Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress  response as well as specialized metabolic programs.Small non-coding RNAs (sRNA) are emerging as major components of the cell’s regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in GramC. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional  and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq.Using RNA deep sequencing (RNA-seq) we examined the sRNome of Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.Our results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in  The prehybridization and hybridization of the membrane with labeled oligo probes was carried using the ULTRAhyb hybridization solution (Ambion) at 42°C.Northern analysis of select sRNAs was performed as described previously using single stranded oligo DNA probes . The proB. subtilis promoter consensus sequences [Lactococcus promoters, we used the promoter HMM model from the PPP tool [http://www.softberry.com). Secondary structures of sRNAs were predicted using Vienna RNAfold [Promoter prediction in the upstream region of the sRNAs were carried out using PSSMs of equences . For thePPP tool . Rho indPPP tool , Erpin [PPP tool  and Find RNAfold ,93.The sRNA nomenclature for the newly identified sRNAs was done in the same manner as described previously .+ reductase; AhrC/ArgR: Arginine repressor; Hfq: Host factor-I protein from Q8 bacteriophage; TFBS: Transcriptional factor binding site; SRP: Signal recognition particle; tmRNA: Transfer and messenger RNA; SAM: S-adenosyl methionine; adhe: Alcohol/aldehyde dehydrogenase 1; ctf: Co-enzyme A transferase; adc: Acetoacetate decarboxylase; SmpB: Small protein B; EF-Tu/Tuf: Elongation factor Tu; rpsA: Ribosomal protein A.SIPHT: sRNA identification protocol using high-throughput technologies; ABE: Acetone-bytanol-ethanol; Q-RT-PCR: Quantitative reverse transcriptase polymerase chain reaction; IOR: Interoperonic region; IGR: Intergenic region; ORF: Open reading frame; UTR: Untranslated region; PSSM: Position specific scoring matrix; HMM: Hidden Markov model; FNR: Ferredoxin-NADPThe authors declare that they have no competing interests.KPV and SWJ carried out the fermentations and RNA isolations. KPV carried out Northern analysis and drafted the manuscript. SGK constructed the libraries for RNA-seq. KPM processed the data and built the web browser. KPV and KPM analysed the data. KPV and MTR carried out the identification of new sRNAs and other computational analyses. BCM and ETP helped with data analysis and interpretation, and participated in the manuscript preparation. ETP conceived the study, participated in its design and coordination. All authors read and approved the final manuscript.Detailing the information on the sequenced libraries and the list of newly identified 46 sRNAs.Click here for fileC. acetobutylicum. Pair-wise and point by point by comparison of each stress level to the no stress control using DEseq at a p-value ≤ 0.05. Table S2: Probes sequences used for Northern analysis.Differential expression of annotated mRNAs of Click here for fileC. acetobutylicum hfq  with the Hfq from E. coli (3GIB_B), reveals conservation in the secondary structure on the Hfq monomeric unit. (A) The α-β1-5 structural unit can be found to be conserved. (B) The corresponding conservation in the protein sequence is displayed below.CBLAST of the Click here for fileContaining the Hfq binding model.Click here for fileHierarchical clustering of the 65 sRNAs belonging to the Hfq constellation (see text for details).Click here for filesolB to the sol (adhE1-ctfA-ctfB) operon.Putative antisense binding of Click here for fileHierarchical clustering of 159 sRNAs under both butanol and butyric acid stress.Click here for file


People often avoid information and situations that have the potential to contradict previously held beliefs and attitudes . According to the motivated social cognition model of political ideology, conservatives tend to have stronger epistemic needs to attain certainty and closure than liberals. This implies that there may be differences in how liberals and conservatives respond to dissonance-arousing situations. In two experiments, we investigated the possibility that conservatives would be more strongly motivated to avoid dissonance-arousing tasks than liberals. Indeed, U.S. residents who preferred more conservative presidents  complied less than Americans who preferred more liberal presidents (Barack Obama and Bill Clinton) with the request to write a counter-attitudinal essay about who made a “better president.” This difference was not observed under circumstances of low perceived choice or when the topic of the counter-attitudinal essay was non-political . The results of these experiments provide initial evidence of ideological differences in dissonance avoidance. Future work would do well to determine whether such differences are specific to political issues or topics that are personally important. Implications for political behavior are discussed. I know that most men…can very seldom discern even the simplest and most obvious truth if it be such as to oblige them to admit the falsity of conclusions they have formed, perhaps with much difficulty – conclusions of which they are proud, which they have taught to others, and on which they have built their lives. , in comparison with liberals. Consistent with this notion, a few studies indicate that selective exposure is more prevalent on the political right than the left Given that political conservatives possess stronger needs for order, structure, consistency, and closure and weaker tolerance for uncertainty and ambiguity compelled by overwhelming force of circumstance to think or act in a way that is contrary to their own beliefs. It would appear that dissonance arousal requires some degree of choice or volition of their own accord. For this reason, we would expect that there would be no ideological differences in compliance with a request to engage in counter-attitudinal thought or behavior under conditions of low perceived choice . However, if conservatives are more strongly motivated to avoid dissonance-arousing situations, they should be less likely than liberals to comply with a request to engage in counter-attitudinal behavior under conditions of high perceived choice . In two experiments we investigated this possibility, using the “classic” method of asking participants to construct counter-attitudinal arguments under conditions of high vs. low perceived choice.People do not experience cognitive dissonance when they are We also sought to determine whether conservatives would eschew dissonance-arousing tasks in general – or only in explicitly political contexts. On one hand, chronic individual differences in cognitive and motivational styles could lead liberals and conservatives to differ in terms of dissonance avoidance even when it comes to nonpolitical topics. On the other hand, political  issues may be of much greater importance or commitment to most people, especially those who identify themselves as either liberal or conservative. For ideologues, political issues would likely possess greater potential for arousing dissonance, in comparison with nonpolitical issues. The discussion of explicitly political  issues would probably also increase the personal salience of ideological commitments, thereby exacerbating social and psychological differences between liberals and conservatives.In Experiment 1, we investigated the hypothesis that political orientation would moderate responses to a dissonance-arousing situation. Specifically, we compared supporters of George W. Bush, a conservative, Republican president, to supporters of Barack Obama, a liberal, Democratic president, when both were instructed to construct counter-attitudinal essays in political and non-political domains. We hypothesized that those who were more politically conservative  would be more motivated to avoid the dissonance-arousing political task than those who were more liberal , especially under circumstances of high (vs. low) choice. We also examined the possibility that ideological differences would arise with respect to non-political (Mac vs. PC) as well as political preferences.The study was approved by the University Committee on Activities Involving Human Subjects (UCAIHS), the Institutional Review Board at New York University. All participants provided written informed consent prior to participation in the study.One hundred and eighty participants  in the United States were recruited online through Amazon's Mechanical Turk between October 2011 and January 2012  to 9 (strongly approve). We also asked participants to indicate their attitudes toward two computer products, Macs and PCs . Responses ranged from 1 (strongly dislike) to 9 (strongly like). Participants were also asked which president they preferred with a binary response option , and which type of computer they preferred . We used the binary outcome to determine which essay would be considered counter-attitudinal for each participant. Computer preferences  were weakly correlated with presidential preferences , r (165)  = −.13, p = .09.Using the classic “induced compliance” paradigm high choice condition were told, “At the moment, we don't have enough of certain kinds of essays, and we need to collect several more. We would really, really appreciate it if you would help us out by writing one. Please, would you be willing to write an essay arguing that Barack Obama is [George W. Bush was] a better president than George W. Bush was [Barack Obama is]? Would you be willing to do this for us?” Participants assigned to the low choice condition were simply instructed: “You are assigned to write an essay arguing that Barack Obama is [George W. Bush was] a better president than George W. Bush was [Barack Obama is].”We used the same instructions employed in classic studies of induced compliance high choice condition were able to respond “yes” or “no” to the request; if they responded “yes,” they were directed to the essay task, and if they responded “no,” they were instead taken to the next section of the experiment. Participants assigned to the low choice condition were simply directed to the essay-writing task.Because we were interested in reactions to dissonance-arousing situations, all participants were asked to write counter-attitudinal essays. Thus, if a participant indicated in the initial survey that he or she preferred George W. Bush and Macs over Barack Obama and PCs, respectively, this participant would be instructed to write essays arguing that Obama is a better president than Bush and that PCs are better computers than Macs. Participants assigned to the extremely liberal) to 11 (extremely conservative).Once they entered the essay task, participants were guided through a “brainstorming process,” with prompts such as: “Please think of a title,” “Please write the first main point of your argument,” “Please write the second main point of your argument,” and “Please state a conclusion to your argument.” At the conclusion of the experiment, participants responded to the same attitude items administered earlier, except that these were now embedded in a list that included filler items. They also provided demographic information, including their political orientation, which could range from 1 .Thirteen participants (6 from the low choice condition and 7 from the high choice condition) were excluded from the main analyses  because they wrote To investigate our hypothesis, we conducted a binary logistic regression that tested for main and interaction effects of ideological preference (support for Bush [coded as 1] vs. Obama [−1]), perceived choice high . It was r (165)  = .56, p<.001. Those who preferred Bush over Obama  were indeed more conservative than those who preferred Obama over Bush , t (154)  = −8.62, p<.001.Furthermore, those who preferred Bush clearly approved of him  and disapproved of Obama , t (50)  = 10.38, p<.001, whereas those who preferred Obama approved of him  and disapproved of Bush , t (115)  = −13.78, p<.001.Consistent with a forced choice dissonance paradigm, ideological asymmetries were assessed in terms of either/or categorical preferences for Bush vs. Obama. Unsurprisingly, categorical preferences were strongly correlated with political orientation for those who reported it, no Bush supporters who were assigned to the high choice condition complied with the request to write an essay favoring Obama over Bush. Because the analysis could not be performed with an empty cell, we selected one participant at random and recoded his compliance status. This recoding entails a more stringent test of our hypothesis.We used generalized estimating equations to determine the effects of ideological preferences, perceived choice, and essay content on compliance behavior. We hypothesized that conservative supporters of President Bush would be more motivated to avoid dissonance and therefore less likely to comply with the request to write a counter-attitudinal essay under conditions of high (vs. low) choice, in comparison with liberal supporters of President Obama. In fact, we found that M = .28, where 1 =  compliance by all participants) were much less likely than those assigned to the low choice condition (M = .89) to write a counter-attitudinal essay, b = −1.88, SE  = .25, Wald  = 56.94, p<.001, setting aside ideological preferences (for the time-being).  A main effect of essay content revealed that participants were also less likely to comply with instructions to write a counter-attitudinal political essay (M = .46) than a nonpolitical essay (M = .59), b = −.66, SE  = .21, Wald  = 10.23, p = .001. A main effect of ideological preferences indicated that Bush supporters (M = .48) were less likely to comply than were Obama supporters (M = .55) in general, b = −.52, SE  = .25 Wald  = 4.29, p = .04. None of the two-way interactions attained conventional levels of statistical significance. The analysis did yield a significant three-way interaction involving ideological preference, choice condition, and essay content, b  = −.49, SE  = .21, Wald  = 5.67, p = .02.The analysis yielded a main effect of perceived choice, indicating that participants assigned to the high choice condition (M = .04) were less likely to comply with the dissonance-arousing, politically relevant task than those who preferred Obama (M = .28), b = −2.36, SE = 1.05, Wald  = 5.03, p = .03, under circumstances of high perceived choice. However, those who preferred Bush (M = .87) were not significantly different from those who preferred Obama (M = .80) under low choice with respect to the political task, b = .51, SE  = .72, Wald  = .50, p = .48. Nor were they less likely to comply with instructions to write a counter-attitudinal nonpolitical essay (about computers) under high choice (M = .25), in comparison with Obama supporters (M = .38), b = −.61, SE  = .50, Wald  = 1.49, p = .22  was dichotomous in nature  .22 see . These rb = −1.70, SE  = .22, Wald  = 61.37, p<.001. As before, there was also a main effect of essay content, such that participants were less likely to write a counter-attitudinal essay in the political than nonpolitical domain, b = −.57, SE  = .15, Wald  = 14.75, p<.001. A main effect of political orientation revealed that greater conservatism was associated with a decreased likelihood of compliance, b = −.14, SE  = .06, Wald  = 6.06, p = .01. This main effect was qualified by a pair of two-way interactions, which suggested that the relationship between conservatism and decreased compliance was stronger for (a) the nonpolitical than the political topic , and (b) under conditions of high (vs. low) choice . In addition, a significant three-way interaction involving political orientation, essay content, and choice condition was observed, b = −.14, SE  = .03, Wald  = 18.99, p<.001. Simple slopes analyses revealed that, consistent with predictions (and the results summarized above), greater conservatism was associated with a decreased likelihood of complying with instructions to write a counter-attitudinal political essay under circumstances of high perceived choice . This pattern did not emerge with respect to the political task under low choice conditions; in fact, there was a marginal tendency for conservatives to comply slightly more often with instructions, b = .18, SE  = .10, Wald  = 3.28, p = .07. There was an even weaker relationship between political orientation and likelihood of compliance with instructions to write a counter-attitudinal essay on a nonpolitical topic (computer preferences) under conditions of high choice, b = −.17, SE  = .10, Wald  = 2.75, p = .10.We also conducted a parallel analysis in which political orientation was treated as a continuous measure of ideological preference . Results were very similar to those summarized above. The analysis yielded a main effect of perceived choice, indicating that participants assigned to the high choice condition were less likely than those assigned to the low choice condition to comply with instructions to write a counter-attitudinal essay, The purpose of Experiment 2 was to replicate and extend the findings of Experiment 1. There are a number of factors in addition to political ideology that might have contributed to the difference in compliance behavior we observed with respect to Bush and Obama supporters. For instance, the fact that Obama was the current President (and would be seeking re-election) may have increased resistance among his ideological adversaries (who were in the midst of selecting his Republican opponent during the time of data collection). Conversely, Obama supporters may have been more willing to argue in favor of Bush because he was no longer running the country, or because of strong national support for him in the aftermath of the terrorist attacks of September 11, 2001. Therefore, we conducted an additional experiment with a different pair of presidents, namely Ronald Reagan and Bill Clinton. These are both former two-term presidents who do not differ in terms of race (as Bush and Obama do) and who left office with similar public approval ratings One hundred fifty-nine participants  in the U.S. were recruited online through Amazon's Mechanical Turk from November 2011 to January 2012. Nine participants (3 from the low choice condition and 6 from the high choice condition) were excluded from the main analyses  because they wrote pro-attitudinal essays despite having received instructions to write counter-attitudinal essays. The general procedure was identical to that of Experiment 1, except that participants were asked to write counter-attitudinal essays on two new topics: (a) whether Bill Clinton or Ronald Reagan was the better president, and (b) whether coffee or tea is the superior drink. Consistent with the results of pilot testing, study participants' beverage preferences  were uncorrelated with their political preferences , r (148)  = .002, p = 98.r (148)  = .43, p<.001. Those who preferred Reagan over Clinton  were indeed more conservative than those who preferred Clinton over Reagan , t (148)  = −5.80, p<.001. Furthermore, those who preferred Reagan approved of him  more strongly than they approved of Clinton , t (53)  = 7.42, p<.001, and those who preferred Clinton approved of him  more than they approved of Reagan , t (95)  = −11.44, p<.001.To assess our predictions, we conducted a binary logistic regression that tested for main and interaction effects of ideological preference (support for Reagan [coded as 1] vs. Clinton [−1]), perceived choice high . As in EM = .25) were much less likely than those under low choice (M = .88) to agree to write a counter-attitudinal essay, b = −1.67, SE  = .21, Wald  = 63.68, p<.001. A main effect of essay content revealed that participants were less likely to comply with the task when it involved politics (M = .45), as compared with beverages (M = .58), b = −.52, SE  = .16, Wald  = 10.02, p = .002. No other main or interaction effects attained conventional levels of significance.We again used generalized estimating equations to determine the effects of ideological preferences, perceived choice, and essay content on compliance behavior. Because we had a strong prediction that those who preferred Reagan would comply less than those who preferred Clinton, we used one-tailed tests to assess simple contrasts but report the results of both one-tailed and two-tailed tests for the sake of clarity and completeness. The analysis yielded a main effect of choice condition, indicating that participants under high perceived choice (M = .10) were less likely to comply with the request to write a counter-attitudinal political essay than were Clinton supporters (M = .22), b = −1.36, SE  = .80, Wald  = 2.91, p = .044, one-tailed . Under low choice, no effect of ideological preference was observed b = −.68, SE  = .68, Wald  = 1.00, p = .16, one-tailed . Those who preferred Reagan (M = .79) were as likely as those who preferred Clinton (M = .84) to write a counter-attitudinal essay about politics under conditions of low choice. No differences were observed either between those who preferred Reagan (M = .30) and Clinton (M = .38) in terms of compliance with instructions to write a counter-attitudinal essay about coffee vs. tea under high choice, b = −.42, SE  = .50, Wald  = .71, p = .20, one-tailed . Replicating Experiment 1, these results lend further support to the notion that there is an ideological difference in reactions to dissonance-arousing situations, insofar as those who preferred Reagan (vs. Clinton) were more likely to avoid a highly dissonant task–but only when it involved a political topic.To probe our focal hypothesis that those who preferred Reagan would be less likely than those who preferred Clinton to comply with a dissonance-arousing task in the political domain, we again conducted simple slopes analyses to spotlight the specific comparisons of interest b = −1.61, SE  = .20, Wald  = 63.58, p<.001. A main effect of essay content revealed that compliance was lower in the political than nonpolitical domain , b = −.41, SE  = .15, Wald  = 7.34, p = .007. No other main or interaction effects reached conventional levels of significance. Simple slopes analyses failed to identify significant effects of political orientation on compliance with instructions to write a counter-attitudinal political essay under conditions of high  or low choice . Political orientation was also unrelated to compliance with instructions to write a counter-attitudinal essay in a nonpolitical domain under high choice, b = .02, SE = .09, Wald = .04, p = .85. Thus, the results of Experiment 2 replicated those of Experiment 1 when ideological preferences were estimated using categorical (either/or) presidential preferences but not when they were estimated using the continuous measure of political orientation administered at the end of the experiment. We will return to this issue in the General Discussion.Once again, we conducted a parallel analysis in which political orientation was treated as a continuous measure of ideological preference, despite the fact that this measure was taken at the end of the experiment, following the experimental manipulation. This analysis yielded a main effect of choice, such that participants assigned to the high choice condition were less likely than those assigned to the low choice condition to write a counter-attitudinal essay, The current research suggests that, in some situations at least, conservatives avoid dissonance-arousing situations to a greater extent than liberals do. Specifically, we found that supporters of Republican presidents were less likely than supporters of Democratic presidents to comply with instructions to write counter-attitudinal essays under circumstances of high perceived choice. This result was specific to the political domain. That is, the ideological difference was observed when participants were asked to write an essay in favor of their less preferred president but not when they were asked to write an essay in favor of their less preferred computer brand or beverage. These results suggest that there are ideological differences in the avoidance of situations that have the potential to arouse cognitive dissonance.single Bush supporter who was willing, when given a choice, to write a counter-attitudinal essay suggesting that Obama is a better president than Bush was. Some conservatives found the task instructions themselves to be extremely distasteful, making post-study comments such as: “Yeah who would actually aprove [sic] of the job Obama has done? I mean really!” and “Not for all the tea in China would I write that.” Similar patterns were observed with respect to Reagan and Clinton supporters, but the ideological difference was weaker, possibly because both presidents left office with high job approval ratings and are now considered to be among the two best presidents in recent history One of the most striking results is that we could not find a relative (rather than an absolute) difference in dissonance avoidance.To be sure, neither liberals nor conservatives were enthusiastic about writing a counter-attitudinal political essay, but several Obama supporters did undertake the dissonance-arousing task under high choice, writing statements such as: “George Bush did what he said he was going to do but Obama filled us with liberal false promises,” and “There were extended periods of economic growth under George W. Bush, while the economy has lagged badly under Barack Obama.” Given an opportunity to provide post-study feedback, one Obama supporter said that she “hated having to brainstorm in support of Bush” but still managed to write a counter-attitudinal essay under high perceived choice. Another liberal apparently enjoyed the exercise, commenting, “This was fun!” In contrast, no Bush supporters agreed to write pro-Obama essays under high choice, nor did any of them state that the exercise was “fun.” The ideological difference we observed appears to be a Our findings are broadly consistent with previous work on selective exposure. Many studies have shown that individuals avoid exposure to potentially dissonance-arousing, attitude-incongruent information, especially when such information is high in personal or ideological relevance not writing an essay supporting a liberal president. Alternatively, liberals may have been more motivated than conservatives to test their “ability to craft logical arguments.” Importantly, however, we observed no ideological differences in willingness to comply with the same instructions when participants were asked to write counter-attitudinal essays on non-political topics.An alternative explanation for the ideological discrepancy in compliance behavior is that conservatives were more sensitive than liberals to cues communicating descriptive social norms. It is conceivable, for instance, that the wording of the instructions in the high choice condition, which suggested that there was a shortage of certain kinds of essays, was interpreted as indicating that very few people were willing or able to argue in favor of President Obama or Clinton. There is indeed evidence that conservatives value conformity more than liberals do choosing one stimulus over another is critical to the experience of dissonance arousal In both experiments, we supplemented our primary statistical analyses, which operationalized ideological preferences in terms of categorical (either/or) preferences for one president over another, with additional analyses using a continuous measure of political orientation . With respect to Experiment 1, the results were virtually identical for the two types of analyses. For Experiment 2, however, the hypothesized pattern emerged when ideological preferences were treated as a categorical variable but not as a continuous measure. It is possible that this difference is attributable to the fact that, as cognitive dissonance theorists have argued, the act of Our finding that political conservatives were less willing than liberals to engage in dissonance-arousing tasks is in line with previous theory and evidence concerning ideological differences in cognitive and motivational styles stronger commitment to their initial beliefs after being exposed to discrediting evidence. Liberals failed to correct their initial misperceptions in the face of disconfirming information, but they did not exhibit a backfire effect.If there are indeed ideological differences in the tendency to avoid dissonance-arousing thoughts, as our data suggest, this may also help to explain why conservatives are more prone to engage in biased forms of moral reasoning Cooper and Mackie It should be noted, at least in passing, that epistemic disadvantages (such as attitude bolstering and avoidance of disconfirming information) might translate into real political advantages  when it comes to the ballot box. At the same time, the refusal to consider opposing points of view in good faith may hinder the functioning of a democratic society


PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of  LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension. In this work, the authors tested if tomato bushy stunt virus (TBSV), which, similar to many (+)RNA viruses, depends on host membrane biogenesis, is affected by deletion of the single lipin gene (PAH1) in yeast model host. They show that pah1Δ yeast supports increased replication of TBSV. They demonstrate that TBSV takes advantage of the expanded ER membranes in lipin-deficient yeast to efficiently assemble viral replicase complexes. Their findings suggest possible positive effect of a genetic disease caused by mutation on the replication of an infectious agent.Genetic diseases alter cellular pathways and they likely influence pathogen-host interactions as well. To test the relationship between a key cellular gene, whose mutation causes genetic diseases, and a pathogen, the authors have chosen the cellular lipins. Lipins are involved in a key cellular decision on using lipids for membrane biogenesis or for storage. Spontaneous mutations in the  Positive-stranded (+)RNA viruses are important and emerging human, animal and plant pathogens. These viruses utilize cellular membranes and lipids during replication to build viral replicase complexes (VRCs) tomato bushy stunt virus (TBSV) (polpol are translated from the genomic (g)RNA and p92pol is the result of translational readthrough of the p33 stop codon pol is the RNA-dependent RNA polymerase Tombusviruses, such as s (TBSV) , are amocucumber necrosis virus (CNV)] in vitro(+)RNA viruses either target existing subcellular membranes or they extensively remodel membranes to support the VRC assembly. Interestingly, tombusviruses can utilize pre-existing membranes, but also remodel subcellular membranes by forming multivesicular body-like structures in infected cells LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension Genetic diseases can alter critical cellular processes, which might affect pathogens that have to take advantage of cellular resources. The host lipins are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage PAH1 (phosphatidic acid phosphohydrolase) gene is the homolog of the mammalian fat-regulating protein Lipin-1 PAH1 codes for a phosphatidate phosphatase (PAP), which dephosphorylates phosphatidic acid (PA), yielding diacylglycerol (DAG)  . Pah1p iPAH1, whose inactivation is responsible for proliferation and enlargement of the ER membrane, facilitates robust RNA virus replication in yeast. Thus, surprisingly, a host gene whose homologs are involved in genetic diseases in humans, greatly affects virus replication.Since (+)RNA viruses likely depend on membrane biogenesis, we examined the relationship between the host lipins and tombusviruses, based on yeast model host. In this paper, we document that deletion of the yeast lipin gene, in vivoin vitroPAH1 gene, which leads to ER membrane enlargement and proliferation pah1Δ yeast. These data suggest that the enlarged ER might provide favorable microenvironment for TBSV and CNV replication or tombusviruses might be able to take advantage of the increased phospholipid content of the cell. Interestingly, the levels of p33 and p92pol replication proteins were increased in pah1Δ yeast  obtained from pah1Δnem1Δ or wt yeast  and (+)-strand RNAs in the CFEs  for TBSV replication, we observed that the replication of TBSV RNA was 2-to-4-fold higher in ER preparations obtained from pah1Δnem1Δ yeast versus the wt yeast, we performed confocal laser microscopy with fluorescently tagged tombusvirus p33 replication protein. When we looked at the localization of YFP-p33 at an early time point (4 hours), we observed that a large portion of YFP-p33 was still cytosolic and a small number of punctate structures were forming in wt yeast RNA, host factors and cellular membranes pah1Δnem1Δ yeast cells resulted in ∼3-fold higher replicase activity in vitro   , a tombuexpected . Based opah1Δ and wt yeasts (PAH1.The second RNA virus tested was Nodamura virus (NoV) , an inset yeasts , suggestArabidopsis Pah2p protein in N. benthamiana leaves using an Agrobacterium-based expression system. Plants have two phosphatidate phosphatase-coding genes, PAH1 and PAH2, which are highly homologous with the yeast PAH1 gene and they can complement the phenotypes in pah1Δ yeast PAH1 and PAH2 in Arabidopsis thaliana causes ER expansion and increased phospholipid synthesis, similar to the phenotypes observed in pah1Δ yeast To obtain evidence if the conserved lipin-like PAP phosphatidate phosphatase also plays a role in tombusvirus replication in plants, we over-expressed the N. benthamiana leaves   , which i AtPah2p . Thus, apah1Δ  yeasts or Dgk1p diacylglycerol kinase over-producing yeast; (ii) increased accumulation and stability of tombusvirus p33 replication protein in pah1Δ yeast; (iii) the enhanced in vitro assembly of the tombusvirus VRCs in a CFE-based assay derived from yeast lacking Pah1p; (iv) detection of highly abundant p33 replication proteins in isolated ER fraction or using confocal microscopy in yeast lacking Pah1p; (v) the higher in vitro activity of the tombusvirus replicase purified from CFE obtained from yeast lacking Pah1p than from wt yeast; and (vi) the stimulatory role of the membrane fraction of pah1Δ yeast in the CFE-based TBSV replication assay. The efficient utilization of the expanded ER membrane is reflected by the rapid localization of the p33 replication protein to the ER membrane at an early time point in yeast lacking Pah1p. In addition, it appears that the tombusvirus replicase assembles faster in yeast lacking Pah1p than in wt yeast. Thus, our model proposes that TBSV and CNV efficiently subvert the expanded ER membranes and utilizes the abundant phospholipids generated in pah1Δ or pah1Δnem1Δ lipin-deficient yeast, leading to robust viral replication. These tombusviruses replicate in the expanded ER membranes in lipin deficient yeast although the peroxisomal membranes are also present (and some of these membranes are still utilized by TBSV and CNV), suggesting that the expanded ER likely contains favorable microenvironment (membrane microdomains) for these tombusviruses. Therefore, it seems that TBSV and CNV are flexible in utilizing peroxisomal and ER membranes depending on cellular conditions. Accordingly, TBSV is capable to assemble the VRCs on ER and mitochondrial preparations in vitroTombusviruses, like many (+)RNA viruses, depend on host membrane biogenesis during replication. Thus, conditions that induce membrane biogenesis in cells might affect replication of some RNA viruses. Accordingly, the key finding in this paper is that tombusviruses (TBSV and CNV) can take advantage of expanded ER membrane surface, which is due to deletion of the cellular Pah1p PAP enzyme leading to massive enlargement of ER membranes and increased phospholipid biosynthesis in yeast pah1Δ yeast. CIRV was also more restrictive in the CFE-based replication assay, utilizing the mitochondrial membranes more efficiently than the ER membranes Similar to tombusviruses, other (+)RNA viruses subvert various intracellular membranes for construction of VRCs, and the ER membrane is often critical for these processes PAH1 in tombusvirus replication based on yeast model host was further supported by data obtained in natural plant host by over-expression of AtPah2p. The over-expression of the yeast Pah1p  in yeast and the AtPah2p in N. benthamiana strongly inhibited tombusvirus replication. This inhibition is likely due to strong competition between cellular phospholipid pathways driven by the over-expressed PAP enzyme and the need for phospholipid-containing cellular membranes in tombusvirus replication The role of PAH1), the role of the PAP enzyme in phospholipid pathways are conserved in yeast, plants and animals. Deletion/mutations of the lipin gene is known to facilitate membrane biogenesis in all these organisms. Therefore, the effects seen with tombusviruses and RCNMV might also manifest with animal viruses possibly leading to exploitation of the expanded ER membranes during infections. Thus, it is possible that one disease state might facilitate the development of robust viral infection at the cellular level.Our findings might also influence the current view on genetic mutations and diseases versus pathogen infections. Mutations in cellular genes frequently alter cellular pathways and they can cause genetic diseases. The same gene mutations/changes might also influence pathogen-host interactions as well. For example, the cellular lipin, which is involved in a key cellular decision on using lipids for membrane biogenesis or for storage Saccharomyces cerevisiae strain RS453 , pah1Δ (pah1Δ::TRP1 derivative of RS453) and pah1Δnem1Δ (pah1Δ::TRP1 nem1Δ::HIS3 derivative of RS453) were published previously We found that the pah1Δnem1Δ yeast was more consistent than pah1Δ yeast in supporting high level of CNV and TBSV repRNA replication for yet unknown reasons. Therefore, most of the experiments were performed with both strains or with only pah1Δnem1Δ yeast strain.LEU2 selection), UpGBK-ADH-His-p33/GAL1-DI-72 (URA3 selection), HpESC-GAL1-His-p33/GAL10-DI-72 (HIS3 selection) HIS3 selection) URA3 selection) URA3 selection), LpGAD-Pho86-CFP (LEU2 selection) URA3 selection), HpYES-GAL1-C95 (HIS3 selection) E. coli were described earlier LEU2 selection) URA3 selection), HpYES-GAL1::C95 (HIS3 selection), UpYES-GAL1::T92 (URA3 selection), HpESC-GAL1::T33/ GAL10::DI-72 (HIS3 selection) The yeast expression plasmids, pYEplac181, pYEplac181-wt-Pah1 and pYEplac181-Pah1-7A have been obtained from Dr. George M. Carman pah1Δ strain was transformed with pESC-CUP1-His-p92 (URA3 selection), pESC-GAL1-His-p33/GAL10-DI-72 (HIS3 selection) and LEU2 based plasmids: pYEplac181 (vector control), pYEplac181-wt-Pah1p (for expression of wt Pah1p) or pYEplac181-Pah1p-7A (for expression of constitutively active phosphorylation-deficient Pah1p), respectively − medium containing 2% galactose and then the cultures were harvested after 24 h 50 µM CuSO4 induction.Yeast wt RS453 and pah1Δnem1Δ strains were transformed with plasmid UpYES-GAL-Hisp33 expressing His6-tagged CNV p33 from the galactose-inducible GAL1 promoter. Yeast transformants were cultured overnight in SC-U− medium containing 2% glucose at 23°C. Yeast cultures were transferred to SC-U− medium supplemented with 2% galactose for 3 h at 23°C. Then, the cultures were shifted back to the SC-U− medium supplemented with 2% glucose and cycloheximide . The amount of p33 was detected by Western blotting with anti- His6 antibody at given time points after cycloheximide treatment. Each sample loading was adjusted based on total protein levels as determined by SDS-PAGE To study the stability of p33 replication protein in yeast, RS453 and pah1Δ or pah1Δnem1Δ yeast cells − (synthetic complete dropout medium lacking leucine and Uracil) medium containing 2% galactose. Replication of TBSV repRNA was induced by adding 50 µM CuSO4 into the medium and, then, the samples were harvested at different time points. The TBSV replication proteins-based assay was similar to that described for CNV above, except yeasts were transformed with plasmids UpYES-GAL1::T92 and HpESC-GAL1:: T33/GAL10::DI-72 were directly grown in 2 ml SC-UH− medium containing 2% galactose for 24 h at 23°C. For CIRV replication assay, yeast strains were transformed with plasmid UpESC-GAL1-C36/GAL10-DI-72 and HpYES-GAL1-C95 and then the yeast was grown at 23°C in 2 ml SC-UH- medium containing 2% galactose for 2 days. Standard RNA extraction and Northern blot analysis was performed as described previously Replication assays were performed by measuring the accumulation of DI-72(+) repRNA relative to the accumulation of the cellular 18S rRNA. For the CNV replication proteins-based replication assay, RS453 (wt), in vivo-assembled tombusvirus replicase complexes. Each membrane fraction preparation was adjusted by the relative amounts of His6-tagged p92 and comparable amounts of replicase from each preparation were used in the subsequent replicase assay. The replicase assay was performed as described in vitro reaction (50 µl) contained 10 µl of the normalized MEFs preparations, 50 mM Tris–Cl pH 8.0, 10 mM MgCl2, 10 mM DTT, 0.1 U RNase inhibitor, 10 mM ATP, 10 mM CTP, 10 mM GTP and 0.1 µl of 32P-UTP (3000 Ci/mmol). Reaction mixtures were incubated for 3 h at 25°C, followed by phenol/chloroform extraction and isopropanol/ammonium acetate (10∶1) precipitation. The 32P-UTP-labeled RNA products were analyzed in 5% acrylamide/8 M urea gels.The membrane fractions were prepared as described previously pah1Δnem1Δ were prepared as described earlier in vitro reaction was performed in 20 µl total volume containing 2 µl of adjusted CFE, 0.5 µg DI-72 (+)repRNA transcript, 0.5 µg purified MBP-p33, 0.5 µg purified MBP-p92pol (both recombinant proteins were purified from E. coli), 30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol, 0.4 µl actinomycin D (5 mg/ml), 2 µl of 150 mM creatine phosphate, 0.2 µl of 10 mg/ml creatine kinase, 0.2 µl of RNase inhibitor, 0.2 µl of 1 M dithiothreitol (DTT), 2 µl of 10 mM ATP, CTP, and GTP and 0.25 mM UTP and 0.1 µl of 32P-UTP. Reaction mixtures were incubated 3 h at 25°C, followed by phenol/chloroform extraction and isopropanol/ammonium acetate (10∶1) precipitation. 32P-UTP-labeled RNA products were analyzed in 5% acrylamide/8 M urea gels 32P-labeled RNA samples were directly loaded onto the gel without heat treatment. Membrane and soluble fractions of these CFEs or in vitro reaction were separated by centrifugation at 35,000 g for 30 min and then mixed them in various combinations (described in the figure legends).CEFs from RS453 (wt) or in vitro TBSV replication assay was performed as described for in vitro replication assay with MEF except that using the purified ER fraction.Yeasts were transformed and cultured as described in the main text for TBSV or CIRV repRNA replication in yeast. The ER fractions were prepared as described earlier 6-tagged recombinant p33. At the end of the in vitro replicase assembly assay, the reaction mixture was diluted with 800 µl solubilization buffer, and the replicase complex was purified followed the procedure described previously 200 µl of the CFE-based replication assay was performed as described above, except that only rATP and rGTP were used. Also, the CFE assay contained the MBP and His− medium containing 2% galactose at 23°C for 24 h and then sample were collected and analyzed by confocal microscopy as described − medium containing 2% glucose and then transferred to media containing 2% galactose, and then, samples were collected for microscopy analysis at given time points.To visualize the ER, Pho86-CFP was used as a marker, while peroxisomes were monitored with the help of Pex13-CFP (a peroxisome membrane marker) Confocal laser scanning micrographs of yeast cells were acquired on an Olympus FV1000 microscope  as described 6 antibody was used as the primary antibody (Invitrogen) and the secondary antibody was alkaline-phosphatase conjugated anti-mouse IgG (Sigma). For cellular protein markers, the following antibodies were used: anti-3-phosphoglycerate kinase (anti-PGK), and anti-heat shock protein 70 (anti-Hsc70) . Sec61p antibody was provided by Tom Rapoport, Harvard Medical School. Fox3p antibody was provided by Daniel J. Klionsky, University of Michigan.To prepare the total protein sample for Western blotting, we followed a previous protocol Arabidopsis thaliana Pah2p in N. benthamiana leaves was performed by agroinfiltration A. thaliana PAH2 (At5g42870) was amplified by PCR using primers #4630 (GCCGGATCCATGAATGCCGTCGGTAGGATC) / #4631 (CGGCTCGAGTCACATAAGCGATGGAGGAGGCAG) and genomic DNA as template. The obtained PCR product was digested with BamHI and XhoI, purified and ligated into pGD-L BamHI and SalI. The resulting plasmid was transformed into Agrobacterium tumefaciens C58C1 N. benthamiana plants were infiltrated with A. tumefaciens (OD600 = 0.8) carrying pGD-L-PAH2 or the control empty plasmid pGD. Two days later, the same leaves were infiltrated with A. tumefaciens (OD600 = 0.2) carrying pGD-CNV to launch CNV replication, or pGD-CIRV to launch CIRV replication 32P-labeled probes complementary to the 3′ end of the viral RNAs Transient expression of N. benthamiana plants were infiltrated with A. tumefaciens (OD600 = 0.8) carrying pGD-L-PAH2 or the control empty plasmid pGD. Two days later, the same leaves were inoculated with infectious saps containing TBSV or RCNMV virions. Leaf samples were collected 3 days later and total RNA was extracted. RNA accumulation was analyzed by agarose gel electrophoresis and Northern blotting using 32P-labeled probes complementary to the 3′ end of the TBSV RNA or RCNMV RNA1 For studies with TBSV and RCNMV, Figure S1PAH1 deletion, leads to ER membrane expansion. Bottom panel: detection of the overproduced Dgk1p in yeast. The His6-tagged Dgk1p, His6-p33 and His6-p92 were detected by Western blotting.Over-expression of Dgk1p enhances TBSV repRNA accumulation in wt yeast as shown by Northern blotting. Note that overproduction of Dgk1p, similar to (EPS)Click here for additional data file.Figure S2PAH1 and double PAH1 and NEM1 deletions on TBSV repRNA accumulation in yeast. Top panel: Replication of the TBSV repRNA in wt, pah1Δnem1Δ and pah1Δ yeasts was measured by Northern blotting 8 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Bottom panel: The accumulation levels of His6-p92 and His6-p33 were tested by Western blotting. Each experiment was repeated.Comparable effects of single (EPS)Click here for additional data file.Figure S3PAH1 and double PAH1 and NEM1 deletions on in vitro TBSV repRNA replication. (A) The scheme of the in vitro TBSV replication assay based on the isolated membrane fraction carrying the tombusvirus replicase and the bound RNA template. (B) Top panel: Comparable in vitro replication of TBSV repRNA in the isolated membrane fraction from pah1Δ yeast when compared with that from pah1Δ nem1Δ yeast. Note that the levels of His6-p92 replication protein were normalized as shown in the second panel. The third, fourth and fifth panels show the accumulation levels of His6-p33, and the cellular Sec61p (an ER marker) and Ssa1p Hsp70. Samples were taken at 24 h time point.Comparable effects of single (EPS)Click here for additional data file.Figure S4pah1Δnem1Δ yeast. (A) The scheme of the CFE-based TBSV replication assay. Purified recombinant TBSV p33 (7 pmol) and p92pol (4 pmol) replication proteins in combination with DI-72 (+)repRNA (0.5 µg) were added to the CFEs made by mixing the soluble and membrane fractions as shown. (B) Denaturing PAGE analysis of the 32P-labeled repRNA products obtained is shown. The TBSV replication assays were based on the mixed soluble and membrane fractions of CFEs prepared from wt or pah1Δnem1Δ yeasts, as shown. The full-length single-stranded repRNA is pointed at by an arrow.Enhanced TBSV replication depends on the membrane fraction of CFE prepared from (EPS)Click here for additional data file.Table S1Viruses used in this study.(DOC)Click here for additional data file.


Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus  Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell's cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites. Enterovirus, are the major causative agent of the common cold. Additionally, they play an important role in the exacerbation of asthma, cystic fibrosis, and chronic obstructive pulmonary disease Human rhinoviruses (HRV), members of the picornavirus family, genus Minor group rhinoviruses, exemplified by the prototype strain HRV2, bind members of the low-density lipoprotein receptor (LDLR) family including LDLR and LDLR-related protein for entry via clathrin-dependent endocytosis in vitro. Exposure to pH<5.8 converts native HRV2 preferentially into A-particles whereas incubation at 50°C–56°C in low ionic strength buffers favours conversion into B-particles (empty capsids) In vivo, and in the presence of liposomes in vitroin vivo uncoating is unknown. It appears highly likely that either the poly-(A) tail at the 3′-end or the VPg peptide linked to the 5′-phosphate of the RNA via a tyrosine ester, begins to emerge from the virion since other modes might be unproductive . Directionality of this process may indicate that the RNA adopts a defined conformation inside the viral shell suggesting a well-organized process of assembly and uncoating. Here, we show that RNA exit does indeed occur in a specific and ordered manner, starting from the 3′-end.These structural modifications can be mimicked, at least partially, r = 12 to 80 kD) were labeled with Cy3 at their N-termini and the change of their diffusion coefficient upon binding to HRV2 (Mr = 8.5 MD) was monitored in vitro-transcribed HRV2 RNA, and Dylight-labeled virus are sufficiently different to allow for determination of their relative concentrations in the mixture; attempts at determining the autocorrelation function for virus with partially-released RNA were hampered by the heterogeneity of the samples obtained after the crosslinking step that was necessary for halting complete egress upon heating (see below). As expected, the values were generally somewhat lower than those for native virus, however, they varied considerably. For this reason, all of the calculations below were carried out using the diffusion coefficient of the native virus instead. Further control experiments . Thus, the slightly different size of the poly-(A) tail, as well as the small VPg that is absent from in vitro-transcribed RNA, had no significant impact on the diffusion properties. The measured and calculated diffusion coefficients of the probes, the free RNA, and the virus are summarized in Fluorescence correlation spectroscopy (FCS) allows measuring the diffusion time, and thus determining the diffusion coefficient, of fluorescent molecules at very low concentrations (down to picomoles/l) in very small sample volumes (down to femtoliters) eriments  demonstrHRV2 was incubated at 56°C in the presence of the respective probes for the times indicated in Measurements were then repeated with additional incubation times. The percentages of free oligonucleotide, oligonucleotide bound to free RNA, and oligonucleotide bound to virus with part of its RNA having become exposed, were obtained by fitting the data to a three-component autocorrelation function using the diffusion coefficients of each constituent obtained in the previous experiment. We assumed that the value for virus and virus with partially-extruded RNA were negligibly different  gave rise, almost exclusively, to particles migrating as a broad peak with a substantially increased migration time . The concentration of empty particles was below the detection limit. Note that R and Rx likely include intermediate particles with and without the typical ‘rod-like’ density  in the non-ionic detergent Thesit time Rx; . Presumaty as in , and S2,x in the presence of the antibody  showed a well-defined band close to the top of the gel. In contrast, the sample that had been heated and incubated with MNase migrated further (Rx). The respective bands were cut out and analyzed for the presence of proteins by SDS polyacrylamide gel electrophoresis followed by silver staining (x) contained VP1–VP4, whereas crosslinked, heated, and MNase-treated virus (Rx) only contained VP1–VP3 indicating that it had been converted into subviral 135S A-particles devoid of VP4. RNA was extracted from the remaining part of the gel slices, reverse transcribed and the cDNA amplified with two primer pairs each, selected to produce fragments derived from the 3′ and the 5′-ends, respectively . Therefore, we determined which part of it remained inside the viral shell and thus protected from RNases. ‘Rod-particles’ were prepared as above at 56°C, accessible RNA was digested with MNase, the nuclease was inactivated with EGTA, and the sample subjected to native agarose gel electrophoresis followed by staining for RNA. As seen in staining . The banBecause of its appealing and suggestive nature, schemes of enterovirus uncoating have long depicted the RNA as exiting at one of the five-fold axes; however, this model was never supported by experimental evidence. Exit at the 5-fold axis was brought into question by results of cryo-EM analysis of intermediate uncoating states of poliovirus In the present paper we demonstrate that RNA egress begins with the 3′-end. Our model is supported by two complementary findings. Firstly, we observed completely different kinetics for the exit of the 3′ and the 5′-ends of the RNA from the viral shell on incubation at 56°C. Deconvolution of the FCS autocorrelation data showed that the 3′-specific oligonucleotide first bound to molecular assemblies with diffusion properties expected for RNA that was still attached to the virus. At later time points, their diffusion rather corresponded to free RNA only, reflecting complete release from the virion. On the other hand, the oligonucleotide specific for the 5′-end never exhibited diffusion times corresponding to such virion-associated RNA. Secondly, in order to halt RNA transit at intermediate states, we used psoralen UV crosslinking. With capillary electrophoresis, we demonstrated that such crosslinked particles heated to 56°C indeed carried partially-externalized RNA that was accessible to a dsRNA-specific monoclonal antibody. We specifically digested this RNA, extracted the fraction that remained protected inside the virion, and, by RT-PCR, we exclusively found sequences close to the 5′-end. In the course of our experiments, we discovered that subviral particles containing ‘rod-like’ density were produced on incubation of native HRV2 at 56°C along with apparently full and empty virions. Similar uncoating intermediates with internal density taking on various conformations had also been observed in heated poliovirus Remarkably, upon closer analysis, the particles visually appearing to be full turned out to be a heterogeneous mixture of substantially distorted virions with a generally higher and broader distribution of RNA content . This coRNA detaching from the inner capsid wall, as is expected to occur on temperature elevation, may not be able to reform its original interactions upon cooling and thus be more prone to tangling. Alternatively, kinetic bottlenecks at regions of high secondary structures could be involved in the RNA condensation observed. ‘Rod-particles’ were only rarely observed on incubation of HRV2 at pH 5 in solution (data not shown); however, when HRV2 was bound to the surface of receptor-carrying liposomes and acidified some virions possessing internal density with a similar appearance were observed Ordered egress of RNA suggests that the viral genome becomes organized during packaging or assembly, which may occur co-transcriptionally Unless otherwise indicated, all chemicals were acquired from Sigma Aldrich. 9-methoxy-7H-furo[1]benzopyran-7-on  was dissolved at 2 mg/ml in DMSO. Oligonucleotides (with and without fluorescent label) were obtained from VBC-Biotech, Vienna, Austria. Dylight 488 and FAM dye were from Thermo Scientific and dissolved in DMSO at 10 mg/ml. YOYO-1 iodide509 was from Life Technologies and dissolved in DMSO at 1 mM. Oligonucleotides for RT-PCR were as follows (denoted as under quotation marks)“5′-end – reverse”dAAGGGTTAAGGTTAGCCACATTCAG-3′5′-“5′-end – forward”dGACCAATAGCCGGTAATCAG-3′5′-“ M-oligo – reverse”dAAGGTGTCAGTGTTATTTATTGGTACTAGGCTG-3′5′-“M-oligo – forward”dGCCCCATGTGTGCAGAGTTTTC-3′5′-“3′-end 3 - reverse”dCCACTCATGCAAAAGCAAATC-3′5′-“3′-end 3 - forward”dCCTTCCCTGAAGATAAATATTTGAATCC-3′5′-“3′-end 5 - reverse”dCTCTGGATCACATCCAACTGCTGATCCAG-3′5′-“3′-end 5 - forward”dGAGTTGACTTACCTATGGTCACC-3′5′-Their positions on the viral genome are indicated in the scheme in in vitro by using the RiboMAX Large Scale RNA Production System – T7 (Promega) and purified by extraction with phenol chloroform followed by ethanol precipitation. For FCS measurements, in vitro-transcribed RNA was labeled with YOYO-1 iodide509; the dye was mixed with RNA (10 nM) at a molar ratio of 400∶1 (5 base pairs per dye molecule) in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and incubated at room temperature for 15 min. With the exception of experiments involving RNase digestions, all samples were supplemented with 2U RNasin/µl (Promega).HRV2 was produced and purified as detailed elsewhere in vitro-transcribed viral RNA or heated HRV2, as indicated in the Figures. O5 was selected to be complementary to a region of low secondary structure by using the Vienna RNA package version 1.8 (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Diffusion coefficients were related to the measured diffusion times by using Rhodamine6G as the standard (DRh6G = 2.8×10−10 m2 sec−1). For easier comparison, the autocorrelation functions were normalized to 1 at autocorrelation time zero (i.e. to one particle in the observation volume). Ten to 50 consecutive measurements on at least three independent samples were carried out with the respective fluorescent analytes at 10 nM  and a data acquisition time of 10 sec each, in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2.FCS was carried out in a Zeiss Confocor 1 instrument using an Argon-ion laser with 488 nm wavelength. The fluorescence autocorrelation function was measured for the labeled oligonucleotides complementary to the HRV2 nucleotide numbers given in brackets: oligo-dT 25-mer  and 5′-end  in the absence or presence of in vitro-transcribed HRV2 RNA at concentrations between 0 and 1000 nM (not shown). This revealed that at least a 25 fold excess (i.e. 250 nM) of heated virus/RNA over oligonucleotide was required for an optimal hybridization signal. The mixture was incubated for 10 min at 56°C and for at least 30 sec at 4°C prior to the measurments. To confirm that the observed change of the diffusion coefficient in the presence of viral RNA was due to hybridization, the sample was treated with a mixture of RNase A  and RNase H  for 15 min in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM DTT at 37°C prior to the measurement (The most favorable molar ratios between oligonucleotide and RNA were determined by mixing labeled oligonucleotide at 10 nM with surement . DyLight−3 Pa·s). Assuming that they adopt a densely-packed sphere with mass density of 1.8 g/cm3For comparison with the measured values, diffusion coefficients of probe and viral RNA were calculated for hypothetic conformational states expected to result in lowest and highest diffusion coefficients  equipped with an uncoated fused-silica capillary  packed in a standard Hewlett Packard cassette and thermostated at 20°C was used throughout. Injection was at 50 millibar pressure for 9 sec. Between all runs the capillary was conditioned by aspirating 100 mM NaOH, water, and the background electrolyte  and 10 mM Thesit) for 2 min each, applying 950 millibar pressure. Detector signals were recorded at 205 nm. For additional measurements at 260 nm, fast spectral scanning mode was employed. Positive polarity mode (negative pole is placed at the capillary outlet) with 25 kV was used for all experiments. HRV2, either psoralen crosslinked or not, was incubated at 56°C for different times as indicated in the Figures. To detect viral RNA outside the virion but still connected to it, crosslinked virus either heat-treated or not (control) was incubated with 1 µl (1 µg)/sample anti-dsRNA mAB J2  for 20 min at room temperature. To remove accessible RNA, crosslinked and heat-treated (10 min at 56°C) virus was incubated with micrococcal nuclease  at 37°C for 20 min. MNase was inactivated by addition of 10 mM EGTA. Subviral particles were quantified as total protein by integration of the peak area at 205 nm and subtraction of the respective calibrated peak area at 260 nm −1.4×peak area (at 260 nm)). The RNA content of the particles was estimated by comparison with native virus (set to 100%); peak areas were recorded at 205 nm and 260 nm for native virus from 12 different preparations giving a ratio of 5.5±0.1 and after subtraction of the contribution of VP4 of 5.3±0.1 (i.e. full 135S particles). For crosslinked, heated, and MNase-digested particles, this ratio was 12.6±1.0 (4 different electropherograms). Based on a ratio of the extinction at 205 nm/260 nm for pure RNA of 1.4, the mean RNA content of these latter particles was estimated to amount to 34.7%.Assuming irreversible release of the RNA (irreversible mass action), the evolution of the concentrations of the three components in time was modelled by using GEPASI v. 3.30 Pfu DNA polymerase (Promega) by using the primer sets given above for 30 cycles. For protein analysis, aliquots of the excised bands were boiled in reducing sample buffer and run on a SDS polyacrylamide gradient gel (12–20%). Proteins were detected by silver staining Crosslinked HRV2 either incubated at 56°C for 10 min or not (control) was MNase treated as above and run on a 0.7% agarose gel in 50 mM Tris-HCl (pH 8.3), 10 mM EDTA. RNA was visualized by ethidium bromide-staining and extracted from the bands with the Zymoclean Gel RNA Recovery Kit (Zymo Research). Reverse transcription (RT) was carried out with SuperScript III reverse transcriptase (Invitrogen). PCR amplification was performed with Psoralen UV crosslinked HRV2 was incubated at 56°C for 10 min. Negative stain EM was carried out at 56,000× magnification (80 kV) in a FEI Morgagni 268D equipped with an 11 megapixel CCD camera (Morada from Olympus-SIS) after staining with 2% phosphotungstic acid. For cryo-EM, samples (4 µl) were deposited on Quantifoil 400 nm copper grids with a 1.2/1.3 holey carbon film ), blotted with an automatic Leica EM Grid Plunger, and shock frozen in liquid ethane cooled in liquid nitrogen. Image acquisition was done as follows: Subviral particles were viewed in a FEI Tecnai F30 Polara cryo-electron microscope (300 kV) with C2 condenser aperture and objective aperture of 70 µm and 100 µm, respectively, at a magnification of 71,949 x. Images were acquired at underfocus between 2.0 µm and 4.2 µm with a Gatan Ultrascan 4000 4k CCD camera, using Leginon automatic image acquisition software Figure S1Spherically averaged radial density plots of 3DR of the three HRV2 subviral particle classes selected by maximum likelihood 3D classification. Radii delimiting the RNA core and the protein shell respectively, as used for the calculation of the relative core density , are ind(TIF)Click here for additional data file.Figure S2Comparison of various native and subviral enterovirus particles with HRV2 subviral particles as obtained on heating to 56°C for 10 min. A) Volumes were computed with bgex (TIF)Click here for additional data file.References S1Supporting information references.(DOCX)Click here for additional data file.Table S1Summary of the 6 ML3D-classes of subviral HRV2 particles that represented more than 5% of the total number of particle images. The resolution of the respective reconstructions refers to the spectral signal to noise ratio (SSNR∧MAP>1) see (DOCX)Click here for additional data file.


Prp19/Pso4, an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)1–4 C2–6 (U/A)1–5, we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEV Prp19/Pso4 complex, which consists of SNEVPrp19/Pso4, CDC5L, PLRG1, SPF27 (BCAS2) and Hsp73 forming the core complex, while also additional proteins are associated Prp19/Pso4 even blocks spliceosome assembly The protein coding information in eukaryotic organisms is split by intronic sequences containing regulatory elements and microRNAs. However, these introns have to be removed during synthesis of mRNAs by pre-mRNA splicing. This process is performed by the spliceosome, a large multi-protein machinery consisting of four small nuclear ribonucleoprotein particles and more than 100 different proteins that assemble dynamically in a step-wise manner on the pre-mRNA Prp19/Pso4Prp19/Pso4 is differentially regulated in replicative senescence of human endothelial cells Prp19/Pso4 is an essential protein in early mouse development Recently we identified Blom7α as another protein, which is associated with this complex by direct interaction with SNEVPreviously, we reported that Blom7α also is involved in pre-mRNA splicing, since besides its co-localization and co-precipitation with other known splicing factors, its addition to nuclear extracts increases the splicing activity in vitro, and co-transfection with splicing reporter minigenes alters the pattern of alternatively spliced variants Here we report the characterization of the RNA binding activity of Blom7α and show that Blom7α co-localizes with RNA in cells. Furthermore, we describe the identification of a splicing inhibitory RNA aptamer, termed AK48 (Aptamer KH-binding 48), which was selected against the KH domains of Blom7α by Systematic Evolution of Ligands by Exponential Enrichment (SELEX).Aptamers have been developed in the early 90's Danio rerio and Arabidopsis thaliana orthologues, as well as with a known KH domain containing protein [PDB ID: 1K1G], we identified two highly conserved KH domains within Blom7α , or its N-terminal half consisting of the KH domains alone (Blom7-KH) using a synthetic RNA library of 30 random nucleotides with 8 rounds of selection including counter selection steps for increased affinity as outlined in Then, RNA was injected over the functionalized surface and resulted in binding as shown by increasing response units (RU) values . Non-speSurprisingly, no dissociation of the RNA-protein interaction was detected indicating a long-lived complex and consequently a high equilibrium binding constant. Dissociation of RNA was achieved only by applying several pulses of 10 mM NaOH, while all other regeneration agents tested failed to disrupt RNA-protein interactions.on (association) of the selected RNAs assembling on the proteins is very low, and in contrast the koff (dissociation) is very high. This leads to the hypothesis that there is a slow RNA-protein-complex formation, but once the complex is assembled and in the correct conformation, it is very stable.As could be concluded from the sensorgram, the kAs SPR analysis confirmed the interaction of selected RNAs with Blom7α, RNA libraries from the last round of selection were reverse-transcribed and TOPO-cloned. The screening of positive colonies resulted in 59 Blom7α wt specific clones, and in 69 positive Blom7-KH specific clones, which were subjected to sequencing.1–4 C2–6 (U/A)1–5(U/A). Furthermore, we computed a consensus sequence, which clearly displays an abundance of A/C rich sequences and poly-C stretches at the 3′-end . Comparison of two other candidates with AK48, the aptamer with the highest response selected against Blom7-KH, demonstrates clearly the higher binding affinity of AK48. . Again, on and koff (data not shown). Furthermore, the association phase has an unusual shape, which might indicate a more complex binding mechanism  and is possibly caused by the two KH domains within Blom7α.AK48 was selected for further analysis and subjected in concentrations of 0.68 and 2 µM to SPR. As expected, with 2 µM a higher response as with 0.68 µM was observed . HoweverThe sequence of AK48 aligned with the putative motif is shown in E. coli. After the purification, fractions 2–4 showed clear bands at the correct heights with minimal degradation products . Therefore, we used recombinant HIS-tagged Blom7α wt and Blom7-KH, expressed in products , and werAs expected, in EMSA we detected a band-shift when using 20 nM biotinylated AK48 and increasing amounts of Blom7α wt , or as wFinally, since we were not able to remove the RNA from Blom7α or the Blom7-KH domain in the SPR-assays above, we tested if 10 minutes pre-incubation with the biotin-labeled AK48 could be competed out by unlabeled RNA. Indeed, not even a 4-fold molar excess of unlabeled AK48 (lanes 2–5) could completely compete out biotinylated AK48 from binding to Blom7 wt . This reSince we described previously that addition of recombinant Blom7α to Hela nuclear extracts increased splicing activity in a dose-dependent manner Although these data suggest that the endogenous RNA binding activity of Blom7α might be essential for its function during the splicing reaction, we cannot rule out that AK48 interacts in-vivo preferably with another unknown protein involved in pre-mRNA splicing , which causes the inhibition of pre-mRNA splicing independent of Blom7α.Prp19/Pso4, a protein predominantly present in the nucleus, was previously shown to physically interact with the C-terminal domain of Blom7α In order to further characterize the novel pre-mRNA splicing factor Blom7α, we here investigated on the ability of Blom7α to bind RNA. Therefore, we performed co-localization studies in Hela cells and found the KH-domain-containing part of Blom7α to be present in the cytoplasm, possibly due to binding of the KH-domains to RNA. Furthermore, we mapped the domain necessary for nuclear retention to a region between amino acids 480 and 549. Since no canonical nuclear localization signal (NLS) is present within Blom7α, the nuclear localization is likely to be driven by interaction with other factors. For instance SNEVAs a next step, we performed SELEX in order to detect putative RNA aptamers binding to Blom7α. Indeed, RNAs binding to full-length Blom7α, as well as to the KH-domain containing part, were enriched during the progress of SELEX. Thus, our hypothesis that the KH domains of Blom7α are responsible for RNA-binding is strengthened.KH domains are besides the RNA recognition motif (RRM) the most abundant nucleic acid-binding domains. Their widespread presence in eubacteria and eukaryotes suggests them to be of ancient evolutionary origin. Proteins containing KH domains are involved in the regulation of gene expression at several levels, such as in transcriptional or translational regulation, splicing and mRNA transport, stability, and localization We observed extremely low dissociation rates of different RNAs in SPR as well as in EMSA, after having bound to Blom7. It might therefore be possible, that Blom7α undergoes conformational changes upon binding to RNA, as was already reported for various other factors interacting with RNA in vivo splicing and in vitro spliceosome assembly As we found Blom7α to be involved in the splicing process, an additional hint for the importance of the found A/C rich motif as common regulatory element is the fact that another nuclear protein (YB-1) binds to an A/C-rich exon enhancer element in CD44 exon v4, which is required for maximal Prp19/Pso4For RRM (RNA recognition motif), the most abundant and best characterized RNA binding module, the beta-sheet is the primary surface for RNA recognition, while additional contacts mediated by N- and C-terminal residues or loops are important in determining substrate specificity In conclusion, small synthetic RNAs as general inhibitors of splicing catalysis might represent the starting point to develop novel therapeutics targeting the process of pre-mRNA splicing.Homo sapiens (Hs), as well as highly similar sequences from Danio rerio [GenBank ID: NP_997758.1] and Arabidopsis thaliana [GenBank ID: NP_566850.3] were aligned with two copies of a part of an already structurally characterized human KH domain protein [PDB ID: 1K1G] using T-Coffee In order to identify conserved protein domains within Blom7α, PSI-BLAST HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 4 mM L-glutamine and 10% (v/v) fetal calf serum.2 and 2 mM EDTA) at room temperature, followed by counterstaining for DNA with DAPI and RNA with Pyronin Y. Microscopy and image analysis were performed using a Zeiss DeltaVision Restoration microscope as described Hela were transfected with Blom7 truncation mutants in pEGFP-C1 using Lipofectamine 2000 (Life Technologies). 48 hours after transfection the cells were washed with PBS and fixed for 5 min in 3.7% (w/v) paraformaldehyde in CSK buffer  flanked by 5′ and 3′ invariant linker sequences (5′-GTGTGACCGACCGTGGTGC-3′ and 5′-GCAGTGAAGGCTGGTAACC-3′) was amplified by PCR with 3′SL downstream primers (TAATACGACTCACTATAGGTTACCAGCCTTCACTGC-3′5′-) containing the T7 transcription promoter (underlined) and P20 upstream primers (5′-GTGTGACCGACCGTGGTGC-3′) and in vitro transcribed. This RNA pool was used for the first round of selection. All steps of in vitro selection were performed in 100 µl SELEX binding buffer  on ice.In brief, a synthetic DNA random library (Proligo) of 30 random nucleotides (46 and Blom7-KH-His6 were coupled to Ni2+-NTA beads (Promega) for the 1st round and washed several times with SELEX binding buffer. After two rounds of counterselection on uncoated and His6- SNEVPrp19/Pso4 coated beads in order to eliminate unspecific binders, the RNA pool was added to Blom7 wt or Blom7-KH coupled beads and incubated for 45 min on ice. Beads carrying protein-RNA complexes were washed with SELEX binding buffer several times and bound RNA was eluted by addition of nuclease-free water and heating for 1 min to 75°C.10 pmol Blom7 wt-HisAfterwards, RNA candidates were reverse-transcribed and amplified by PCR. Following rounds of selection were performed as before.After 8 cycles of selection against Blom7 wt or Blom7-KH, candidates were cloned using the TOPO TA Cloning Kit (Life Technologies) according to the manufactures' instructions. Plasmids of 100 positive clones of each selection were Sanger-sequenced.SPR (surface plasmon resonance) experiments were performed using the BIACORE 3000  system and Sensor Chip CM5  at 23°C.Activation of the chip surface with a 1∶1 mixture of N-hydroxysuccinimide (NHS) (50 mM) and 1-ethyl-3-(3-dimethylpropyl)-carboimide (EDC) (200 mM) and immobilization of proteins (500 nM in 10 mM NaAc pH 5) were performed following the manufacturer's instructions. Afterwards the chip surface was saturated with ethanolamine  and the system primed with SELEX binding buffer or HEPES buffer containing 3 mM magnesium acetate. RNAs were refolded by heating (95°C for 1 min), freezing (4°C for 3 min), and keeping at room temperature for 5 min, diluted  in SELEX binding buffer and injected . Unspecific binding was considered by subtracting blank runs on a surface not loaded with protein and on a surface loaded with a non RNA-binding control protein. The association and dissociation rates were monitored for 600 s each. Regeneration of the chip surface was performed using pulses of 10 mM NaOH.The computation of the consensus sequence of selected RNAs was done with MEME 5′-UGCCCCAAUCCUACACGACCCAAGGCCCCC-3′-Bi) and AK48scr (5′-CACGCACUCACCACGCACGCAUCCGCUCAC-3′-Bi) were synthesized and PAGE-purified by Dharmacon.Biotinylated versions of AK48 (6 and Blom7-KH-His6 were PCR-cloned into pET30A (Qiagen) and transformed into BL21. After purification over Ni-NTA-beads (Qiagen) and dialysis against PBS, fractions 1–4 were loaded onto 4–12% PAGE gels (Invitrogen) and stained with PAGE-Blue (Fermentas) according to the manufacturer's instructions. Fractions 2–4 were pooled and used for subsequent experiments. Quantification of proteins was performed by OD280 measurement and calculation of concentrations by using the specific molar extinction coefficients (41870 for Blom7 wt and 13850 for Blom7-KH).Blom7 wt-His2, 1 mM DTT and 5% glycerol, as well as RNAs and proteins as indicated in the figure legends. Biotinylated RNAs were always added last and reactions were incubated for 20 min at room temperature, if not indicated otherwise. Afterwards the samples were carefully mixed with 5 µl 5× REMSA loading dye (Pierce) and loaded on a 6% polyacrylamide DNA Retardation Gel (Invitrogen). Electrophoresis was performed in 0.5× TBE at 100 V for 90 min at 4°C. The RNAs were transferred to positively charged nylon membranes (Roche) by tank blotting in 0.5× TBE at 400 mA for 40 min at 4°C and crosslinked at 120 mJ/cm2 at 254 nm. For the detection of RNAs the Chemiluminescent Nucleic Acid Detection Module (Pierce) was used according to the manufacturer's instruction. Bands were visualized on a Lumi-Imager (Roche) in chemiluminescence mode.For EMSAs, RNAs were refolded by heating (95°C for 1 min), freezing (4°C for 3 min), and keeping at room temperature for 5 min. 20 µl reactions were set up in PBS (pH 7.5) containing 1 mM MgClNuclear extracts used in the splicing assays were obtained commercially from Dundee Cell Products Ltd . Splicing assays and analysis of spliceosome assembly were done as described previously Supporting Methods S1Detailed protocols for SELEX and SPR-analysis.(DOCX)Click here for additional data file.