Nanoarchaeum equitans contains several fragmented genes and revealed unusual RNA processing pathways. These include the maturation of tRNA molecules via the trans-splicing of tRNA halves and genomic rearrangements to compensate for the absence of RNase P.The minimal genome of the tiny, hyperthermophilic archaeon N. equitans cell are analyzed using RNA-Seq deep sequencing methodology. All tRNA half precursor and tRNA termini were determined and support the tRNA trans-splicing model. The processing of CRISPR RNAs from two CRISPR clusters was verified. Twenty-seven C/D box small RNAs (sRNAs) and a H/ACA box sRNA were identified. The C/D box sRNAs were found to flank split genes, to form dicistronic tRNA-sRNA precursors and to be encoded within the tRNAMet intron.Here, the RNA processing events in the N. equitans lost many essential metabolic pathways but maintains highly active CRISPR/Cas and rRNA modification systems that appear to play an important role in genome fragmentation.The presented data provide an overview of the production and usage of small RNAs in a cell that has to survive with a highly reduced genome.  Nanoarchaeum equitans is a 400 nm small archaeon isolated from hot submarine vent microbial communities whose growth relies on its attachment to the cell surface of the archaeon Ignicoccus hospitalis [N. equitans into a novel phylum termed 'Nanoarchaeota'. However, different phylogenetic studies focused on ribosomal proteins and concluded that N. equitans represents a member of a fast-evolving euryarchaeal lineage related to the Thermococcales [N. equitans Kin4-M revealed a minimal, compact genome of only 490 kilobases and an extremely high gene density with little noncoding DNA or pseudogenes [N. equitans was the first organism shown to require the assembly of tRNA halves to generate six essential functional tRNA isoacceptors [trans-splicing reaction [N. equitans is also the only currently identified organism that can survive without an RNase P molecule [N. equitans tRNA genes, ensuring proper transcription initiation conditions.spitalis . Phylogecoccales . The genudogenes . This hiudogenes ,4. Exampudogenes . Other ucceptors . A heterreaction -8. N. eqmolecule . RNase PN. equitans and I. hospitalis [N. equitans genome encodes a fairly complete set of proteins for replication, transcription and translation. In addition, surprisingly extensive sets of genes with proposed roles in DNA repair and RNA modification are annotated. Finally, two clustered regularly interspaced short palindromic repeats (CRISPR) arrays and a complete set of CRISPR associated (Cas) proteins are present. These systems are mainly characterized as adaptive antiviral defense systems even though the viral threat towards N. equitans is not known [The loss of many essential pathways has to be compensated by the transfer of metabolites between spitalis . It is aspitalis . The N. ot known ,15. In tN. equitans. Depending on the chosen method of RNA isolation, library preparation and employed RNA sequencing platform, different RNA species are selectively enriched [N. equitans cell, small RNAs were enriched but not depleted of ribosomal RNAs. All RNA samples were treated with T4 polynucleotide kinase and tobacco acid pyrophosphatase before adapter ligation. These steps were employed to ensure that RNAs with 5'-triphosphate or 5'-OH termini and 2',3'-cyclic phosphate termini were captured. Illumina HiSeq2000 sequencing was employed, which yielded over 12 million reads that could be mapped onto the N. equitans genome  and linked to a putative amino acid permease. This observation provides a starting point for the analysis of the highly abundant protein NEQ026 potentially being involved in the uptake of amino acids from I. hospitalis.RNA-Seq methodology was used to obtain a global overview of the production and processing of small RNA molecules in enriched . To obtaN. equitans was the first organism to be identified to generate six tRNA isoacceptors via a trans-splicing reaction using tRNA half molecules. Earlier studies characterized the mature spliced tRNA molecules in the cell, yet only a single tRNA half transcript could be identified [N. equitans cell. The tRNA half molecules contain the sequence that will fold into the mature tRNA body and a GC-rich stretch that is complementary to a sequence only found in the matching tRNA half. These sequences are proposed to facilitate the identification of the matching halves upon which the tRNA body folds [trans-spliced mature tRNAs. The termini of all tRNA halves were identified  for the 3' tRNAHis half and shortened by one nucleotide (A) for the 3' tRNALys half. In some cases, the obtained tRNA precursor sequences merge with the mRNA of the adjacent gene. One example is the 5' tRNAHis half that is located directly upstream of the gene encoding the valyl-tRNA synthetase and that defines its 5' untranslated region. It is tempting to speculate that this structured tRNA half might play a role in mRNA stability or regulation of valyl-tRNA synthetase synthesis. The 5' tRNAGlu (UUC) half and 3' tRNAiMet half sequences extend into adjacent genes of different orientation, which might cause difficulties for proper termination signals. These sequences indicate that a defined 3' end might not always be a necessity for the assembly of tRNA halves into functional tRNA molecules. Finally, both 5' tRNA halves for the tRNAiMet and the tRNAHis already contain the additional -1 base that leads to an extended acceptor stem in the mature tRNA.entified ,17. The dy folds . Subsequd Figure . The 5'-Tyr, which requires a C1 base for its recognition by the tyrosyl-tRNA synthetase. However, without RNase P, such tRNAs could not start with a pyrimidine residue and it was reported that an unsual G-1 extension solves the need for both a C1 base and a purine residue at the transcription start [Tyr locus did contain the G-1 extension.Potential low abundance structural RNA molecules were searched in intergenic regions but the otherwise universal RNase P RNA molecule could not be detected, which is in agreement with previous studies . Genomicon start . As thisN. equitans contains two CRISPR clusters whose crRNAs were found to be highly abundant in the cell. It should be noted that both CRISPR clusters are annotated in the reverse orientation in popular CRISPR databases [N. equitans are not known. Nevertheless, the reduced genome still allows the presence of 41 different spacer sequences distributed onto two CRISPR clusters. The CRISPR clusters are transcribed and cleaved within identical 28 bp repeat sequences. Processed crRNAs were detected for all spacers and contained a 5'-terminal 8 nucleotide tag containing the 3'-terminal repeat nucleotides ATTGAAAG that is usually generated by Cas6 cleavage  as the protein that catalyzes the degradation of the viral DNA [atabases ,19. Thesatabases . Virusesopensity . The lowopensity  and a coiral DNA ,24. All N. equitans maintains a large arsenal of RNA modification enzymes, and during genome annotation, 14 small nucleolar (sno)-like RNAs that serve as guide RNAs for the methylation of rRNAs were described [N. equitans and uses S-adenosylmethionine as a cofactor to catalyze the formation of 5-methyl uridine in tRNAs and rRNAs [escribed . The datescribed  were verescribed   and NEQ392 . A H/ACA box sRNA guides the pseudouridylation of rRNA targets. The identified compact H/ACA box sRNA contains two extended hairpins that each contain a bulge with kink-turn (k-turn) bulges  automatically generates the 5' terminus of C/D box sRNA , tRNAGly(TCC)) are located adjacent to the C/D box sRNAs3 and sRNA14.Alignment of the DNA stretches upstream and downstream of the identified small RNA termini enables the analysis of potential promoter and terminator elements. Previously, the conserved elements of nanoarchael tRNA and tRNA half gene promoters were identified . The proN. equitans, C/D box sRNA can be located within tRNA introns [N. equitans.Most C/D box sRNAs overlap with mRNA sequences  of rRNAs in hyperthermophilic growth conditions. In conclusion, analysis of the RNA fractions in the minimal N. equitans cell revealed the loss or degeneration of universal RNA molecules  while other, seemingly less essential RNA species  are found to be highly abundant. The identification of C/D box sRNAs adjacent to split protein encoding genes and tRNAs as well as within a tRNA intron suggests their involvement in genome fragmentation.In the presented study over 12 million RNA sequence reads were mapped to the minimal 0.49 million bp genome of bacteria ,38. N. eN. equitans Kin4-M cells were a kind gift of D Söll. The organism was grown in the Archaeenzentrum Regensburg  in a 300 liter fermenter in simultaneous culture with I. hospitalis KIN4-I and purified by gradient centrifugation as described [escribed . Total Rescribed  and smalN. equitants/I. hospitalis RNA (3 μg) was treated with T4 polynucleotidekinase to ensure proper termini for ligation. A protocol for the dephosphorylation of 2'3' cyclic phosphate termini was modified from [ied from : 1 μg ofN. equitans reference genome (GenBank: NC_005213) with CLC Genomics Workbench 5.0 . The following mapping parameters were employed: mismatch cost, 2; insertion cost, 3; deletion cost, 3; length fraction, 0.5; similarity, 0.8). This program was also utilized to determine the coverage of individual RNA molecules. All predicted RNA molecules and their termini were manually verified and all intergenic regions were checked for the presence of RNA molecules with coverage of less than 1,000 reads. The following algorithms were used for the computational analysis of the data: RNA folding (Mfold [Sequencing reads were trimmed by (i) removal of Illumina TruSeq linkers and poly-A tails, and (ii) removal of sequences using a quality score limit of 0.05. A total of 16,614,433 reads with an average length of 62.3 nucleotides were obtained after trimming. Of these, 626,555 reads below 15 nucleotides were removed, and 12,178,737 reads were mapped to the g (Mfold ), tRNA gg (Mfold ), snoRNAg (Mfold ), C/D bog (Mfold ), crRNA g (Mfold ), RNA alg (Mfold ), RNA viCircular C/D box sRNAs 23 and 24 were amplified from the small RNA purification sample via the Thermoscript RT-PCR system  with Thermoscript reverse transcriptase and Platinum Taq DNA polymerase according to the manufacturer's instructions. The RNA was denatured at 100°C for 5 minutes and snap-cooled on ice for 5 minutes to facilitate reverse transcription at 70°C through potential secondary structures of the RNA. The following oligonucleotides were employed: sRNA23For, 5'-CTGAATTTATGATGAAGAGCCTGGATGCAG-3'; sRNA23Rev: 5'- CATCATAAATTCAGAGTAGCGGCTTTCTTC-3'; sRNA24For, 5'- GCTGAACATCGGGTATACTGAATAGTGATG-3'; sRNA24Rev, 5'- CCGATGTTCAGCATTTTTAATATTGCTCTCAG-3'. The oligonucleotides partly overlap to ensure proper annealing to the sRNA template. PCR amplificates were cloned into a pCR2.1 TOPO vector (Invitrogen) and subjected to DNA sequencing .The RNA-Seq data are available at NCBI's Gene Expression Omnibus (GEO) website as series GSE38821.Cas: CRISPR associated protein; CRISPR: clustered regularly interspaced short palindromic repeats; crRNA: CRISPR RNA; PCR: polymerase chain reaction; RT: reverse transcriptase; snoRNA: small nucleolar RNA; sRNA: small RNA.The author declares that they have no competing interests.LR planned and initiated the project, analyzed the data, wrote the manuscript and approved the final version of the manuscript for publication.N. equitans mapped RNA reads. Illumina HiSeq2000 sequencing reads were mapped to the N. equitans reference genome . The excel file contains the read coverage for the entire genome mapping. The genome region 449944 to 449989 (AAAAAAAGAAGAAAGAAAAAAGAAAGAAATAAAAAA) causes poly-A mapping artifacts.Click here for fileN. equitans C/D box sRNAs and H/ACA sRNAs. This excel file contains detailed analysis of the C/D box sRNAs and H/ACA sRNAs. Indicated are genomic location, genomic context, termini, box C and box D structures and potential sites of action.Click here for file


Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted Arabidopsis Rrp44/Dis3 homologs. AtRRP44A could functionally replace S. cerevisiae Rrp44/Dis3, but AtRRP44B/SOV could not. rrp44a knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but rrp44b mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which rrp44a did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that  Eukaryotic 3ʹ to 5ʹ exoribonucleolytic activities are important for a wide range of reactions of RNA metabolism and maturation, including processing of small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and ribosomal RNA (rRNA), and mRNA decay. The main pathway of 3ʹ to 5ʹ exoribonucleolytic activity is the RNA exosome .The exosome is evolutionarily conserved among eukaryotes. The exosome targets a vast range of RNAs at many stages of their lives, from maturation through constant quality control to final turnover. The exosome of eukaryotes is composed of eleven polypeptide components that can be divided into two major groups according to their structure and function -4.The first group of exosome components comprises nine proteins that form the ‘exosome core’. Six of the subunits, Rrp41, Rrp42, Rrp45, Rrp43, Rrp46 and Mtr3 (each containing an RNase PH domain), are organized into a hexamer, capped on one side by a trimer of subunits Rrp40, Rrp4 and Csl4 . In yeast, the loss of any one subunit of the exosome core is lethal, showing similar rRNA processing defect profiles .The exosome core itself has no catalytic activity in yeast and humans ,8. The aArabidopsis thaliana, using tandem affinity purification of tagged AtRRP4 and AtRRP41, Chekanova et al. . Se. SeE. coA AT2G383 sequence context . rrp44aKD-1#7-3-1, rrp44aKD-2#6-2-1 and gusKD-2#2-10-3, respectively. The expression of amiR_RRP44A-1 and amiR_RRP44A-2 was confirmed in rrp44aKD-1#7-3-1 and rrp44aKD-2#6-2-1, respectively, by small RNA gel blot analysis . Levels of the mRNA decreased to 5% in l plants . These prrp4KD-2 and rrp4KD-3, and rrp41KD-1#3-3 (hereafter referred as rrp41KD-1) and rrp41KD-1#9-2, respectively.AtRRP4 and AtRRP41, the exosome core components, were also knocked down in the same way as AtRRP44A to compare their biological functions. As shown in Arabidopsis AtRRP44A and/or AtRRP44B/SOV are similarly involved in 5.8S rRNA 3ʹ end processing, we performed northern blot analysis using a probe  tails to its RNA substrates in yeast and human, is involved in 5.8S rRNA 3ʹ end processing  and the plasmid pBS3269: [pFL36]lys2∆: DIS3wt-TEV-PA (for expression of ScRrp44/Dis3 in S. cerevisiae) were kindly provided by Bertrand Séraphin [−1). Ten-fold dilution series of each transformant were spotted on LEU and TRP minus . The DNA oligonucleotide probes specific for rRNAs were end-labeled with γ-32P-ATP using T4 polynucleotide kinase . Hybridization was performed at 42°C using PerfectHyb Plus Hybridization buffer . See For northern blot analysis, 5 µg of total RNA was separated on 7 M urea-6% polyacrylamide gels in TBE buffer . RNA was electroblotted onto Hybond-Nrrp44aKD-1 and rrp44aKD-2 mutants, three rosette leaves of 20 to 30 plants at 25 days after germination were pooled for RNA extraction. Total RNA for qRT-PCR analysis was extracted using RNAiso Plus reagent  according to the manufacture’s protocol. cDNA was synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). Any genomic DNA in the total RNA was eliminated with gDNA Eraser. The RNA was then reverse transcribed by PrimeScript RT with random hexamer primers according to the manufacturer’s instructions. Following the reaction, the cDNA was diluted 1/5. All qPCR reactions were performed as previously reported [For quantification of AtRRP44A mRNAs in the WT and the reported . The prireported .pTCV-t1d1, a plasmid containing full-length cDNA of TCV downstream from T7 RNA polymerase promoters, was kindly provided by Dr. Jack T. Morris. Virions were prepared as described previously . VirionsFigure S1cerevisiaerrp44 doxycycline (DOX) repressible mutant.AtRRP44AHA does not complement the S.  (A) Growth phenotypes resulting from expression of plasmid-borne AtRRP44A-HA, S. cerevisiae Rrp44 (ScRrp44) and negative control alleles were assessed in the presence  or absence  of DOX after incubation for 120 h at 17, 22, 23, 30, 34 or 37°C. –LEU, without leucine. (B) AtRRP44AHA expression was determined by western blot analysis in the S. cerevisiae repressible rrp44 mutant without DOX. AtRRP44AHA protein was extracted from yeast cultured at 30 °C.(TIF)Click here for additional data file.Figure S2Activity of the CAB3 promoter in leaf tissue.Arabidopsis CAB3 promoter activity was determined by staining of Arabidopsis transformants expressing the GUS reporter gene under the control of about 1.5 kb genomic sequence containing the CAB3 promoter. 20 days post-germination (dpg) 5th rosette leaf (Left panel) and a transverse section of the leaf (Right panel).(TIF)Click here for additional data file.Figure S3Levels of mRNAs in knocked-down mutants.The amounts of AtRRP4 mRNA in gusKD-2#2-10-3, rrp4KD-2#2-6 and rrp4KD-3#4-2, and the amounts of AtRRP41 mRNA in gusKD-2#2-10-3, rrp41KD-1#3-3 and rrp41KD-3#9-2 were analyzed by qRT-PCR. Total RNAs were isolated from 25 dpg rosette leaves. Error bars represent standard errors. Six biological replicates and two technical replicates were performed. * indicates significant difference .(TIF)Click here for additional data file.Figure S4TCV genomic RNA accumulation was not affected by AtRRP41, AtRRP44A or AtRRP44B/SOV.(A) TCV genomic RNA structure. qRT-PCR of TCV accumulation in rrp41KD-1#3 and #9 (B), in rrp44aKD-1 and -2 (C), and in rrp44b-1 and -2 (D). TCV virions were inoculated at 16 dpg onto 4th and 5th rosette leaves, which were then collected at 3 days post-inoculation (dpi) for total RNA isolation. Error bars represent standard errors. Six biological replicates and two technical replicates were performed. Each biological replicate included six plants. EF1a mRNA was used as an endogenous control.(TIF)Click here for additional data file.Table S1rrp44a transmission in the selfed progeny of heterozygous rrp44a/+ mutants.(XLSX)Click here for additional data file.Table S2rrp44a/+ mutants.Germination of the selfed progeny of heterozygous (XLSX)Click here for additional data file.Table S3Oligonucleotides used in this study.(XLSX)Click here for additional data file.Table S4Plasmids used in this work.(XLSX)Click here for additional data file.Method S1Plasmid construction.(DOCX)Click here for additional data file.


Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5′ and 3′ UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5′ end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3′ end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle. Flavi2.The DENV genome is 11 kb long, has a type 1 cap (m7GpppAmp) structure at the 5′ end, and lacks a poly(A) tail at the 3′ end. Besides encoding viral proteins, the genome contains RNA structures that regulate different viral processes. Upon infection, the incoming genome serves as mRNA for translation, and subsequently, as template for RNA synthesis. The newly synthesized RNA can be used for new rounds of translation or as substrate for encapsidation. Efficient utilization of the genome during these processes must be temporally regulated to ensure viral spread. This regulation is mediated by RNA elements present in the coding and non-coding regions of the viral genome acting as promoters, enhancers, and repressors of the viral processes. In addition, during infection the viral RNA participates in triggering or avoiding the antiviral host response ,9.DENV 5′UTRs are between 95 to 101 nucleotides long. They contain two RNA domains with distinct functions during viral RNA synthesis. The first domain of ∼70 nucleotides is predicted to fold into a large stem-loop SLA, . A similSpecific structures at the 3′ end of the viral genome also play crucial roles in viral RNA synthesis. The approximately 450 nucleotide long DENV 3′UTR can be divided into three domains . Domain A conserved feature of DENV and other flavivirus genomes is the presence of inverted complementary sequences at the ends of the RNA that mediate long-range RNA-RNA interactions ,30,49–51per se for viral replication [Studies from many different laboratories using infectious clones and replicon systems provided compelling evidence for the essential role of genome cyclization during flavivirus replication ,49,54–57lication ,55,58.More recently, it has been shown that the DENV genome is a dynamic molecule that acquires different conformations during viral replication . In this3.An essential role of genome cyclization for replication of DENV and other flaviviruses has been demonstrated (for review see ). Molecuin vitro polymerase assays with recombinant proteins, it has been demonstrated that an RNA molecule carrying the SLA was able to promote RNA synthesis by NS5 only if templates up to 500 nucleotides in length were used. It was proposed that the polymerase was unable to reach the 3′ end initiation site of longer RNA molecules. However, RNAs of about 2 kb carrying the 5′ and 3′ end viral sequences, which resemble the viral genome, were efficient templates for polymerase activity provided that the cyclization sequences were intact. This observation supported the notion that long range 5′-3′ RNA-RNA interactions in the template are necessary for the polymerase to reach the 3′ end of long RNA molecules. Mutagenesis analysis in the context of DENV infectious clones confirmed the requirement of both the SLA structure and the complementary sequences for viral RNA replication in cell culture. A correlation between the RNA structural elements required for polymerase activity in vitro and viral RNA replication in infected cells was observed, confirming the central role of the SLA promoter in viral RNA synthesis [Using ynthesis ,15.cis-acting RNA signals along the genome, and in some cases, thousands of nucleotides away from their site of action, indicates high flexibility of viral RNA molecules.While it is not surprising to find a core promoter for RNA synthesis at the 3′ end of a viral genome, it is still intriguing why certain plus strand RNA viruses, such as DENV, would have promoters or enhancer elements for RNA replication at the 5′ end of the genome. Location of 4.in vitro data indicating a primer-independent (de novo) mechanism of initiation of flavivirus polymerases [NS5 is the largest and the most conserved of the flavivirus proteins. It contains an N-terminal methyl transferase domain (MTase) and a C-terminal RdRp domain. The structure of the NS5 C-terminal domain of DENV revealed a classical polymerase fold, bearing palm, thumb, and finger motifs . The preymerases –63.The viral protein NS5 has the ability to bind RNA with high affinity. Using mobility shift and filter binding assays, specific interaction of the full length NS5 and the RdRp domain of DENV with the 5′ terminal region of the viral genome was observed ,64. ThesAlignments of the SLA sequence from different flaviviruses together with probing analysis, indicated the presence of conserved structural RNA elements . The SLA62U63 bulge between the helical regions S1 and S2, indicated that this element is essential for replication, and that at least one U residue in the bulge is required. Substitution of the UU bulge for an AA bulge gave rise to revertant viruses with a UA bulge, confirming the requirement of at least one U in that position. Deletion of the side stem loop abolished viral replication, however, changing the sequence of the stem, the sequence of the loop, or modifying the length of the stem resulted in RNAs that were able to replicate [Infectious DENV RNAs with mutations at each side of stems S1 and S2, disrupting stem formation, showed impaired replication. Revertant viruses carrying spontaneous mutations were found to partially reconstitute the helical regions. In addition, mutations at both sides of the stems, maintaining the structure but changing the nucleotide sequence, replicated efficiently. These observations, together with the sequence conservation, and the co-variations observed in different positions within S1 and S2, indicated a requirement of the bottom part of the SLA for viral replication. Deletion or substitution of the conserved Ueplicate . RegardiIn vitro RNA binding assays using mutated RNAs with deletions of the top loop or the side stem loop showed a requirement of these two elements of the SLA for efficient ribonucleoprotein complex formation and polymerase activity in vitro [in vitro polymerase activity. The essential role of this U bulge in viral replication suggested an additional function of this element in infected cells. Substitutions of nucleotides present at the top loop of SLA inactivated the promoter for in vitro polymerase activity, which correlated with the lack of viral replication of RNAs carrying these mutations in transfected cells. Moreover, when the spontaneous changes obtained in the revertant viruses in cell culture were introduced in the SLA used for the in vitro assay, promoter activity was restored. Interestingly, RNA-protein binding studies with the top loop SLA mutants indicated that NS5 was able to form high affinity complexes that were inactive for RNA synthesis. This allowed dissociation of polymerase binding from polymerase activity, providing evidence for a post-binding activation of the polymerase [Footprinting studies carried out to investigate the interaction of NS5 with the viral 5′UTR RNA were recently reported . Both NSin vitro . Mobilitlymerase .A mutagenesis analysis of DENV NS5 to identify specific requirements for promoter-dependent polymerase activity was recently reported. In this study, a battery of 19 amino acid substitutions of basic residues present on the surface of the protein were designed in a recombinant protein and in the context of a DENV infectious clone . Evaluat5.de novo RNA synthesis copying from the 3′ end of the genome. Although 3′ nucleotides of the 3′SL must be recognized by the polymerase during the initiation process, the 3′UTR by itself does not function as template for in vitro RNA synthesis [in vitro polymerase assays, revealed that the large and stable stem of the 3′SL, represses RNA synthesis.The NS5 polymerase binds the SLA promoter and initiates ynthesis ,66. In aynthesis . MutagenIn the context of the viral genome, hybridization of the complementary sequences 5′-3′UAR induces conformational changes within the SLB at the 5′UTR, and the bottom half of the 3′SL at the 3′UTR . In thistrans initiation polymerase assay [cis and the 3′UTR molecule in trans. The trans initiation of RNA synthesis was demonstrated to be absolutely dependent on RNA-RNA hybridization [It has been reported that the DENV RdRp has a narrow template channel, which would only accommodate a 3′ end of an RNA in a single stranded form . Thus, tse assay ,15,55,66dization ,55,58. Udization . Togethe6.in vitro assays showed that the oligo(U) track functions as spacer that enhances viral RNA synthesis [Between the SLA structure and the 5′UAR sequence at the viral 5′UTR there is an oligo(U) track conserved in DENV and other flavivirus genomes . Differeynthesis . Deletioynthesis .trans initiation assay described above  spacer was shortened (The mechanism by which the oligo(U) track enhanced DENV RNA synthesis was investigated by testing the ability of the 5′UTR with or without the U track to interact with the 3′ end of the genome, and to promote polymerase activity using the ed above . These shortened . A direcRecent footprinting studies indicated that binding of the DENV RdRp to the 5′UTR induces large conformational changes downstream of the SLA promoter, within the oligo(U) track spacer . Nucleot7.in vitro studies have demonstrated the binding of cellular proteins to different regions of flavivirus 3′UTRs. Binding of EF-1α, TIA, the related protein TIAR, PTB, YB-1, Mov34, human La autoantigen, and NF90 to the viral RNA has been demonstrated [A sequence at the 5′ end of the 3′SL, which includes the 3′UAR cyclization element, forms a small hairpin (sHP) . Thus, nnstrated –79. It iIt is possible that the presence of competing structures in the viral genome could provide a way to modulate different RNA conformations. To test this idea in the DENV genome, the effect of mutations displacing the equilibrium towards the circular or the linear form of the RNA on viral replication was evaluated . Mutatio8.A great deal has been learned in the last years about the role of RNA signals present in the DENV genome. However, mechanistic aspects that explain how these structures participate in the viral processes are still lacking. How does the SLA interact with the polymerase to promote RNA synthesis on the authentic 3′ end of the genome? How does the 3′SL change its conformation during the initiation process in the infected cell? What is the role of helicases and RNA chaperones in modulating the architecture of viral genomes? It is of great interest to define the interplay between the promoter SLA, the 3′SL, the viral NS5 polymerase, and RNA structures that work as enhancers of viral RNA synthesis. It is likely that these RNA signals interact with other RNA molecules and/or proteins. Therefore, effort towards identifying functionally relevant binders of these RNA structures will be necessary to fully understand their mechanism of action. Another important question is whether the functional structures identified as regulators of RNA synthesis are necessary for both minus and plus strand RNA amplification.It has been widely accepted that flavivirus genome cyclization is essential for viral replication, however, recent observations support the idea that linear and circular conformations of the viral RNA co-exist and that dynamic RNAs are required for viral replication. This new view of the viral genome, as a flexible molecule, will help to understand the function of alternative RNA structures formed in different stages of the viral life cycle. Finally, because the DENV RNA acquires complex secondary and tertiary structures, the challenge is to study these high order structures in the context of the whole viral genome.


Polypyrimidine Tract Binding Protein (PTB) is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES) activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understanding of the mechanism of RNA binding by RNA Recognition Motif (RRM) domains. In the present review, we will describe the structural properties of PTB, its paralogs and co-factors, the role in post-transcriptional regulation and actions in cell differentiation and pathogenesis. Defining the multifunctional roles of PTB will contribute to the understanding of key regulatory events in gene expression. In eukaryotic cells, multiple and coordinated processes contribute to the fine-tuning of the transcription and translation of selected genes in the nucleus and the cytoplasm. The initial steps take place in the nucleus where promoter recognition, mediated by transcription factors, requires chromatin and nucleosome modifications driven by epigenetic events, and allows the regulation of the RNA polymerase transcriptional activity. When pre-mRNAs emerge from the transcription sites, they are associated with The RNPs assembled on each mRNA diversify their composition by recruiting and reassorting selected components. During mRNA maturation, the RNPs are further recombined and modelled, regulating the subsequent stages of RNA biogenesis, which include the 5′-end capping, splice site recognition, RNA editing, 3′-end cleavage, polyadenylation, nuclear pores export, sub-cellular localization, ending to protein translation and RNA degradation –3 Figur. AlternaTwo major RNA binding protein families, the heterogenous nuclear ribonucleoproteins (hnRNPs) and the serine/arginine (SR)-rich proteins (SR proteins) contribute to AS events within RNP complexes –10. BothOne of the most investigated exon recognition repressors, belonging to the hnRNPs family, is the polypyrimidine tract-binding protein (PTB) that binds to intron pyrimidine-rich elements and mediates tissue-specific regulation of exon splicing ,18.PTB is recruited not only in AS, but in additional steps of RNA processes eliciting various biological effects, including transport, stabilization, and mRNA translation. The cell type and the position of the specific RNA binding sites are determinant factors for the variety of PTB effects on RNA destiny . StartinThis review will focus on the established roles of PTB in splicing regulation and protein translation highlighting the most recent contributions to the understanding of its multi-functionality.2.PTB (accredited gene name PTBP1), also known as hnRNP I, is a shuttling protein that moves rapidly between nucleus and cytoplasm, with a predominant nuclear localization [N-terminus of the protein [PTB gene maps on chromosome 19 at position p13.1, and is organized in 15 exons that are subjected to alternative splicing, producing at least four isoforms [lization . The 57 lization ,24, and  protein . The PTBisoforms . PTB1 isisoforms . In spit3.C-terminal domain (PABP), and the WW domain [The members of the hnRNP protein family were initially discovered in the late 1980s and were attributed to the same protein family by the presence of the RRM, which is the most conserved domain among the RNA binding domains (RBDs). The RRM is a functional domain broadly distributed in hundreds of proteins encoded by eukaryotic genomes. Compared to other known RBDs, such as the K-homology domain, the RGG (Arg-Gly-Gly) box, the Sm domain, the DEAD/DEAH box, the Pumilio/FBF (PUF domain) and the Piwi/Argonaute/Zwille (PAZ), for review see , RRM is W domain . The strW domain ,30. CanoW domain . In a tyW domain ,32 and cRRM3 can compete with the pre-mRNA splicing factor U2AF65 (U2 auxiliary factor) at polypyrimidine stretches located at the 3′ splice site of target introns . In addiThe peculiarity of the structural features of the RRMs allows PTB to be involved in the splice site choice in alternative splicing events, but also to be recruited in several additional events in post-transcriptional regulation, including RNA stability and translation. The consensus sequences for RNA-PTB binding are represented by sequences containing 15–25 pyrimidines, with a preference for pyrimidine tracts containing UCUU, CUCUCU –36. The et al. [RRM1 and RRM2 are separated by flexible linkers from the rest of the protein, which allow them to adopt different conformations and to favour contact with specifically structured RNA. A novel interaction with U1 snRNA has been recently attributed to RRM1 and RRM2 and this represents the first demonstration of a direct interaction between PTB and the spliceosome . Detaileet al. , have hiIn addition to RNA, RRM2 can interact with the amino acid sequence (S/G)(I/L)LGXXP present in the co-repressor proteins Raver1 and Raver2 –46, enab4.Two PTB paralogs are generally expressed in mammalian tissues: the nPTB/brPTB/PTBP2 which is expressed at high levels in adult brain, muscle and testis ,48 and RPTB and its paralogs share more than 70% amino acid sequence identity organized in the four RRM-type domains. The most extensively studied is the nPTB, initially described as a neuron specific splicing factor . Furthercis-acting sequences at the 3′ untranslated region (3′UTR) of CD154. In activated T lymphocytes, the alternatively spliced isoforms of PTB expressed at different amounts act as trans-factors competing with each other for binding to the CD154 3′UTR, and modulating CD154 mRNA stability [In T lymphocytes, both PTB and the alternatively spliced isoform lacking RRM1and RRM2, called PTB-T, participate in humoral and cellular immunity by targeting the CD154 cytokine mRNA. The activity is elicited in the cytoplasm by binding to pyrimidine tability . These dRaver1 gene is ubiquitously expressed in tissues at different levels and encodes a 748 amino acid protein containing three RRMs, two nuclear localization signals (NLSs), one nuclear export sequence and a proline-rich region at its C-terminal region [raver1 null mutant mouse, the loss of Raver1 is associated with a reduction of synaptic plasticity [As a splicing repressor factor, PTB may require the binding to selected factors that act as co-repressors. Raver1 and Raver2 are two well characterized PTB co-repressors –60. Ravel region ,62,63. Sl region . During l region ,63 and ml region . In a raasticity . A recenasticity .Raver1 and Raver2 promoters and to the identification of cis-acting signals [Raver2, the additional PTB co-factor is a 625 aa protein of 72 kDa with a similar overall organization of the functional domains when compared to Raver1. Raver2 expression is restricted to differentiated neurons and glia cells. We have recently contributed to the characterization of  signals . In cont signals .5.PTB is implicated in the control of alternative exon selection during mRNA processing of many different transcribed genes, including its own pre-mRNA. The exon typologies include short alternative exons named “cassette exons”, “mutually exclusive exons” or alternative 3′ terminal exons ,54,61,68A-γ2) exon 9 regulation, in which a single PTB molecule is required to loop out a branch-point adenosine [These models describe a looping-out RNA mechanism in which PTB binds distant pyrimidine tracts, resulting in a new position in which the polypyrimidine tracts are close to each other ,68. All denosine . In the Additional factors, other than U2AF65, may antagonize the PTB repressive role such as the ETR3-like factor (CELF) proteins CUG-BP, RBM4, TIA-1, Nova, and Fox-1 . Recentlcis-acting splicing regulatory elements recognized by PTB has been derived by high-density oligonucleotide splice-sensitive microarray analyses in HeLa cells, where PTB was knocked down [Microarray-based genome wide approaches and CLIP-seq methods have shown that PTB not only acts as a splicing repressor, but can also enhance the inclusion of exons –92. A glked down . The resked down . Two difked down . The altked down . The datked down . Althougked down ,96,97.6.PTB has the property to shuttle between nucleus and cytoplasm. Whereas in the nucleus PTB acts as an alternative splicing factor, in the cytoplasm it is involved in post-transcriptional regulation processes that require cap-independent translational controls, RNA localization and stability.PTB participation in internal ribosome entry site (IRES)-mediated translation initiation of both cellular and viral mRNAs is extensively documented. IRESs require the formation of specific RNA structural conformations at the 5′UTR of the mRNA that help the recruitment of the translational machinery. Additional cellular factors, known as initiation of translation accessory factors (ITAFs), may bind the IRESs and lead to a more efficient translation initiation . PTB hasPTB is a determinant of tissue and host tropism for rhinovirus and poliovirus –107. PTBin vitro that PTB binds specifically to the insulin mRNA 5′UTR, when cap-independent insulin biosynthesis is produced in response to nitrosative stress [The mouse homologue of PTB is involved in the regulation of early mouse development through IRES dependent translation of CDK11 (p58) protein kinase and modulates the cell cycle in mouse embryonic stem cells . In the e stress . PTB pose stress  and regue stress .N-ras) translation regulatory factor [As a chaperone, PTB influences apoptotic peptidase activating factor 1 (Apaf1) translation by recognizing the Apaf1-IRES structure  and posiy factor . Cytoplay factor ,133.7.mper2, a circadian core clock gene, by binding to the 3′UTR CU-rich sequences [PTB has been found to be associated with pyrimidine-tracts present at 5′ and/or 3′UTRs of mRNAs. By binding to UTR sequences, PTB may be involved in the 3′-end processing, localization, and stability of several mRNAs ,134–138.equences .Drosophila and Xenopus development. These studies demonstrate that PTB plays a role in the remodeling of RNP, promoting RNA localization and translation regulation of critical determinants of the embryo development. In the cytoplasm, Drosophila PTB, known as the product of the hephaestus locus, binds to multiple sites in the oskar 3′UTR leading to mRNA oligomerization that causes oskar translation repression. The model proposed in this study is that PTB may promote the formation of densely packed RNP particles with oskar RNAs, thereby preventing access for the translation machinery [Xenopus PTB ortholog. In Xenopus oocytes, PTB is required for transport and localization of the Vg1 RNA, a critical factor for the correct patterning of the embryo. Lewis et al. [An additional PTB functional role that deserves to be highlighted derives from the studies on achinery . A simils et al.  have dem8.Drosophila and in development of Xenopus [ptb knockout mice the absence of PTB results in embryonic lethality and severe defects in proliferation and differentiation of embryonic stem cells [et al. [In vivo and in vitro results demonstrate that the reduced expression of PTB derives from a caspase-dependent cleavage of the protein during the myocardium development. The process is regulated by histone deacetylases and the caspase inhibitor FLICE-like protein (cFLIP), which induce the PTB degradation during cardiac muscle differentiation.Based on the extensive literature supporting the relevance of PTB in multiple biological processes, including development, as mentioned in section seven for germ cell differentiation in  Xenopus ,155, it em cells . As wellem cells ,157. Widem cells ,93. Bothem cells . Splicinem cells . An estiem cells . An inteem cells ,160. Theem cells . Ye et a [et al.  have demversus nPTB seems to occur in post-mitotic lens fibre cells [et al. [A relevant role played by PTB in splicing regulation during cell differentiation is highlighted by studies on neuronal differentiation, reviewed in . PTB andre cells . A recen [et al.  in a mou [et al. . A compr9.et al. [et al. [The levels of PTB and nPTB expression are subject to microRNA control. Given the estabilished role of microRNAs (miRNAs) in post-transcriptional gene expression regulation, their involvement in PTB and nPTB expression during cell differentiation was expected to be demonstrated. A number of pieces of evidence are now available supporting the function of tissue specific microRNAs in the regulation of PTB expression. The reduced level of PTB and the almost complete absence of nPTB during myoblast differentiation, have been demonstrated to be associated with the induction of the muscle-specific microRNA miR-133 expression . During et al.  demonstr [et al.  have sho10.FGFR1, FGFR2), apoptosis , invasion (CSRC), motility , and multi-drug resistance (ABCC1). In the cytoplasm, PTB increases IRES-mediated translation or RNA stability of transcribed genes involved in proliferation (IGFR1), angiogenesis (VEGF), and apoptosis (APAF1) [N-terminal truncated isoform by binding to IRESs cis-acting elements [PTB expression may affect cancer initiation and progression ,169. Per (APAF1) ,126–128.elements . The preelements .PTB forms complexes with transcripts encoding focal adhesion scaffolding proteins which may affect cell spreading ,174. Kno11.PTB by activating its promoter region. In congenital myasthenic syndrome, the aberrant inclusion of muscle nicotinic acetylcholine receptor α subunit (CHRNA1) exon P3A is caused by an intronic mutation. PTB overexpression by tannic acid has been demonstrated to foster CHRNA1exon P3A skipping, opening new possibilities of therapeutic approaches based on PTB expression regulation [The contributions derived from the intense studies on RNA binding proteins confirm that the unique properties of PTB RRM domains are the key elements enabling its multiple cellular functions. The relevant role played by PTB in most post-transcriptional processes is indeed based on its ability to recognize specific RNA sequences, modifying the local RNA structural arrangements. Competition in RNA binding between PTB and other regulators of the RNA metabolism appears critical to its diverse functions. As a consequence, the levels of PTB expression as well as the rate of nuclear import and export may become censorious to the cell fate. Thus, while the identification of new PTB targets is rapidly progressing by means of genome-wide investigations , an effogulation . We expe


An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use  RNA polymerase II (Pol II) is a molecular machine that is responsible for transcribing all protein coding genes in the eukaryotic genome. Transcription by Pol II is a highly regulated process consisting of several rate-limiting steps. During transcription elongation, a number of transcription factors are essential to modulate Pol II activity. One of these factors is the Negative Elongation Factor (NELF), and it plays a major role in promoter-proximal pausing, a widespread phenomenon during early transcription elongation. NELF-E, a protein subunit of the NELF complex contains a conserved RNA binding domain that is thought to regulate transcription through its interaction with newly transcribed RNA made by Pol II. However, the function of the RNA binding activity of NELF-E remains unresolved due to prior conflicting studies. Here, we clarify the RNA binding properties of NELF-E and provide insight into how this protein might facilitate promoter-proximal pausing of Pol II in transcription. Moreover, we identify the precise region of NELF-E binding in one of its known regulatory targets, HIV-1. Taken together, the results presented indicate a dynamic interplay between NELF and specific RNA sequences around the promoter pause region to modulate early transcription elongation. RNA polymerase II (Pol II) is a molecular machine responsible for transcribing all protein coding genes in the eukaryotic genome in a highly regulated multistep process. With the help of specific and general transcription factors, it binds to promoters, rapidly initiates transcription, transcribes approximately 20–60 nucleotides of nascent RNA, and then can pause before entering productive elongation Negative elongation factor) and DSIF [DRB  sensitivity inducing factor], form a stable complex with Pol II and inhibit its elongation shortly after initiation. In contrast, P-TEFb (Positive transcription elongation factor b), a complex of CDK9 kinase and CyclinT, overcomes the influence of these factors and promotes the release of Pol II into productive elongation Three protein complexes have a major role in Pol II pausing. Two of these, NELF  that is essential for its ability to bind RNA and inhibit elongation in vitroin vitro cross-linking study by Gilmour and coworkers suggested that RNA binding by Drosophila NELF-E may not be involved in promoter-proximal pausing, but instead may interact with longer nascent transcripts at a location further downstream NELF consists of four protein subunits  Studies investigating the regulation of HIV-1 transcription implicate how NELF-E functions Drosophila and human NELF-E  and identify the presence of an NBE within TAR RNA, which is located in a different region than previously thought to be bound by NELF-E. Finally, we found that NBEs are enriched at promoter-proximal pause regions in the Drosophila genome. This implies a functional role for NELF-E RNA binding in Pol II pausing.Here, we characterize the RNA binding specificity of NELF-E and attempt to clarify its role in promoter-proximal pausing. We demonstrate that NELF-E is capable of binding to RNA with high affinity and specificity. Moreover, we define the NELF-E binding element (NBE) for both Drosophila NELF-E. To identify the sequence specificity of dNELF-E, a microcolumn-based SELEX  experiment was performed with full-length dNELF-E or its RRM domain 15 unique molecules) used contained a 70-nucleotide randomized region flanked by two constant regions that allowed for amplification of selected RNAs and in vitro transcription to generate subsequent aptamer pools. This affinity-based approach utilized modular, custom-made microcolumns that permit high-efficiency selection of aptamers by exploiting optimal fluidic parameters CUGAGGA(U). Examination of the pool 6 sequencing results suggests that the more conserved 3′ position in the NBE from the RRM domain selection is due to faster convergence of NBE containing sequences during earlier SELEX cycles (unpublished data).No published studies have investigated the nucleotide specificity of Analysis of the most enriched RNA aptamers containing an NBE revealed a common secondary structure consisting of a putative non-canonical kink-turn (K-turn)  [29]. K-d) of dNELF-E binding to Napt1min: a fluorescence electrophoretic mobility shift assay (F-EMSA) and a fluorescence polarization (FP) assay, each relying on different physical properties of the protein/RNA complex d; F-EMSA = 44±22 nM and FP = 21±7 nM) (Two approaches were used to quantitatively measure the equilibrium dissociation constant (K21±7 nM) ; Table 1d; F-EMSA = 880±170 nM and FP>2000 nM), demonstrating the importance of the NBE . The binding affinity of dNELF-E to Napt1NBEmut is much weaker  compared to that of Napt1min  compared to the parent minimal aptamer Napt1min  Drosophila homolog, dNELF-E, binds specifically and with high affinity to its RNA targets. Interestingly, a closer examination of the TAR sequence reveals the sequence CUGGGA within the loop region, which is very similar to the NBE sequence CUGAGGA found in Napt1min.The NELF-E RRM is conserved between tructure  that incd; F-EMSA = 350±40 nM and FP = 130±10 nM)   . Since d82±1 nM) . Based od; F-EMSA = 300±20 nM and FP = 200±10 nM)  and dNELF-E specifically targets the hNBE, it is plausible that hNELF-E actually binds this region of TAR, instead of the lower stem as previously reported. The wild-type TAR RNA sequence was first tested for binding with hNELF-E and found to bind with a higher affinity than previously reported (K0±10 nM)  [25]. ThTo test if hNELF-E requires the hNBE in the loop region for its interaction, binding to the isolated dsRNA stem of TAR was examined. We generated a stem that lacks the hNBE (TAR-ΔhNBE) by annealing together two ssRNA sequences of TAR. No significant binding was detected with concentrations up to 2 µM hNELF-E protein . This wad; F-EMSA = 250±20 nM and FP = 250±20 nM) as unmodified TAR , while hNELF-E binds the two targets with similar affinities and has a small difference in binding free-energy . The results for dNELF-E(mut) show that, like hNELF-E, it does not discriminate between the two targets . This analysis was repeated comparing the binding of each NELF-E variant to TAR+A and TAR RNA , that substitutes the seven amino acid region of dNELF-E with the human counterpart . We then TAR RNA . A simil hNELF-E . This imDrosophila genomic regions near annotated transcription start sites (TSSs). The conserved seven-nucleotide NBE (CUGAGGA) that was characterized in this study  increase of the NBE similarity index among paused genes compared to non-paused genes  −16, Fisher's Exact Test)  from transcriptionally engaged Pol II in ct Test) . This suCUGAGGA(U) for Drosophila). NELF-E requires that the consensus be accessible in single-stranded RNA, and the binding can be enhanced with more complex secondary structures, such as the K-turn of Napt1min or the loop region of HIV-1 TAR RNA.RRM-domain proteins are known to have diverse modes of target recognition that can include a variety of specific RNA, DNA, and protein interactions This work reveals that hNELF-E binds specifically to the HIV-1 TAR RNA stem loop that is closely related to the dNBE. These results have important implications for transcriptional regulation of HIV-1 by NELF and the P-TEFb-Tat complex. The hNBE overlaps the binding site for the P-TEFb subunit CycT1 and is adjacent to the TAR bulge region where Tat binds It is fitting that the NBE would be enriched in pause regions (+20 to +60 base pairs from the TSS) seeing that NELF plays a critical role in promoter-proximal pausing for many genes. Binding of NELF-E to this element might stabilize paused Poll II, working together with other pausing factors including DSIF An intriguing observation is the increased probability of NBE-like sequences >100 base pairs downstream of the TSS into the gene body . This agIn addition to its role in promoter-proximal pausing, evidence suggests that NELF may coordinate a number of mRNA processing steps during transcription Drosophila and human NELF-E, and the RRM domain of dNELF-E (amino acid residues 147–247) were subcloned into pHIS-parallel1 to generate N-terminal hexahistindine-tagged recombinant proteins E. coli cells (Agilent Technologies). Liquid cultures were grown at 37°C and induced in mid-log phase with IPTG. Cultures were induced with either 1 mM IPTG at 37°C for 3 hours or 0.2 mM IPTG at 18°C overnight before collecting cells by centrifugation. Harvested cells were purified in batch according to the manufacturer's instructions for Ni-NTA Superflow (Qiagen) resins. Buffers used for the purification included lysis buffer , wash buffer (lysis buffer with 200 mM NaCl), and elution buffer (wash buffer with 20% glycerol and 250 mM Imidazole). When necessary, eluted protein samples were subject to a mono Q column  for further purification as described elsewhere Full length 5′-AAGCTTCGTCAAGTCTGCAGTGAA-N70-GAATTCGTAGATGTGGATCCATTCCC-3′. This template allows for amplification and transcription using primers that are complementary to the constant regions and one primer encoding a T7 promoter. The starting RNA pool used in this selection had a complexity of >5×1015 unique molecules. Microcolumn SELEX was performed on dNELF-E and its RRM domain using a 20 µl column for each protein. A detailed method was previously described by Latulippe and Szeto et al. with some modifications 2 for round 1 and 1 mM MgCl2 for each subsequent round), and 0.02% Tween-20. Wash buffer includes 20 mM Imidazole in the binding buffer.A 120 nucleotide RNA library was generated as described http://cores.lifesciences.cornell.edu/brcinfo). Analysis of the sequencing data, which includes filtering and clustering analysis are described in detail by Latulippe and Szeto et al. A purified PCR product from cycles 4 and 6 were re-amplified with barcoded primers and sequenced on the HiSeq 2000 (Illumina) sequencing platform using a standard single-end, 100 nucleotide sequencing protocol at the Cornell University Life Sciences Core Laboratory Center (in vitro transcribed from synthetic DNA templates (Integrated DNA Technologies), PAGE purified, and eluted into DEPC treated 10 mM Tris-Cl pH 7.5. Napt1min includes an additional GC base pair on end to accommodate for the additional guanosine designed in the Napt1min template containing a T7 promoter. Purified RNA were then 3′-end labeled with fluorescein 5-thiosemicarbazide (Invitrogen) as described 2, and 0.02% Tween-20, 0.01% IGEPAL CA-630, and 10 µg/ml yeast tRNA) to a final volume of 50 µl in black flat-bottom 96-well half-area microplates (Corning). It is recommended to use DEPC-treated water and SUPERase-In RNase inhibitor (Invitrogen) according to the manufacturers directions to prevent RNA degradation. Reactions were equilibrated for 1–2 hours before taking FP measurements on a Synergy H1 Microplate Reader (BioTek) with the appropriate filter cube for fluorescein (Ex: 485/20 Em: 528/20). After taking FP measurements, the same experiment was loaded on a pre-chilled 5% slab acrylamide gel (0.5X TBE) and electrophoresed at 4°C for approximately 1 hour and 10 minutes. Gels were imaged immediately on a Typhoon 9400 imager . The fluorescence intensity of bound and free RNA was measured with ImageQuant and the data was fit to a Hill equation in Igorpro software (Wavemetrics), which includes the Levenberg-Marquadt algorithm and statistical analysis tools Fluorescence electrophoretic mobility shift (F-EMSA) and fluorescence polarization (FP) assays were performed as described previously Drosophila S2 cells as described previously 7 nuclei and GRO-seq libraries were prepared as in Core et al. 5′- AAGCTTCGTCAAGTCTGCAGTGAA-3′) and the library was amplified using this oligo and a forward oligo that recognizes the 5′ constant region and contains the T7 promoter (5′-GATAATACGACTCACTATAGGGAATGGATCCACAT CTACGA-3′), allowing the final library to utilize the same reagents that are used for preparation of SELEX pools between cycles. The final GRO-RNA library had an average size of ∼150–200 nucleotides including the constant regions.Nuclei were isolated from non-heat-shocked RNA aptamer isolation via dual-cycles) which has been shown to significantly reduce the time and cost of isolating RNA aptamers and to improve enrichment rates, by systematically omitting amplification steps Due to the relatively low complexity of the GRO-RNA library, a total of two selection cycles were completed using a method we call RAPID (http://cores.lifesciences.cornell.edu/brcinfo). Following sequencing, reads were partitioned according to 5′-end barcode using the fastx_barcode_splitter.pl script from the FASTX-Toolkit v0.0.13.1 (http://hannonlab.cshl.edu/fastx_toolkit/). Barcodes were then trimmed using the fastx_trimmer utility from the FASTX-Toolkit. After trimming, the 5′ library preparation adapter was removed using the semi-global alignment and adapter removal utility cutadapt v1.1 (parameters: -g -e 0.20 -m 26 -O 18 –match-read-wildcards) Control and experimental libraries were multiplexed and sequenced on an Illumina HiSeq 2000 instrument using a standard single-end, 100 nucleotide sequencing protocol at the Cornell University Life Sciences Core Laboratory Center (D. melanogaster genome (assembly dm3) using 64-bit bowtie v0.12.7 allowing 2 possible mismatches and requiring unique alignment Trimmed sequences were then mapped to the Drosophila10 p-value) within +10 to +30 base pair region for each gene was used as the test value, and the two groups were compared using a Kolmogorov-Smirnov test or two-sample unequal variance t-test. Sequence logos were generated as described previously DNA sequence motifs were analyzed as described previously Figure S1Secondary structure predictions of putative dNELF-E aptamers. The mfold web server was used to generate each structure; shown are the most thermodynamically stable predictions for each sequence analyzed. Nucleotides that make up the NBE are colored red. A sequence identification name is given below each putative aptamer.(TIF)Click here for additional data file.Figure S2dNELF-E binds to NBE containing aptamers. (a) Predicted secondary structure of Napt25 min with the NBE nucleotides shown in bold. (b) A representative F-EMSA of full length dNELF-E binding to Napt25 min. Below the gel is a plot of the fraction of bound Napt1min against protein concentration with a fit to the Hill equation. The equilibrium dissociation constant is shown in the graph for this individual experiment.(TIF)Click here for additional data file.Figure S3Drosophila. (bottom) A representative plot of the fraction bound of either HIV-1 TAR (black line) or HIV-1 TAR+A (red line) RNA bound to hNELF-E(mut). A visual representation of each RNA tested is shown, with the inserted ‘A’ of TAR+A colored red.hNELF-E(mut) binds to HIV-1 TAR and HIV-1 TAR+A with a similar binding affinity. (top) A summary of the mutagenesis performed on hNELF-E. The seven amino acid region boxed as in (TIF)Click here for additional data file.Figure S4−22, 10 to 20 = 1.1×10−32, 20 to 30 = 7.4×10−43, and 30 to 40 = 0.72 (not significant).The NBE enriches +20 to +30 nucleotides downstream the TSS. A boxplot of -log10 for the NBE near the TSS in (TIF)Click here for additional data file.Figure S5Enrichment of NBE-like sequences within genomic RNA. The abundance of sequences containing NBE-like motifs identified from a NELF-E SELEX compared to a resin only control. The bar graph shows the number of sequences containing an NBE where the variable position is indicated. Reads are plotted as multiplicity per million mapped reads (MPM).(EPS)Click here for additional data file.


Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Parameciumbursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated  The addition of a 5’ cap structure to RNA transcripts synthesized by RNA polymerase II is fundamental to eukaryotic gene expression . The N7-RNA cap synthesis occurs in 3 consecutive enzymatic steps. An RNA triphosphatase (RTase) first cleaves the 5’ terminal phosphate of the RNA molecule, to form a diphosphorylated end. An RNA capping enzyme, or RNA guanylyltransferase (GTase), transfers a GMP moiety onto the diphosphorylated end, followed by an RNA guanine-N7) methyltransferase (MTase) which adds a methyl group at the N7 position to form the classical RNA cap structure . Cry. Cry-. In cellulo translation assays, were done with the firefly luciferase RNA bearing a 60 nt long polyA tail at its 3’ end, which was synthesized from an HpaI digested plucA60 plasmid . Following transcription, the RNA molecules were purified on denaturing 8M UREA-PAGE and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantified by spectrophotometry at 260 nm.RNA substrates were synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. For the evaluation of RNA capping efficiencies, a 50 nt long RNA was synthesized using a modified version of the T7 promoter that will allow the initiation of the transcription with a single guanosine residue  and TriLink Biotechnologies (USA).2 and 1.5 µg/ml of inorganic yeast pyrophosphatase (Roche) for 1hr at 30°C.The first step of the GTase reaction was carried out by incubating the purified PBCV-1 GTase (10 µM) with the appropriate substrates  in a buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 5 mM MgCl32P] GTP and 2 mM of either unlabelled GTP or each unlabelled nucleotide analogue separately. The IC50 of nucleotide analogues was determined by carrying out the standard first step reaction in the presence of 0.2 pmol of [α-32P] GTP and increasing concentrations of up to 2 mM of each nucleotide analogue. The reactions were stopped by the addition of EDTA to 10 mM and SDS to 1% and analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS. The radiolabelled proteins were visualized by autoradiography of the gel. Radiolabelled covalent complex formation was quantified by scanning the gel with a PhosphorImager (Amersham Biosciences).Inhibition of the first step of the GTase reaction was evaluated by carrying out the standard first step GTase reaction in the presence of 0.2 pmol of  GTP to the enzyme. A single SDS-stable GMP-enzyme complex that migrated as a 39.9 kDa species was detected following SDS-PAGE (32P] GTP and the PBCV-1 capping enzyme (10 µM). The presence of non-labelled GTP (2 mM) led to a maximum loss of the radiolabelled EpG signal  as an inhibitor is shown in 50 values ranged from 100 µM (high inhibition e.g. GTP) to 2.0 mM or more (low inhibition e.g. 2’dGTP). The fact that some nucleotide analogues led to inhibition of the PBCV-1 GTase activity at high concentrations (> 2.0 mM) raised the possibility that they might be acting as chelator for the essential divalent metal ion co-factor, and therefore these analogues were not included in subsequent analyses.We initially monitored the ability of a library of 22 synthetic nucleotide analogues to inhibit the PBCV-1 GTase. The first step of the GTase reaction entails the nucleophilic attack of the α-phosphate of GTP by the enzyme and the subsequent formation of a covalent enzyme-GMP intermediate. The ability of the purified PBCV-1 GTase protein to form a covalent GMP-enzyme intermediate was detected by label transfer from . Re. Re24]. sibility  and 2D. 9 and A10) were clearly not transferable onto RNA. We conclude that the formation of the covalent E-NMP intermediate does not necessarily imply the completion of the second step of the GTase reaction A second assay was performed to demonstrate the transfer of nucleotide analogues onto RNA. A purified 32P-internally labelled RNA was incubated with the PBCV-1 GTase, the PBCV-1 RTase, magnesium ions and each nucleotide analogue separately. The reaction products were analyzed by Urea-PAGE. The addition of GTP or a nucleotide triphosphate (A2) analogue led to the formation of a slower migrating species corresponding to the capped RNA , an evolutionarily conserved subunit of the heterotrimeric eIF4F initiation complex . ,32. 9,32reaction -column 2 to GTP) -column 418) cannot be hydrolyzed by the PBCV-1 GTase  GTP in the presence of either GTP (0.5 mM) or A19 or A20 (0.5 mM) in a standard GTase buffer ranging from a pH of 5 to 9. The reactions were resolved by SDS-PAGE and analyzed by a Phosphorimager. The formation of the radiolabelled E-GMP complex was quantified and its relative ease of formation is plotted as a function of the pH. High ease of formation implies low inhibition by either GTP or the nucleotide analogue, while low ease of formation implies high inhibition by the unlabelled nucleotides.(TIF)Click here for additional data file.Figure S3Binding of GTP to the wild-type and K188A mutant of the PBCV-1 GTase. Increasing amounts of GTP were added to a 2 µM solution of the enzyme in binding buffer  and the emission spectrum was scanned from 310 to 440 nm, following excitation of tryptophan residues at 290 nm. (●) indicates wild-type enzyme and (▲) indicates the K188A mutant enzyme.(TIF)Click here for additional data file.Table S1RNA (guanine-N7) methyltransferase activities with RNA capped with nucleotide analogues.(TIF)Click here for additional data file.


As an RNA virus, hepatitis C virus (HCV) is able to rapidly acquire drug resistance, and for this reason the design of effective anti-HCV drugs is a real challenge. The HCV subgenomic replicon-containing cells are widely used for experimental studies of the HCV genome replication mechanisms, for drug testing in vitro and in studies of HCV drug resistance. The NS3/4A protease is essential for virus replication and, therefore, it is one of the most attractive targets for developing specific antiviral agents against HCV. We have developed a stochastic model of subgenomic HCV replicon replication, in which the emergence and selection of drug resistant mutant viral RNAs in replicon cells is taken into account. Incorporation into the model of key NS3 protease mutations leading to resistance to BILN-2061 , VX-950  and SCH 503034  inhibitors allows us to describe the long term dynamics of the viral RNA suppression for various inhibitor concentrations. We theoretically showed that the observable difference between the viral RNA kinetics for different inhibitor concentrations can be explained by differences in the replication rate and inhibitor sensitivity of the mutant RNAs. The pre-existing mutants of the NS3 protease contribute more significantly to appearance of new resistant mutants during treatment with inhibitors than wild-type replicon. The model can be used to interpret the results of anti-HCV drug testing on replicon systems, as well as to estimate the efficacy of potential drugs and predict optimal schemes of their usage. Hepatitis C virus (HCV) chronifies in ≈80% of infections, causing hazardous liver diseases, most notably liver cirrhosis and hepatocellular carcinoma. Current success in treating HCV is associated with the use of such drugs as ribavirin, peginterferon, protease inhibitors, and their combinations. However, the drug resistance of virus remains an important problem. Substantial efforts therefore are made to understand the mechanism of HCV infection, as well as to develop new antiviral therapies de novo synthesis of minus-strand RNA. This minus-strand RNA serves as a template for the synthesis of new plus-strand RNA, presumably directly from the double-stranded RNA intermediate. Newly synthesized plus-strand RNA can then either be used for re-initiation of minus strand synthesis, or is exported from the membrane vesicles to the cellular cytoplasm, where it can again be translated or incorporated into membrane vesicles for the next replication round The replication cycle of the HCV subgenomic replicon in Huh-7 cells includes: 1) the IRES-mediated translation of the plus-strand RNA with the formation of polyprotein, processing of which by HCV NS3 protease yields the viral nonstructural proteins NS3, NS4A, NS4B, NS5A, NS5B; 2) the formation of membrane vesicles induced by the viral protein NS4B with the participation of the HCV NS5B protein and the cellular proteins PI4K-IIIα, cyclophilin A/B, hVAP-A/B among others; 3) the replication of the viral genome in membrane vesicles. Inside of the membrane vesicles, the RNA-dependent RNA polymerase NS5B associates with the 3′-end of the viral plus-strand RNA and initiates the The HCV NS3 protease is one of the promising candidates for the design of new potential anti-HCV drugs. New α-ketoamide inhibitors of NS3 protease, namely telaprevir (VX-950) in vitroIt is well known that HCV is able to quickly acquire drug resistance. The drug resistant mutant RNAs appear during the wild-type RNA replication, partly, because the HCV NS5B polymerase lacks a proofreading function Two sources of the mutant RNAs that determine the acquisition of drug resistance by virus are described in the literature. The first one is the RNA quasi species that are resistant to a specific drug and preexist both in cell culture and patient liver or plasma prior to therapy cis/trans isomerase, that directly interacts with the NS5B and NS5A proteins of the viral replicase complex and stimulates the RNA binding activity of this complex, was recently proposed as a prospective target for antiviral therapeutic strategies Due to the rapid development of resistance in HCV, increasing attention was recently paid to inhibitors of host factors involved in HCV replication. Cyclophilin A/B, the cellular peptidyl-prolylMathematical models can improve our understanding of antiviral action and may predict the efficacy of potential drugs. Several models were developed to describe the HCV RNA dynamics and drug effects in patients Modeling of the intracellular HCV replication processes appears to be a very complicated mathematical problem. Dahari H. et al. Binder et al. Nakabayashi J. Here we develop a minimal stochastic mathematical model, describing the kinetics of the viral RNA suppression by the NS3 protease inhibitors in replicon cells. In contrast to the previous model in vitro replicon studies results from the outgrowth of preexisting NS5B mutations rather than from the generation of mutations after the onset of antiviral suppression It was demonstrated previously that the resistance observed during We also use our model to study the efficacy of potential inhibitors targeted to the integral cell factor. These inhibitors can either stop the cell factor formation or reduce its activity in the viral replication complex. Our analysis revealed that the NS3 protease inhibitors are more effective when combined with cell factor inhibitors reducing activity of the viral replication complex.cis-complementation mechanism, i.e. complexes containing proteins translated from a particular viral genome can mainly replicate it p) and mutant (pm) viral nonstructural proteins do not mix when forming the replicase complexes pcf and pcfm with the cellular factor cf. For simplicity we assumed the equality of the rate constants kp for the interaction of cf with either the mutant or wild-type viral proteins. In turn, pcf and pcfm can bind to wild-type or mutant plus-strand RNA in the cytoplasm. Binding of pcf to the wild-type plus-strand RNA R results in the formation of vesicles V with the rate constant kv, whereas binding of pcfm to the NS3 protease mutant plus-strand RNA Rm results in that of vesicles Vm with the same rate constant. In agreement with our previous model cf restricts the number of active replicases and, hence, of the viral RNA in the cell. Thus, the proposed minimal model is an extension of our previous model, incorporating vesicles and mutants.We propose the following scheme describing the subgenomic replicon replication in the model . The repV and Vm vesicles produce and release the wild-type R and mutant Rm plus-strand HCV RNAs into cytoplasm with rate constant kout. According to available data, the HCV NS5B polymerase lacks a proofreading function, which results in an estimated erroneous base substitution rate of ∼1 in 10,000 replicated bases V can produce the mutant RNAs Rm with the rate constant kout_v_vm, and, vesicles Vm with regard to back mutations can produce the wild-type RNAs R with the rate constant kout_vm_v. The quantities R and Rm are translated into wild-type (polyp) and mutant (polypm) polyproteins, respectively, with the rate constant kt.Instead of incorporating the rather complicated processes of new RNA strand synthesis inside vesicles explicitly, we assumed that the polyp can reversibly interact with the NS3 protease inhibitor inh, giving rise to the polypi complex with the rate constants ki and ki_rev for the forward and reverse processes, respectively. Similarly to the wild-type polyprotein, the mutant polyprotein polypm can interact with the NS3 protease inhibitor inh forming the polypmi complex with the rate constants kim and ki_rev_m for the forward and reverse processes, respectively. The transformation of the noncovalent complex to the covalent adduct is modeled in wild-type and mutants with the rate constants ki2, ki_rev2, and ki2_m, ki_rev2_m, respectively.Based on available information about mechanisms governing the action of known NS3 protease inhibitors, we introduced in the model a reversible process of NS3 protease/inhibitor noncovalent complex formation and a slow transformation process of this complex to the covalent adduct p when cleaved from the polyp polyprotein, and pm otherwise. The mutant NS3 protease is processed from polypm protein. The wild-type and mutant polyproteins can have different processing rates, indeed, differences of the catalytic constant catk were reported between the mutant NS3 protease as compared to the wild-type polyp and polypm is described in the model via the rate constants kc and kcm, respectively. According to the study reported in V or Vm with an active replicase complex contains one minus-strand RNA molecule, 6–12 plus-strand RNA molecules, and up to 1000 molecules of each nonstructural protein translated from these plus-strand RNAs. Therefore, kc and kcm in the model are rate constants of production of 1000 wild-type NS3 proteins and 1000 mutant NS3 proteins, respectively. We note that the wild-type and mutant RNA translation rate constant kt also corresponds to synthesis of 1000 polyproteins.The processing of both wild-type and mutant polyproteins results in the formation of nonstructural proteins, denoted by ijRm, where i stands for replication capacity of a group and j denotes its sensitivity to inhibitor. Both i and j can take values from 1 to 3 and so encode low, moderate and high values of the ijRm parameter for the group. We assumed that each RNA group replicates in specific vesicles ijVm. For example, 11Vm vesicles produce 11Rm mutant RNAs with low replication capacity and sensitivity to inhibitors, 21Vm produce 21Rm RNAs with moderate replication capacity and low sensitivity to inhibitor, and 32Vm produce 32Rm with high replication capacity and moderate susceptibility, etc. The mutant proteins  and replicases (ijpcfm) are also specific to the group. Each mutant group is characterized by the set of parameters ij, kimij,kcm and ijki_revm. The parameters kout_vm_v and kout_vm_vare assumed the same for all mutants. We showed only one mutant group in Different NS3 protease mutants have different replication capacity and sensitivity to inhibitors, defining their selective advantage during inhibitor action The stochastic approach is particularly useful for modeling biological processes going on at low concentrations of molecular substances Each run of the stochastic model gives an individual dynamics of concentrations of all molecular substances, which differs from run to run. To compare the viral RNA concentrations obtained in the model to the data we averaged the simulation results over 100,000 runs of the model.R, and values of the ij, Vmij, polyp, polypmijV, Rm, ij, pcfp, pm, and ijpcfm variables were set to 0.Two problems were solved in correspondence with the model: first, the viral RNA should reach a correct stationary state in the absence of inhibitors, and, alternatively, it should be inhibited in the presence of inhibitors according to the data. Action of inhibitors was modeled in two steps. First, the dynamics was calculated in the absence of inhibitors up to the steady state, and then we continued this dynamics after the addition of the inhibitor to the system. Therefore, the initial conditions were fixed at the transfection moment and were identical for the presence of inhibitors as well as for the absence of them. We assumed that initially there are 100 molecules of the wild-type viral RNA ij, ki2, ki_rev2, ki2mij, ki_rev2mij, kout_v_vm, kout_vm_v, kt, mr and kc were determined from the literature data, while values of parameters mp, mpolypkout, kp, kv, kcf, mcf, kimij, and kcmij were found by the model fitting to the experimental data  mr was set to 0.5 h−1. On the basis of the duration of the rate-limiting step of processing of polyprotein (wild-type) kc was set to 1 h−1. The rate constant of translation of 1000 molecules of polyprotein kt was set to 0.1 h−1The initial estimates of ki, ki_rev, ki2, and ki_rev2, characterizing the rates of the two stages of covalent adduct formation, were taken from i*KIn order to describe the interaction of inhibitors BILN-2061, VX-950, and SCH 503034 with the wild-type polyprotein, values for constants ijkim for the non-covalent mutant polyprotein/inhibitor complex was estimated via the ratio of ki to the relative sensitivity of mutant ij to the inhibitor , ijkcm  for considered mutant groups resistant to BILN-2061 and VX-950. Therefore, the deterministic model has 11 parameters; 7 of these parameters were optimized and 4 were estimated from literature. The stochastic model has 22 parameters, including 11 parameters from the deterministic model and 11 mutant-related parameters. For the stochastic model, only mutant-related parameters were optimized, while all the rest parameter values were taken from the optimization results in the deterministic model. Thus, totally 18 parameter values were optimized, including 7 parameters from the deterministic model and 11 mutant-related parameters from the stochastic model. The data consisted of 13 experimental observations of the viral RNA concentration after 3 days of treatment with different doses of BILN-2061 and VX-950 In this paper we used the enhanced Differential Evolution Entirely Parallel method. The original DEEP kp, kv, mp, mpolyp, kout_v, kcf, mcf and 6 sets for ijkim, ijkcm were selected as the best results of the fitting  and for parameter Rinit defining the initial number of virus RNA molecules. First, we calculated values of the objective functional including all penalizing terms (‘full cost’) when each parameter was varied around its optimal value with all the rest parameters being fixed at the optimal values. We allowed the full cost function to increase up to 150% of its optimal value and determined corresponding variation ranges for all parameters. Five parameters  had these ranges equal to 15–35% of their optimal values, while these ranges were more than 50% for all other parameters  after the subgenomic HCV replicon transfection are shown in One feature of the plus-strand viral RNA kinetics at initial time is worthy of special mention. According to The HCV RNA-dependent RNA polymerase NS5B lacks a proofreading function ijkcm). One can see in The dynamics of production of the NS3 protease mutant plus-strand RNAs after transfection with wild-type replicon in the absence of inhibitors is shown in kcm 0.95 h−1) and T54A (kcm 0.95 h−1) was about two months, and for A156T (kcm 0.42 h−1) this time was only a few days. The total fraction of all drug resistant mutants in the resultant RNA pool at day 60 post-transfection equals about 1% in our model that is close to the experimental estimate of the fraction of the preexisting drug resistant mutant RNAs for the same time period, that is approximately 1% The steady-state concentrations of viral RNAs for all the mutants are given in The inhibitor action is modeled after the system reaches the steady-state. The experimental kinetics of the viral RNA suppression in the presence of the BILN-2061  and VX-9The predictive power of the model was examined using the experimental kinetics of the viral RNA suppression with the SCH-503034 inhibitor  [22], anThe experimental dependencies of the viral RNA suppression rate shown in Consequently, the dependencies of the viral RNA suppression rate on the increasing concentration of the SCH-503034, SCH-900518 and ITMN-191 inhibitors  were calin vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor. It was shown that depending on the concentration of TMC380765, distinct mutational patterns emerged. Besides, the analysis of the selective advantage and replication capacity of the known NS3 protease mutants was carried out at various concentrations of the BILN-2061 and VX-950 inhibitors The ability of viruses to develop resistance to drugs is one of the central problems in pharmacology. The selection of drug-resistance mutants under inhibitor pressure depends on many factors, including the concentration of the inhibitor in the cell. For example, in In According to the figure, our model suggests the the maximum abundance of each mutant type is observed at different concentration of the inhibitor. The peaks of abundance curves for mutants with higher susceptibility to inhibitor are usually shifted towards lower inhibitor concentrations. For example, in case of the BILN-2061 inhibitor  the firsThe obtained results are in agreement with experimental data on relative replication capacity and selective advantage of the NS3 protease mutants resistant to the BILN-2061 and VX-950 inhibitors The curves of mutant abundance at different inhibitor concentrations allow us to draw the hypothesis on how the viral RNA pool for each kinetic curve in in vitro replicon studies results from the outgrowth of preexisting mutations rather than from the generation of new mutations after the onset of antiviral suppression. Significantly extending the limit of detection as compared with previous approaches in silico modeling provides the only tool to address this question. We use our model to assess the relative frequency of preexisting and newly emerging drug-resistance mutations within a single cell and compare our estimates with those obtained by Robinson et al. (2011) The impact of preexisting and newly emerging under drug pressure mutants in the drug resistance acquisition by the HCV replicon is widely discussed in the literature We estimate the contribution of preexisting and newly emerging drug-resistance mutants into total RNA pool as the ratio of the amount of mutants of each type to the whole viral RNA at the 9th day post-treatment. As preexisting mutants we consider all the mutants descending directly from mutants that existed at steady-state before the treatment starts. The newly emerged mutants are mutants that arise during the wild-type viral RNA replication under drug treatment, as well as their offspring.Due to the low fidelity of the polymerase NS5B and the high rate of turnover of the viral RNA, drug resistance is and will continue to be a major problem in the development of specific HCV inhibitors. The use of combinations of antiviral compounds that have different targets could be effective in preventing the emergence of drug-resistant viruses. In literature the effects of the combined action of the NS3 protease inhibitors and inhibitors of host factors such as cyclophilins that are also essential for viral replication are described kout, while the action of an inhibitor that blocks the cellular factor production was modeled by variation of the parameter kcf. In order to compare these effects the values of the parameters kout and kcf were set in a way that the levels of the viral RNA suppression in the case of the individual action of each inhibitor coincide , and hepatitis B virus (HBV) is complicated by drug-resistant variants of these viruses. The HCV RNA-dependent RNA polymerase, as well as the HIV and HBV reverse transcriptases, are intrinsically error-prone and have no proofreading function. These properties of the virus enzymes promote the appearance of multiple genetic variants of the viruses (quasispecies) that co-exist with the dominating wild-type virus. These quasispecies may contain mutants much less sensitive to antiviral drugs. In the absence of drugs, the drug-resistant variants appear rarely in patients, but the antiviral drug treatment leads to fast selection of the drug resistant mutants The subgenomic HCV replicon cell culture systems are widely used in studies of the HCV drug resistance. These systems are applied for selection of the drug-resistant replicon clones in the presence of drugs and subsequent sequencing of viral genes in order to determine mutations and investigate replication capacity of mutants, as well as their sensitivity to inhibitors Many cellular proteins are known at the moment that participate in HCV replication in hepatoma cells Here we present a minimalistic stochastic model of subgenomic HCV replicon replication in Huh-7 cells which takes into account the existence of drug-resistant mutants. The constructed model is based on the previous modeling attempts This model reproduces well the viral RNA kinetics in the cell from the moment of the subgenomic HCV replicon transfection until steady state, as well as the viral RNA suppression kinetics in the presence of NS3 protease inhibitors. The model was fitted to the experimental kinetics of viral RNA suppression in the presence of inhibitors BILN-2061 and VX-950 In contrast to our previous deterministic model, the suggested here stochastic model describes the two-phase dynamics of viral RNA suppression on prolonged time intervals of inhibitor action. This fact deserves special attention. The deterministic model developed earlier was extended to incorporate the emergence and selection of drug-resistant mutants. The extended deterministic model as well as the stochastic one was able to correctly reproduce the establishment of the viral RNA steady-state after transfection in the absence of inhibitors ; mr, degradation rate constant for the wild type and mutant RNAs (not shown); mpolyp, degradation rate constant for the wild type and mutant polyproteins (not shown); mp, degradation rate constant for the wild type and mutant nonstructural proteins; mcf, degradation rate constant for the cellular factor.(TIF)Click here for additional data file.Figure S2Viral RNA kinetics in a cell (a) after the subgenomic HCV replicon transfection and (b) after adding 200 nM BILN-2061 inhibitor. The kinetics was calculated by using deterministic (black line) and stochastic (red line) models.(TIF)Click here for additional data file.Figure S3Best fitted results for kinetics of the viral RNA decay in the presence of 50 nM BILN-2061 inhibitor in case of the new model with only wild-type HCV RNA (red line), Binder et model(blue line) and new model with drug resistant mutant HCV RNAs (black line).(TIF)Click here for additional data file.Table S1Propensity functions used in the stochastic model.(DOCX)Click here for additional data file.Text S1Model construction.(DOC)Click here for additional data file.Text S2Sensitivity to variation of parameter values.(PDF)Click here for additional data file.Text S3Method of sensitivity analysis.(PDF)Click here for additional data file.Text S4Parameters optimization.(PDF)Click here for additional data file.