (in parentheses).