Although cellular RNA is subjected to the same oxidative insults as DNA and other cellular macromolecules, oxidative damage to RNA has not been a major focus in investigations of the biological consequences of free radical damage. In fact, because it is largely single-stranded and its bases lack the protection of hydrogen bonding and binding by specific proteins, RNA may be more susceptible to oxidative insults than is DNA. Oxidative damage to protein-coding RNA or non-coding RNA will, in turn, potentially cause errors in proteins and/or dysregulation of gene expression. While less lethal than mutations in the genome, such sublethal insults to cells might be associated with underlying mechanisms of several chronic diseases, including neurodegenerative disease. Recently, oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease, Parkinson disease, dementia with Lewy bodies, and prion diseases. Of particular interest, oxidative RNA damage can be demonstrated in vulnerable neurons early in disease, suggesting that RNA oxidation may actively contribute to the onset of the disease. An increasing body of evidence suggests that, mechanistically speaking, the detrimental effects of oxidative RNA damage to protein synthesis are attenuated, at least in part, by the existence of protective mechanisms that prevent the incorporation of the damaged ribonucleotides into the translational machinery. Further investigations aimed at understanding the processing mechanisms related to oxidative RNA damage and its consequences may provide significant insights into the pathogenesis of neurodegenerative and other degenerative diseases and lead to better therapeutic strategies. Neurodegenerative diseases are common and are strictly age-associated; for example, the prevalence in the United States per 1,000 elderly is 65 for Alzheimer disease (AD) and 9.5 for Parkinson disease (PD), while the annual incidence per 1,000,000 of general population is 1.6 for amyotrophic lateral sclerosis (ALS) –9. IndeeAlthough RNA is subject to the same oxidative insults as DNA and other cellular macromolecules, oxidative damage to RNA has not been a major focus in oxidative stress studies. This is somewhat surprising since RNA is largely single-stranded and, its bases not being protected by hydrogen bonding or binding proteins, it is, in theory, more susceptible to oxidative insults than DNA –13. It mIt is now evident that RNA molecules are not only intermediates for the transfer of genetic information but also key players in many mechanisms controlling the expression of genetic information –23. GiveRecent progress in genetics has revealed an expansion of the role of RNA beyond its classical function in the “Central Dogma.” It is now evident that only a minority of genetic transcripts (2–3% in human) code for proteins. Non-coding RNA (ncRNA), rather than being cast aside as “junk,” functions directly in structural and catalytic activities and also plays a critical role in regulating the timing and rate of gene expression –23. Of pIn familial forms of two of the most common neurodegenerative diseases, AD and PD , germlinThe disruption of transcriptional or translational fidelity in neurons leads to the accumulation of aberrant or misfolded proteins and neuronal death , 28. Oxiin situ examination of nucleoside oxidation in postmortem brain tissue [The availability of specific antibodies to 8-OdG and 8-OG has enabled us to perform n tissue , 38. In n tissue , 39. In n tissue . This idn tissue .xeroderma pigmentosum, a condition that manifests clinically in its hypersensitivity to sunlight and progressive neurological disease [Similar RNA oxidation was also observed in brain samples of patients with Down syndrome , dementi disease . Additio disease , a neuro disease , which a disease . These fet al. [et al. [et al. [et al. [Our immunocytochemical studies of neuronal RNA oxidation were followed by biochemical detection of the oxidized nucleoside in AD brain with immunoblot analysis –55. Shanet al. , 55 usedet al. . Althoug [et al. , 55 focu [et al.  and Ding [et al. , 54 repo [et al. .Of note, both immunocytochemical , 39, 42 In addition to the brain, significantly increased levels of the oxidized RNA nucleoside, 8-OG, have been identified in cerebrospinal fluid from patients with AD and PD –58 as wenegatively correlate with levels of DNA oxidation [Experimental studies in rodents have shown that neuronal RNA oxidation (8-OG) and spatial memory deficit are observed in older animals  as well xidation .Animal models of neurodegeneration via neurotoxins demonstrate RNA oxidation. Animals treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), for example, show degeneration in nigrostriatal dopaminergic neurons  and a si2•−) to hydrogen peroxide (H2O2) [A strong genetic link between oxidative damage and neurodegeneration has been suggested by the finding that about 20% of patients with familial ALS carry a mutation in SOD1, a metalloenzyme that catalyzes the dismutation of the toxic superoxide (Oe (H2O2) . Althouge (H2O2) , a transe (H2O2)  shows ine (H2O2) , 68.Cell culture experiments further suggest an association between increased RNA oxidation and neurodegeneration , 70. In Various neurodegenerative diseases including AD, PD, and ALS are associated with defects in the ubiquitin-proteasome system which has been shown to affect multiple aspects of RNA metabolism . AnotherThe involvement of RNA oxidation in a variety of neurological diseases raises the possibility that this mechanism is epiphenomenal. In this respect, it should be noted that RNA oxidation is seen in early-stage AD , 40 as wOf note, neuronal RNA oxidation is seen not only in early stage neurodegeneration but also in cases with subacute sclerosing panencephalitis, a condition caused by persistent measles virus infection and accompanied by neurofibrillary tangles . From thThe relatively early chronological appearance of neuronal RNA oxidation is further suggested by a primary rat cortical culture model . In the While protein carbonyls, lipid peroxidation products and glycoxidation products are relatively stable due to the formation of cross-links, oxidized RNAs are likely turned over more rapidly. On the other hand, RNA oxidation reflects the “steady-state balance” of oxidative damage at a “snapshot” point , 80. In 2•− with nitric oxide (NO•), and is not known to accumulate in cells. As such, it is not surprising that intracellular level of 3-nitrotyrosine parallels the level of 8-OG in AD and Down syndrome brains [3-Nitrotyrosine may be another steady-state marker of oxidative damage. 3-Nitrotyrosine is formed by a modification of tyrosine residue of proteins by an attack of peroxynitrite (ONOO−), a powerful oxidant produced from the reaction of Oe brains , 41.2•−, H2O2, and hydroxyl radical (•OH) that are produced by cellular respiration [2•− is minimally permeable through cell membranes [2O2 [The brain is especially vulnerable to oxidative damage because of its high content of unsaturated fatty acids, high oxygen consumption rate and relative paucity of antioxidant enzymes compared with other organs , 86. Givpiration –87. •OH,piration  and O2•−embranes . Generalnes [2O2  reacts wnes [2O2  to damagIn the AD brain, disrupted mitochondria likely also play a central role in producing abundant ROS and supplying redox-active iron into the cytosol , 91–93. More than 20 different types of oxidatively altered purine and pyrimidine bases have been detected in nucleic acids , 36, 99.in vitro by expressing oxidized mRNA species in cell lines. Oxidized mRNAs lead to loss of protein level and function, and potentially produce defective proteins leading to protein aggregation [in vitro using translation assays with oxidized ribosomes from rabbit reticulocytes and which demonstrate a significant reduction of protein synthesis [The biological consequence of oxidatively damaged mRNA species has been investigated regation . In a reregation . When oxregation . In anotregation . Coincidregation . The bioynthesis . Notablyynthesis , 54. Isoynthesis . These fIt is noteworthy in this respect that studies on some anti-cancer agents have shown that RNA damage can lead to cell-cycle arrest and cell death, via a p53-dependent mechanism associated with inhibition of protein synthesis or p53-independent mechanism as yet uncharacterized .Degradation of RNA plays a central role in cellular metabolism and damaged RNA can be removed through degradation by ribonucleases (RNase), but selective degradation of oxidized RNA has not been established for known RNases , 104. Oxet al. [Until relatively recently, damaged RNA was thought to only be degraded and not repaired. However, Aas et al.  have suget al. , 109. Inet al. . Alk B aet al. . DNA damet al. , 110. HoCells have mechanisms of dealing with nucleotide damage other than direct excision and repair, which seems to be useful for defense against oxidative damage to both DNA and RNA. Because oxidation of nucleotides can occur in the cellular nucleotide pool, and oxidized nucleotides can potentially be incorporated into DNA and RNA, the mechanism preventing incorporation of the oxidized nucleotide comes into play in coping with nucleic acid damage , 12, 103In addition to the hydrolyzing action of MTH1 and NUDT5, several other enzymes involved in nucleotide metabolism show discriminatory activity against the oxidized nucleotides. Guanylate kinase (GK), an enzyme that converts GMP to GDP, is inactive on 8-OH-GMP. Similarly, ribonucleotide reductase (RNR), an enzyme that catalyzes reduction of four naturally occurring ribonucleoside diphosphates, is inactive in converting 8-OH-GDP to 8-OH-dGDP thus preventing incorporation of the oxidized nucleotide into DNA synthesis . On the One important unanswered question is whether cells have machinery to deal with oxidatively damaged nucleotides that are contained in RNA, since RNA can be directly oxidized even if the incorporation of oxidized nucleotides into RNA is blocked. Recently, proteins that bind specifically to 8-OG-containing RNA have been reported, namely, Escerichia coli polynucleotide phosphorylase (Pnp) protein and human PNP , 117 as The human PNP protein binds preferentially to 8-OG-containing RNA and cellular amounts of human PNP protein decrease rapidly by exposure to agents inducing oxidative stress, while amounts of other proteins in the cells do not change after these treatments . RecentlIt is possible that RNA quality control mechanisms are defective or inefficient in cancer cells as well as cells of neurodegenerative diseases. Further elucidation of the mechanisms of repair or avoidance of RNA damage and their potential role in preventing human diseases might provide new approaches to therapy in a number of conditions that are so far untreatable.in vitro and in vivo. There are presently only a small number of studies suggesting the existence of coping mechanisms for RNA damage. The known mechanisms and the implications of their failure may be only the “tip of iceberg” of the role of sublethal RNA oxidation in chronic disease. Understanding the consequences and cellular handling mechanisms of oxidative RNA damage may provide clues to the pathophysiology of neurodegenerative diseases and form the basis for better therapeutic strategies.Involvement of RNA oxidation in the process of neurodegeneration has been demonstrated in vulnerable neuronal population in neurodegenerative diseases such as AD and PD as well as in several cellular and animal models of neurodegeneration. Particular emphasis should be placed on the early-stage involvement of RNA oxidation in the process of neurodegeneration, which suggests a primary role of RNA oxidation in the disease process. Indeed, oxidized RNA is associated with a disturbance in protein synthesis


In spite of many sources of inaccuracies, it seems possible to conclude that n is not very high, probably smaller than in many normal tissues.Twenty-one patients, each having at least one metastasis per lung were investigated. A single dose of 1000 rad was delivered to the metastasis located in one lung. while the metastasis located in the other lung received 2 doses of 500 rad separated by a 3-hour interval. The changes in volume of the irradiated metastases were followed at least until the metastases reattained their initial volume. By comparing in each patient the effects of the 2 types of exposure it was possible to estimate the extrapolation number, Furthermore this work demonstrated, in practically all the tumours studied, an acceleration of the growth rate of the metastases after irradiation.


Agrobacterium tumefaciens to transiently express a hairpin RNA homologous to Potato virus Y (PVY) in plant tissues. A complete and specific interference with aphid transmission of PVY was achieved by inducers of RNA silencing, as demonstrated by specific siRNAs accumulation in agroinfiltrated tissues. To our knowledge, this is the first report of successful interference with non-persistent transmission of a plant virus using RNA interference.Hairpin RNAs have been used to confer resistance to viruses in plants through RNA silencing. However, it has not been demonstrated that RNA silencing was effective against inoculation by aphids of non-persistently transmitted viruses, the major route of plant virus spread in nature. As a proof-of-principle strategy, we made use of  One of the most efficient mechanisms by which plants protect themselves from viruses is the specific RNA-dependent silencing pathway termed post-transcriptional gene silencing (PTGS). In certain circumstances, the RNA silencing machinery recognizes several features of viral infections involving the formation of double-stranded (ds) RNA and initiates a response that degrades viral RNA and eventually enables the plant to recover from virus infection . This prPotyvirus, the largest group of plant viruses. These viruses are transmitted in a non-persistent manner, a category of non-circulative transmission also known as stylet-borne [Agrobacterium tumefaciens to transiently express hairpin RNA molecules in plant tissues. The Agrobacterium-mediated transient expression system has previously been used to deliver RNA silencing inducers and suppressors into plants [Potato virus Y (PVY) for its ability to interfere with the non-persistent transmission of this virus by aphids.Aphid transmission is the main method of spread for most plant viruses in nature including members of the genus et-borne . Since to plants . We prevo plants . Howeverin planta the inverted-repeat RNA corresponding to coat protein (CP) gene sequences of PVY. First, the cauliflower mosaic virus (CaMV) 35S promoter was extracted from pAVA393 [EcoRI, and ligated to a derivative of pCAMBIA2300 in which the 3' terminator region of the nopaline synthase gene (NOSt) was inserted between PstI and HindIII sites. The viral sequences were then extracted from pBKS-IRCPPVY using KpnI and BamHI and introduced into the pCAMBIA2300 derivative to generate pIRCPPVY. To prepare pBKS-IRCPPVY, a fragment of plasmid pBSK-CPPVY [TCTAGACACTGAAATGATGG-3' and 5'-CTTGGTACCGGAGAGACACTACATC-3', corresponding to positions 8509–8523 and complementary to 9374–9390, respectively , in the PVY genome [XbaI and KpnI restriction sites (underlined) were created in both primers, respectively, to facilitate further cDNA cloning in a triple-ligation reaction: the PCR product was cut with XbaI-KpnI; pCB278, a plasmid harbouring the phleomycin resistance gene [HindIII-XbaI and both fragments were ligated to the KpnI-HindIII cut pBSK-CPPVY to obtain pBKS-IRCPPVY. Sequencing confirmed the correct ligation of the three components. pBKS-IRCPPVY then incorporates a bacterial gene as a spacer sequence between the sense and the antisense orientations of the PVY CP sequence. We took advantage of the positive selection conferred by this prokaryotic resistance gene to reduce the risk of intramolecular homologous recombination in bacteria. Upon transcription, RNA from pIRCPPVY is intended to fold into a stem-loop structure consisting of 881 bp of PVY dsRNA and approximately a 500 bp spacer sequence  polymerase gene (IR54PMMoV) [Agrobacterium tumefaciens GV2260 by triparental mating and the infiltration of plant tissues was performed essentially as described by Tenllado et al. [The commercial plasmid pCAMBIA2300 (CAMBIA) was used to express  pAVA393  using EcSK-CPPVY  was amplY genome . An XbaINicotiana benthamiana plants were infiltrated with A. tumefaciens cultures carrying IRCPPVY or the empty vector. At 4 days post-infiltration, plants were challenge-inoculated by applying 20 μl of PVY sap inoculum (1/10 w/v) on the infiltrated leaves dusted with Carburundum. Plants were kept in growth chamber with a 16 h light/8 h dark cycle at 25°C. By 7 days post-inoculation (dpi), mosaic symptoms were displayed by PVY-inoculated plants that had been infiltrated with Agrobacterium carrying the empty vector. In contrast, inoculated plants that had been infiltrated with IRCPPVY remained symptomless throughout the entire testing period (2 months). Analyses of viral CP accumulation were performed by dot blot analysis  clone were collected, starved for a period of 2–3 h and allowed to probe for 5–10 min on leaves of PVY-infected N. benthamiana plants. Aphids (10–15 per plant) were released onto an agroinfiltrated leaf covered with a plastic bag at 4 days post-infiltration for inoculation over a 2 h period before spraying with pirimicarb at 0.05% (w/v). Plants were transferred to the growth chamber for observation. The results  that had been agroinfiltrated with the IRCPPVY construct were free of symptoms until their life cycles were completed. Dot blot analysis confirmed the visual observations of the accumulation of PVY CP in upper leaves at 14 dpi -hairpin RNA to the transmission of this virus by aphids. BYDV is a persistently transmitted, circulative virus and thus represents a different transmission mechanism, with distinctive impact on plant virus epidemiology, to that of PVY [We have demonstrated that transient expression of an antiviral hairpin RNA by anically ,3. Howevke place . As a reke place  reportedt of PVY . Since iOnce having established the principle that RNAi is a highly efficient approach to interfere with the non-persistent transmission of plant virus by aphids, we envision extending the non-transgenic, RNAi-based approach to confer protection against aphid-borne viruses by directly delivering dsRNA to plants . SeveralThe author(s) declare that they have no competing interests.JRDR, FT; Design and conception of study. MV;cloning cDNA constructs and dot blot analysis. MV, BM-G; transmission assays. FT; manuscript preparation. All authors read and approved the final manuscript.


The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their  in vitro assay to study their activity. SARS-CoV genomic RNA and all eight subgenomic mRNAs were synthesized in this in vitro reaction. By centrifugation, RTC activity could be isolated from the cytoplasm, together with membrane structures, viral enzymes, and RNA. The activity of these isolated RTCs was dependent on a cytoplasmic host factor. RTC activity was destroyed by detergent treatment, suggesting a critical role for membranes that appeared to protect the complex against protease and nuclease digestion. Our data establish a functional connection between viral RNA synthesis and intracellular membranes and show that host factors play a crucial role in SARS-CoV RNA synthesis.The SARS-coronavirus (SARS-CoV), which causes the life-threatening severe acute respiratory syndrome, replicates in the cytoplasm of infected host cells. A critical early step in the SARS-CoV life cycle is the formation of a replication/transcription complex (RTC) that drives viral genome replication and subgenomic mRNA synthesis. Virus-encoded enzymes form the core of this RTC, which is believed to be associated with characteristic virus-induced membrane structures derived from modified host cell membranes. To investigate the connection between these membrane structures and SARS-CoV RNA synthesis, and to characterize RTC composition and function, we isolated these complexes and developed the first  Following infection and genome translation, positive-strand RNA (+RNA) viruses establish a cytoplasmic enzyme complex that directs the amplification and expression of their genome. The viral RNA-dependent RNA polymerase (RdRp) is the central enzyme of this ‘replication/transcription complex’ (RTC), but it also may include other viral non-structural proteins (nsps) and host factors that cooperate to synthesize viral RNA. Over the past decade, it has become clear that +RNA virus RTCs are invariably associated with virus-induced membrane structures, which are poorly characterized but presumably provide a framework for RNA synthesis by facilitating the concentration and cooperation of viral macromolecules on a dedicated membrane surface. They may also protect the viral RNA from nucleases in the cytoplasm of the host cell, aid in shielding the double-stranded RNA intermediates of virus replication from the host cell's innate immune system, or contribute to the coordination of the viral life cycle in time and space. These membrane-bound RTCs are the molecular machines that drive the RNA synthesis and evolution of +RNA viruses. Clearly, unraveling their structure and function will be critical to understand the biochemistry of +RNA virus replication and develop novel antiviral control strategies.e.g. the nsp12-RdRp e.g. the nsp8-primase The RTC of coronaviruses, including that of SARS-coronavirus (SARS-CoV), the causative agent of the life-threatening severe acute respiratory syndrome  detergents, which also released replicase subunits and (mainly) sg RNA from the membrane fraction. Protease and nuclease protection experiments indicated that viral RNA and nsps were protected by membranes, thus further substantiating the functional connection between SARS-CoV RNA synthesis and virus-induced membrane structures that appear to be essential for RTC activity.Immunofluorescence microscopy previously revealed that newly synthesized SARS-CoV RNA and several nsps colocalize in perinuclear foci in SARS-CoV-infected cells in vitro RNA synthesis assay (IVRA) to study their activity in vitro. In this reaction, the incorporation of [α-32P]CTP into viral RNA was analyzed in a mixture containing NTPs, Mg2+, an ATP-regenerating system, and an inhibitor of cellular transcription (Actinomycin D). The RTC activity in cytoplasmic extracts prepared from SARS-CoV-infected Vero-E6 cells produced a set of 32P-labeled RNA molecules with sizes corresponding to those of the SARS-CoV genome and all eight sg RNAs , which is in line with the large excess of positive over negative strands that is commonly observed in vivo.To determine the polarity of the nded RNA . A strontrol RNA . This in35S-labeled amino acids were incorporated into proteins during a 100-min reaction. When using the PNS of uninfected cells, SDS-PAGE revealed a smear of 35S-labeled material  showed no RTC activity , lane 2,The P10 fraction, which contained the bulk of RTC activity, was analyzed by electron microscopy (negative staining) in combination with an immunogold labeling for the (putative) transmembrane proteins nsp3, nsp4, and nsp6  and nsp4The distribution of newly synthesized SARS-CoV RNAs between the RTC-containing P10 and cytoplasmic S10 fractions was analyzed by fractionation of PNS after an IVRA . The bulFor selected nsps, for which suitable antisera that are reactive in Western blot experiments were available, the distribution between the cytoplasmic S10 fraction and the RTC-containing P10 fraction was analyzed. This revealed that these RTC subunits were enriched or mainly present in the P10 fraction . The bulin vitro synthesized radiolabeled RNA in each fraction was analyzed  generated in infected cells. A similar in vitro system was recently developed for the distantly related arterivirus equine arteritis virus (manuscript in preparation), suggesting that our method may be generally applicable to nidovirus RTCs.Although RdRp activity in cell lysates was previously reported for the coronaviruses mouse hepatitis virus and transmissible gastroenteritis virus in vitro RTC activity  have been made. Still, a potential complication may be the inability of such exogenous templates to enter the membrane-protected RTC, as also observed in this study for molecules like Bal31 nuclease and proteinase K , but the isolated RTCs had to be supplemented with an S10 fraction from infected or uninfected cells to regain activity . This inFree RNA of transmissible gastroenteritis virus was previously found to be susceptible to nuclease treatment, whereas most negative-stranded RNAs, and a small fraction of (probably nascent) positive-stranded RNAs, were present in membrane-protected complexes The bulk of newly synthesized SARS-CoV genome remained associated with the RTC-containing heavy membrane structures, while sg RNAs appeared to be more readily released from the structures in which they had been synthesized. In previous studies with transmissible gastroenteritis virus, it was also found that preferentially sg RNAs were no longer associated with the membrane-associated complexes If SARS-CoV RTCs, as this study suggests, are enclosed by membranes that may provide an optimal environment for viral RNA synthesis, this raises the question of how newly synthesized RNA products are released from these structures. Moreover, the fact that RTC activity depends on a cytoplasmic host factor that does not cosediment with the complex is an additional indication that crosstalk between cytoplasm and RTC-containing membrane structures must occur, e.g. via channels that may facilitate transport across membranes. Taken together, our data support the existence of a functional link between SARS-CoV RNA synthesis and the unusual membrane structures induced upon coronavirus infection.Vero-E6 cells were infected with SARS-CoV (strain Frankfurt 1) at a multiplicity of infection of 5 as described previously 2 flasks) were harvested by trypsinization at 10 hours post infection. To inhibit cellular transcription, 2 µg/ml actinomycin D was present in all solutions used for harvesting and washing of the cells. After washing with PBS, cells were resuspended in 2 ml ice-cold hypotonic buffer  and 2 µg/ml actinomycin D, pH 7.4) and incubated for 10 min at 4°C. Cells were disrupted using a Dounce homogenizer by giving 30 strokes with a tight fitting pestle. Isotonic conditions were restored by adding HEPES, sucrose, and DTT, which resulted in a final lysate containing 35 mM HEPES, pH 7.4, 250 mM sucrose, 8 mM KCl, 2.5 mM DTT, 1 mM MgOAc2, 2 µg/ml actinomycin D, and 130 U/ml RNaseOUT. Nuclei, large debris, and any remaining intact cells were removed by two successive 5-min centrifugations at 1,000×g, and the resulting PNS was either assayed immediately for RTC activity or stored at −80°C. The SARS-CoV titer present in PNS was approximately 108 plaque-forming units per ml. Plaque assays performed before and after IVRAs revealed that no measurable de novo virus production occurred during this assay (data not shown). A 10,000×g pellet (P10) and supernatant (S10) fraction were prepared from PNS by centrifugation at 10,000×g for 10 min. The pellet was resuspended in dilution buffer , in 1/10 of the original PNS volume from which the pellet had been prepared. In some experiments, PNS was incubated for 15 min at 4°C with 0.5% TX-100 prior to the preparation of P10 and S10 fractions.SARS-CoV- or mock-infected cells , 10 U/ml creatine phosphokinase (Sigma), 1 mM ATP, 0.25 mM GTP, 0.25 mM UTP, 0.6 µM CTP and 0.12 µM and 10 µCi [α-32P]CTP . Unless otherwise indicated, IVRAs were performed for 100 min at 30°C. Reactions were terminated by adding 60 µl of a mixture containing 5% lithium dodecyl sulfate, 0.1 M Tris-HCl, pH 8.0, 0.5 M LiCl, 10 mM EDTA, 5 mM DTT, and 0.1 mg/ml proteinase K, and incubating at 37°C for 10 min. When protein synthesis was tested, [α-32P]CTP was replaced with 14.3 µCi of Promix , containing a mixture of [35S]methionine and [35S]cysteine. To assess the effect of translation inhibition, 70 µg/ml of cycloheximide or 350 µg/ml of puromycin were added.Assays were performed using either 25 µl PNS, 20 µl S10, 5 µl P10, or 5 µl P10 supplemented with 20 µl S10. When required, the total volume was adjusted to 25 µl with dilution buffer. The subsequent addition of reaction components yielded a 28 µl final reaction volume, containing 30 mM HEPES pH 7.4, 220 mM sucrose, 7 mM KCl, 2.5 mM DTT, 2 mM MgOAcin vitro synthesized RNA was detected by exposing a PhosphorImager screen directly to the dried gel, after which screens were scanned with a Personal Molecular Imager FX (Bio-Rad) and data were analyzed with Quantity One version 4.5.1 (Bio-Rad). Unlabeled endogenous SARS-CoV RNA was detected by hybridization with a 32P-labeled oligonucleotide SARSV002 (5′-CACATGGGGATAGCACTAC-3′), which is complementary to a sequence present in the 3′-end of all SARS-CoV RNAs RNA was isolated from IVRA reaction mixtures by acid phenol extraction and isopropanol precipitation. Reaction products were analyzed by denaturing formaldehyde agarose gel electrophoresis essentially as described previously, except that a 1% agarose gel was used In vitro transcribed RNAs (0.75 µg) corresponding to nt 29,364-29,727 of the 3′-terminal region (3′-TR(+)) or complementary to nt 1-378 (3′-TR(−)) of the SARS-CoV genome were immobilized to Hybond N+ membrane . As negative controls, RNAs corresponding to nt 12,313–12,660 (ctrl. a) of the equine arteritis virus genome or its complementary sequence (ctrl. b) were included. The membrane with the immobilized probes was prehybridized for 4 hours in a hybridization mixture containing 5×SSPE , 0.05% SDS, 5x Denhardt and 100 µg/ml homomix I. Subsequently, the membrane was hybridized with half of the 32P-labeled RNA recovered from a 28-µl IVRA in 0.8 ml hybridization mix, which was first heat denatured at 70°C for 15 min. After hybridization for 16 h at 56°C, membranes were washed twice for 20 min at 56°C with 4 ml of 5x SSPE, 0.05% SDS, and the hybridization signal was quantified by PhosphorImager analysis.Proteins were separated by SDS-PAGE and transferred to Hybond-P PVDF membrane  by semi-dry blotting. After blocking with 1% casein in PBS containing 0.1% Tween-20 (PBST), membranes were incubated with anti-nsp3, anti-nsp5 or anti-nsp8 rabbit antisera, diluted 1∶2000 in PBST with 0.5% casein and 0.1% BSA. Peroxidase-conjugated swine anti-rabbit IgG antibody (DAKO) and the ECL-plus kit  were used for detection.Protease protection experiments were done by incubating PNS (50 µl) for 10 min at 20°C with 20 µg/ml of proteinase K either in the absence or presence of 0.5% TX-100. After inactivation of the protease by addition of 2 mM PMSF and fractionation into a 10,000×g pellet (P10) and supernatant (S10), samples were analyzed by Western blotting. For nuclease protection assays, a standard 100-min IVRA was performed with the PNS, after which 5U of Bal31 nuclease was added, either in the presence or in the absence of 0.5% TX-100. After a 10-min incubation, samples were fractionated into S10 and P10 fractions. Radiolabeled RNA was isolated from the fractions and analyzed as described above.2, 10 mM EGTA, pH 6.9 was added to P10 fractions. Formvar-coated grids were placed on 10-µl drops of these fixed P10 fractions and incubated at room temperature for 1 min. After blocking with 1% BSA in PBS, grids were incubated for 30 min with rabbit antisera directed against nsp3, nsp4 or nsp6 (1∶200) in PBS containing 1% BSA. Bound rabbit IgG was detected with protein A carrying 15-nm gold particles. After negative staining with 2% phosphotungstic acid, grids were viewed in a FEI T12 transmission electron microscope at 120 kV.One volume of 6% paraformaldehyde in 60 mM PIPES, 25 mM HEPES, 2 mM MgCl


Xenopus laevis egg extracts [in vitro assay.Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies  involvinextracts , althougextracts ,4. To auWe found that Tpr binds strongly and specifically to importin α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin α/β import pathway.Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export. Molecules are transported between the cytoplasm and the nucleus through nuclear pore complexes (NPCs), massive proteinaceous structures that span the double membrane of the nuclear envelope (NE). Molecules smaller than ~20-40 kDa in size can passively diffuse through the NPC. However most protein, and nucleic acid is transported by receptor and energy dependent mechanisms (reviewed in -8).Nucleocytoplasmic transport is mediated by shuttling transport receptors termed karyopherins or importins/exportins . In tThe small GTPase Ran, which binds directly to both importins and exportins, plays a key role in determining the directionality of nuclear transport. The GTP-bound form of Ran is concentrated in the nucleus and the GDP-bound form predominates in the cytoplasm, due to the nuclear localization of the Ran guanine nucleotide exchange factor RCC1 (RanGEF) and the cytoplasmic localization of the Ran GTPase- activating protein (RanGAP). The binding of RanGTP to karyopherins modulates the affinity of the receptors for cargo. When an importin-cargo complex encounters RanGTP in the nucleus, RanGTP promotes the dissociation of cargo from the receptor as well as dissociation of the importin from nucleoporins, and the importin-RanGTP complex is recycled back to the cytoplasm. The converse is true for exportins: intranuclear RanGTP promotes the binding of cargo to exportins, and when the RanGTP-containing export complex encounters RanGAP in the cytoplasm, GTP hydrolysis results in release of the cargo and regeneration of the free exportin -11.The framework of the NPC consists of eight central spokes flanked by nuclear and cytoplasmic rings, forming a ring-spoke assembly that surrounds a central transport channel. Extending outward from the ring-spoke assembly are ~50-100-nm-long nuclear fibrils, which are joined in a basket-like structure ("nuclear basket"), and ~35-50-nm-long cytoplasmic fibrils . TheIn addition to undergoing reversible disassembly during mitosis in higher eukaryotes, NPCs are assembled throughout interphase in concert with NE growth . MoreoveXenopus laevis [Saccharomyces cerevisiae  [Drosophila melanogaster [Arabidopsis thaliana [Drosophila Tpr has been linked to mitotic spindle organization and spindle checkpoint control [A conserved component of the NPC is the protein Tpr (for translocated promoter region)  and its s laevis , Sacchard Mlp2p) , Drosophnogaster  and Arabthaliana . In mammthaliana  and is sthaliana . Mammalithaliana , whereasthaliana . Numerouthaliana -30, nuclthaliana , silent thaliana -34, spinthaliana , unsplicthaliana  and locathaliana ,38. In a control .Xenopus Laevis egg extracts demonstrates that importin β and importin α co-immunoprecipitate with Tpr [in vitro nuclear import assays indicate that the nuclear import of Tpr is efficiently mediated by the classical importin α/β pathway.In mammalian cells, classical nuclear protein import is not detectably affected in Tpr depleted cells ,40, wherwith Tpr . WhetherTo investigate the interactions of Tpr with the karyopherins CRM1, importin β and importin α, we used quantitative solid phase binding analysis. To this end, we expressed recombinant full-length mammalian Tpr using a baculovirus system, and expressed the C-terminal domain of Tpr  in bacteria. The expression and purification of these Tpr constructs was analyzed by SDS-PAGE Fig. . Full-led = 83 nM, Table Kd = 176 nM, Table We first analyzed the interactions of Tpr with CRM1 -44, we eXenopus egg extracts demonstrated that importins α and β co-immunoprecipitate with Tpr [d values between 9 nM and 225 nM depending on the nucleoporin [Kd = 50 nM, Table In studies conducted in our laboratory and others, import of classical NLS cargoes was found to be unaffected in Tpr depleted cells ,40. Howewith Tpr . This stleoporin , reflectSince the interaction of importin β both with cargo and with nucleoporins is dissociated by RanGTP , as a spImportin β is a superhelical solenoid of tandem HEAT repeats, which binds to nucleoporin FG repeats on its outer surface, and to RanGTP and importin α and other cargoes on its inner surface (reviewed in ). Since d = 21.5 nM, Table d for Tpr) and the ΔIBB-importin α concentration increased from 0-600 nM. As shown in Fig. We next measured the affinity of importin α for Tpr and C-Tpr Fig. . The resPrevious studies showed that the C-terminal domain of Tpr is responsible for its nuclear localization ,28,48. AThe potential involvement of importin α and importin β in the nuclear import of C-Tpr was studied in digitonin-permeabilized cells reconstituted with recombinant transport factors Fig. . We usedOur quantitative binding studies showing that Tpr selectively binds to CRM1 present in a trimeric export complex supports the notion that Tpr plays a direct role in protein export [Escherichia coli containing the viral backbone, where recombination generated a recombinant bacmid containing His-Tpr inserted into the viral backbone. Mammalian C-Tpr encompassing amino acids 1626-2348 was constructed by inserting HindIII fragment into HindIII site of pET30c vector, and the construct was analyzed for correct orientation.Recombinant baculovirus particles containing the mammalian Tpr cDNA were prepared using the Bac-to-Bac Baculovirus Expression System (Gibco BRL). The cDNA of Tpr was cloned into the Sal I site of the donor plasmid pFastBac HTc containing an N-terminal 6×His tag. The recombinant donor plasmid was transformed into DH10Bac Escherichia coli BL21+ cells and grown at 37°C in 2 × LB medium to an OD600 of 0.5-0.6. Expression was induced by the addition of 0.2 mM IPTG for 3 h at 37°C. Cells were resuspended in 50 mM Tris buffer, pH 7.5 supplemented with 300 mM NaCl, 2 mM DTT, 1 mg/ml lysozyme, 10 μg/ml DNase, and the protease inhibitor cocktail. The suspension was sonicated (3 × 20 s), and centrifuged at 100,000 × g for 30 min. The supernatant was loaded on a Sepharose Q column, washed extensively with the lysis buffer containing 300 mM NaCl, and then eluted with a gradient of 300-530 mM NaCl for 1 hour. Fractions containing C-Tpr were pooled; ammonium sulfate precipitated and run on a Superdex 75 column equilibrated in transport buffer. Expression and purification of His-importin α, ΔIBB-importin a, importin β, His-S-tag-importin β, Ran, nuclear transport factor 2 (NTF2) and GST-M9  was performed as previously described [N-terminal His-tagged Tpr was expressed in SF9 cells at 27°C for 45 hours. Cells were lysed in RIPA buffer  with mammalian protease inhibitor cocktail . Cell lysates were cleared at 14,000 × g and supernatants were purified on Talon affinity resin (Clontech). Only micorgram quantities of full-length Tpr could be obtained by this method. From Western blot analysis, we found that a significant amount of an immunoreactive band comigrating with purified full-length Tpr did not bind to the Talon resin, suggesting either that the His-tag was cleaved from this fraction, or that the His-tag was inaccessible, possibly because Tpr in the unbound fraction was present in small aggregates. Recombinant C-Tpr (comprising residues 1626-2348) was expressed in escribed  before uSolid phase binding assays were carried out on microtiter plates  coated with 25 ng of Tpr or C-Tpr. Assays were conducted as previously described . BindingCompetition assays between ΔIBB-importin α and importin β for binding to Tpr were carried out on microtiter plates  coated with 25 ng Tpr. Different ratios of ΔIBB-importin a and importin β were mixed together and incubated with immobilized Tpr. The experiments were carried out in duplicates. One repeat was probed with antibodies against S-tag to detect importin β and the other repeat was probed with antibodies against ΔIBB-importin α. Results are presented as percentage of highest level of binding.in vitro assays, substrate visualization, and preparation of HeLa cytosol (by digitonin lysis) were carried out essentially as in [For analysis of nuclear import in digitonin-permeabilized adherent NRK cells, nuclear import ly as in . 5 mg ofly as in . Import BSA: bovine serum albumin; CRM1: chromosome region maintenance 1; FG: phenylalanine glycine; Mlp1p: myosin-like protein 1; NE: nuclear envelope; NES: nuclear export signal; NLS: nuclear localization sequence; NPC: nuclear pore complex; NTF2: nuclear transport factor 2; pAb: polyclonal antibody; RanBP1: RanGTP binding protein; RanGAP1: RanGTPase activating protein; RanGEF: Ran guanine nucleotide exchange factor; RCC1, regulator of chromosome condensation 1; Tpr: translocated promoter region; WGA: wheat germ agglutinin.IBE designed and performed the experiments, analyzed and interpreted data, and drafted the figures and manuscript. PDF prepared recombinant full-lengthTpr, performed preliminary versions of the binding assays and contributed intellectually to this development of the experiments. LG conceived and oversaw the conduct of the research and was involved in writing the manuscript. All authors read and approved the final manuscript.


The isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize, e.g. cartilage samples. Additionally, cartilaginous tissues are characterized by a low cellularity and an abundance of extracellular matrix (ECM) molecules. But given the growing interest in understanding pathogenesis of degenerative diseases, e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), studies have to consider expression pattern of cells in its natural environment.Aqueous™ kit) and the combined protocol (TRIzol®/RNeasy Mini™ kit), working in a reproducible and reliable manner.We compared the current RNA isolation methods for the extraction of high-quality RNA of snap-frozen biopsies from limited amounts of hypocellular cartilaginous tissue. The focus of the study was to gather information about procedure-related differences in RNA quality and yield. Here, we describe two protocols, the phenol/chloroform-free filter-based method (RNWe conclude that preparation, storage, homogenization, and quality control are altogether critical steps for in-depth analysis of differential gene expression, especially in hypocellular tissues with highly crosslinked ECM like cartilage. The isolation of sufficient amounts of high quality RNA from articular cartilage is a persistent problem for the research addressing cartilage molecular biology -5. This queous™). In contrast to former studies, a solid proof of RNA quality using capillary electrophoresis was included. Therefore, our main objective was to compare this new approach with standard protocols using TRIzol® reagent and/or the RNeasy™ system with respect to procedure- and species-related differences in RNA quality and yield.Our objective was to develop a detailed code of practice for successful high quality RNA isolation from cartilage samples. While it is generally accepted that RNA purification from chondrocytes is quite simple resulting in large amounts of high quality RNA Figure , there iHuman cartilage samples were obtained from patients undergoing total knee replacement surgery. Tissue samples were used with patients' informed consent and institutional approval . Adult bovine (cow) and immature bovine  cartilage was directly obtained from the slaughterhouse. Sterile cuts  were prepared with a cutter  from cartilage samples. Immediately after removal, the cartilage cuts were frozen in liquid nitrogen and thereafter stored at -80°C (for long-time storage).®/RNeasy Mini™ kit are provided in the Additional files . In contrast, cartilage samples derived from cows or calfs are characterized by an enhanced cell number. For these samples, even other extraction methods are suitable. In addition, the RNA extraction from cartilage samples of healthy donors appears much easier in comparison to material obtained from diseased donors.Concerning an adequate storage condition for maintaining RNA integrity recent studies suggest the incubation of freshly obtained cartilage samples in the stabilization reagent RNAlater™ -13. Here® reagent and RNeasy™ resulted in a significant RNA degradation [see Figure queous™ system, however, was not negatively influenced by milling regarding both RNA yield and quality [see Figure queous™ kit system further increases the quality and especially the amount of isolated RNA in any circumstance [see Figure Concerning the method of choice for cartilage homogenization, we have to conclude that the respective method should be chosen in dependency of the subsequent isolation method. For example, milling before RNA isolation with TRIzolA matter of special importance is the mandatory check of RNA integrity of each sample by using capillary electrophoresis [see Figure In order to exclude that RNA degradation in human material was due to the pathologies of the patients from whom the samples originated, we also examined "healthy" cartilage derived from young  and old cows. We found species-related and quality-related differences [see Additional file Aqueous™-based method should be useful for identifying genes differentially expressed by chondrocytes in its natural environment. In spite of this advance, there is still room for improvements and recent studies have to be interpreted with care.As expected, RNA isolated from cartilage does never reach yield and purity of RNA isolated from cultured chondrocytes [see Additional file ECM: extracellular matrix; gDNA: genomic DNA; MD: microdismembrator ; OA: osteoarthritis; PCR: polymerase chain reaction; QC: quality control; RT-PCR: quantitative Real-time-PCR; RA: rheumatoid arthritis; RIN: RNA integrity number; RS: rotor-stator; RT-PCR: reverse transcription polymerase chain reaction; RT: room temperature; SC: scalpel.The authors declare that they have no competing interests.AR designed the study. AR and SN planned and conducted the laboratory experiments. AR, RH and BW analyzed the data. The manuscript was drafted by AR and edited by AR and RH. All authors approved the final manuscript.Comparative analysis of current methods for RNA isolation from cartilage/chondrocytes. This table provides RNA quality control parameters (detected with the NanoDrop) and parameters of cell yields after chondrocyte extraction from cartilage.Click here for filequeous Midi™ kitProtocol 1 - RNA isolation from cartilage using RNA. This data file provides a complete protocol for using the RNAqueous Midi™ kit. It enables the reader to start immediately with RNA isolation. This protocol is the best one for RNA isolation from human cartilage samples.Click here for fileProtocol 2 - Combined method for RNA isolation from cartilage. This data file provides a complete protocol for using the combined method. It enables the reader to start immediately with RNA isolation. This protocol is acceptable for RNA isolation from bovine cartilage samples.Click here for filePrimers, product length, and specific amplification conditions for (q)RT-PCR. This table provides additional information about primers and amplification conditions for qRT-PCR and RT-PCR.Click here for fileCharacterization of special parameters during RNA isolation from bovine articular cartilage. In this figure we compare special parameters during RNA isolation based on Agilent analysis.Click here for fileQuality control of total RNA from human cartilage explants from one typical donor using RT-PCR. In this figure, we present the results of an typical gel electrophoresis image after RT-PCR.Click here for file


The genome-scale data collected by the FANTOM4 collaborative research project are presented as an integrated web resource. In FANTOM4, an international collaborative research project, we collected a wide range of genome-scale data, including 24 million mRNA 5'-reads (CAGE tags) and microarray expression profiles along a differentiation time course of the human THP-1 cell line and under 52 systematic siRNA perturbations. In addition, data regarding chromatin status derived from ChIP-chip to elucidate the transcriptional regulatory interactions are included. Here we present these data to the research community as an integrated web resource. The Functional Annotation of the Mammalian Genome (FANTOM) consortium is an international collaborative research project initiated and organized by the RIKEN Omics Science Center ) -5 focusiThe fourth round, FANTOM4, aimed at the elucidation of the transcriptional regulatory network controlling the differentiation of a human macrophage-like cancer cell line . As our To understand the transcriptional changes that facilitate this state transition, we measured mRNA expression changes using microarrays and used next-generation deepCAGE tag sequencing to monitor the dynamics of transcriptional initiation at the promoter level. We then used bioinformatic approaches to predict the transcription factors that regulate these promoters. To test these predictions, we carried out small interfering RNA (siRNA) knockdown of 52 transcription factors expressed in THP-1 cells and monitored their effect on the predicted targets and all other genes using microarrays. The majority of these knockdowns focus on transcription factors that are themselves transcriptionally repressed in response to PMA. Finally, we complemented these datasets with chromatin immunoprecipitation with microarray (ChIP-chip) for several key factors and markers of active transcription  acetylation and RNA polymerase II).The data obtained will serve as an essential resource for further analyses for the scientific community. Thus, we accumulate all of these data into a single web resource and make it publicly accessible. An easy-to-use graphical interface facilitates the integrated visual inspection for the scientific community and a collection of all underlying data files enables further bioinformatics analyses. We complemented the resource by incorporating CAGE data previously obtained from a wide-range of cell types. This public resource offers one of the most extensive perspectives of promoter activities to date. Here, we describe the web resource with the experimental details.CD11b (ITGAM), is up regulated in all three biological replicates  overlap with the promoter sets defined by deepCAGE , while t studies ,11.CD11b gene upon knockdown of SP1 and PU.1, which is consistent with their binding to the CD11b promoter region as well as with previous studies [We predicted transcriptional regulatory interactions employing a hybrid approach of: transcription factor binding site (TFBS) predictions within evolutionarily conserved regions that are proximal to the CAGE defined promoter regions (-300 bp to +100 bp) using TFBS matrices; and microarray derived gene expression. To validate the predicted interactions, we performed large-scale siRNA perturbation knockdown experiments for 52 transcription factors that are expressed in the undifferentiated state, including the two transcription factors used in the ChIP-chip experiments, SP1 and PU.1. We monitored the effects on gene expression with microarrays at 48 h after transfection. All transfection and subsequent microarray experiments were conducted in biological triplicates. We observed down-regulation of the CD11b and many other genes that are up-regulated during THP-1 differentiation, indicating that Myb can work directly or indirectly as a transcriptional repressor [Interestingly, our large-scale perturbation study revealed that knocking down of Myb induced the expression of epressor . The expAdditional data, in-depth sequencing of small RNA and large-scale quantitative RT-PCR (qRT-PCR) of 2,000 transcription factors were performed subsequently to complement the analysis. The deep sequencing of small RNAs, ranging from 10 to 82 nucleotides, uncovered a novel class of transcription initiation-associated short RNAs with a length around 18 nucleotides . MoreoveIn addition to the above data focusing on THP-1, we updated the genome mapping of the 5 million FANTOM3 CAGE tags obtained from 41 human samples .CD11b promoter' produces a small window presenting quantitative promoter activity, in particular, up-regulation of this promoter in THP-1 cells in response to PMA. A mouse click on the microarray probe corresponding to this gene produces a small window depicting its expression, which also shows increased abundance of the mRNA of CD11b. The parallel display of these two windows enables the examination of the relationship between promoter activities identified by CAGE and gene expressions measured by the microarray  for direct access using the DAS protocol , which iA wide-range of large-scale experiments with several replicates was performed for the THP-1 differentiation in response to PMA stimulation. Analyses and findings are being reported in a series of separate publications, each of them employing parts of the complex data. Understanding interconnections between experiments and the data as a whole is challenging. To describe the whole set of experiments in a consistent way, we employed the MAGE/ISA-tab file format ,20, a stWe accumulated a wide-range of experimental data focusing on one particular biological system, THP-1, into a unique resource to promote the understanding of transcriptional regulation on a genome-scale. The number of CAGE tags derived from the THP-1 cell line is approximately fivefold higher than the number of all human CAGE tags derived from 41 RNA samples taken in FANTOM3 .An independent collection of 5'-end reads of human transcripts, DBTSS , comprisThe ENCODE database  collecteThe presented FANTOM web resource updates and integrates data and analysis results of previous FANTOM activities as well as of the ongoing FANTOM4 activity. Containing 40 million CAGE tags (29 million from 127 human RNA samples and 11 million tags from 206 mouse RNA samples), the FANTOM web resource is one of the most complete resources of TSSs available. The focus of the FANTOM4 project on the comprehensive and detailed characterization of the differentiation of THP-1 cells makes the FANTOM web resource the largest experimental data repository for the very well studied THP-1 cell line model system.We are continuously producing and collecting CAGE data for various organisms and experimental conditions and successively integrate it into the FANTOM web resource. Our explicit goal is to making this web resource the central repository for CAGE data in the world.The THP-1 cell line was subcloned by limit dilution and one clone (clone 5) was selected for its ability to differentiate relatively homogeneously in response to PMA. THP-1 cells were used for all subsequent experiments. THP-1 cells were cultured in RPMI, 10% fetal bovine serum, penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol. THP-1 was treated with 30 ng/ml PMA  over a time course of 96 h. Total cell lysates were harvested in TRIzol reagent  at each time point. Undifferentiated cells were harvested in TRIzol reagent at the beginning of the PMA time course. Total RNA was purified from TRIzol lysates according to the manufacturer's instructions.et al. [et al. [The preparation of the CAGE library from total RNA was a modification of methods described by Shiraki et al.  and Kodz [et al. , adapted [et al. . The mapTHP-1 samples were identical to those used for deepCAGE libraries, and RNA was purified for expression analysis by RNeasy columns , FastPure RNA Kit or TRIzol . RNA quality was checked using the Nanodrop  and Bioanalyser . RNA (500 ng) was amplified using the Illumina TotalPrep RNA Amplification Kit, according to the manufacturer's instructions. cRNA was hybridized to Illumina Human Sentrix-6 bead chips Ver.2, according to standard Illumina protocols. Chip scans were processed using Illumina BeadScan and BeadStudio software packages and summarized data were generated in BeadStudio (version 3.1).3. Beads were vortexed for 60 minutes at room temperature. The supernatants were incubated for 3.5 h at 65°C to reverse the cross-links, and incubated with RNaseA, and then proteinase K, followed by a phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation to recover the DNA. Immunoprecipitated DNA was amplified by either linker-mediated PCR or in vitro transcription followed by synthesis of double-strand cDNA. Amplified DNA was end-labeled with biotin-ddATP and was hybridized to Affymetrix whole genome tiling for Polymerase II (GeneChip Human Tiling 1.0R Array) and H3K9Ac (GeneChip Human Tiling 1.0R Array), and promoter arrays (GeneChip Human Promoter 1.0R Array) for PU.1 and SP1.THP-1 cells were cross-linked with 1% formaldehyde for 10 minutes and cells were collected by centrifugation and washed twice in cold 1× phosphate-buffered saline. The cells were sonicated for 5-7 minutes with a Branson 450 Sonicator to shear the chromatin . Complexes containing DNA were immunoprecipitated with antibodies against H3K9Ac , PU.1 , SP1 , and RNA Polymerase II . The immunoprecipitated sample was incubated with magnetic beads/Protein G  for 1 h at 4°C followed by washing. The complexes were eluted from the magnetic beads by addition of 1% SDS and 100 mM NaHCO6 cells/dish for transfection. Transfection was performed with 1.6 μg/ml  of Lipofectamine 2000 (Invitrogen) and 20 μM  of stealth siRNA (Invitrogen) or 20 μM  of pre-microRNA  by reverse transfection protocol in accordance with the manufacturers' instructions. Total RNA for Illumina microarray analysis was extracted 48 h after transfection, using the FastPure RNA kit  in accordance with the manufacturer's instructions. All microarray experiments were conducted in biological triplicate.THP-1 cells were seeded in 6 cm dishes at a density of 1 × 10The graphical user interface, DAS API, the original GFF files of the genome browser, and all the additional data files are publicly available at .API: application interface; CAGE: cap analysis of gene expression; ChIP-chip: chromatin immunoprecipitation with microarray; FANTOM: Functional Annotation of the Mammalian Genome; H3K9: histone H3 lysine 9; PMA: phorbol myristate acetate; qRT-PCR: quantitative RT-PCR; siRNA: small interfering RNA; TFBS: transcription factor binding site; TSS: transcriptional starting site.HK, JS, and ARRF organized the data stored in the database. HK and ML configured and set up the genome browser database. JS, ARRF, and AW designed, implemented and set up the FANTOM4 EdgeExpressDB instance. SK integrated all the available CAGE tags as tag clusters. HS, YH, PC, KI and DH constructed the original datasets. HS, PC, YH, and CD were involved in conceptualization and supervision. HK wrote the manuscript, and ARRF, PC, and CD improved the manuscript.The following additional data are available with the online version of this paper: a figure showing the experimental steps of cell and RNA preparations (Additional data file An investigation design graph of RNA preparation showing the cell batches are prepared from the same original cells, treated with several conditions, and RNA is extracted. These steps are described in the MAGE/ISA-tab file as meta-data, which include all of our experimental details, and the content is visualized as an investigation design graph by SDRF2GRAPH .Click here for fileAn extended version of Table Click here for file


E. coli is known to bind to RNA polymerase interfering with transcription initiation. Because 6S RNA concentrations are maximal at stationary phase and binding occurs preferentially to the holoenzyme associated with σ70 (Eσ70) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for σ70-dependent promoters characterized by a weak -35 recognition motif or extended -10 promoters. There are many exceptions to this precept, showing that other types of promoters, including stationary phase-specific (σ38-dependent) promoters are inhibited.6S RNA from To solve this apparent ambiguity and to better understand the role of 6S RNA in stationary phase transition we have performed a genome-wide transcriptional analysis of wild-type and 6S RNA deficient cells growing to mid-log or early stationary phase. We found 245 genes at the exponential growth phase and 273 genes at the early stationary phase to be ≥ 1.5-fold differentially expressed. Up- and down-regulated genes include many transcriptional regulators, stress-related proteins, transporters and several enzymes involved in purine metabolism. As the most striking result during stationary phase, however, we obtained in the 6S RNA deficient strain a concerted expression reduction of genes constituting the translational apparatus. In accordance, primer extension analysis showed that transcription of ribosomal RNAs, representing the key molecules for ribosome biogenesis, is also significantly reduced under the same conditions. Consistent with this finding biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S RNA is apparently compensated by an increase of the basal ppGpp concentration, known to affect growth adaptation and ribosome biogenesis.70-dependent promoters. Moreover, the results indicate that 6S RNA is embedded in stationary phase adaptation, which is governed by the capacity of the translational machinery.The analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to σ While most bacterial regulatory RNAs are acting at the level of translation [E. coli is transcribed together with the gene ygfA, whose protein product shows sequence similarity to methenyltetrahydrofolate synthetase. Despite the fact that this bi-cistronic arrangement is highly conserved among enterobacterial and g-proteobacterial ssrS transcription units its functional significance is presently not known [in vitro and in vivo [70 (Eσ70) and this interaction is thought to be brought about by the particular RNA secondary structure, which is highly conserved and mimics an open promoter DNA [70 region 4.2 have been identified, which are crucial for 6S RNA binding [6S RNA encoded by the gene nslation ,2 6S RNAnslation -6. 6S RNot known ,8. The cot known ,10, reacot known . Only inot known . Since t in vivo ,12,13. Boter DNA ,15. Bind binding .70 RNA polymerase is considered to be bound to 6S RNA and thus should generally be prevented from binding to σ70-dependent promoters. The mechanism of transcriptional inhibition must be more complex, however. Original observations had suggested that only a subset of σ70 promoters were affected and σ38-dependent promoters were generally not believed to be 6S RNA sensitive. Later studies indicated that preferentially extended -10 promoters and promoters with a weak -35 consensus element were responsive to 6S RNA [in vitro and in vivo and in several cases results were obtained, which are inconsistent with the above supposition [Some uncertainty exists regarding the promoter specificity of 6S RNA-dependent transcriptional regulation. Clearly, not all promoters are sensitive to 6S RNA. During late stationary phase most of the Eσo 6S RNA ,15. Thiso 6S RNA . Althougposition ,17. ObviTo gain more insight into the complex growth phase- and promoter selectivity we have performed a global transcription analysis of wild-type and 6S RNA deficient strains using microarrays. Until now no global transcription data have been collected under exponential growth conditions. Therefore total RNA was analysed from cells grown to either mid-exponential or early stationary phase. The results reveal more than 500 differentially expressed genes, which are ≥ 1.5-fold up- or down-regulated in a 6S-dependent manner. Apparently, there is no strict correlation to a specific set of promoters, such as exponential or stationary phase-specific. The significance of the microarray data was substantiated for selected examples by qRT-PCR and quantitative primer extension analysis. The list of mRNAs with enhanced expression levels at exponential growth in the absence of 6S RNA comprise enzymes involved in guanine metabolism, a number of stress proteins and transcriptional regulators mainly involved in stress adaptation. The list of genes repressed under the same conditions in the absence of 6S RNA again includes many transcriptional regulators. During early stationary growth we find enhanced mRNA levels for several aromatic amino acid transporters, transcriptional regulators, genes involved in stress adaptation, guanine metabolism or proteins that cope with defective translation. However, the most outstanding effect during stationary growth is a concerted reduction in the expression of genes, which constitute the translational apparatus, as well as several co-transcribed genes in operons for ribosomal proteins and genes encoding translation factors. Consistent with the reduction of ribosomal protein mRNA levels in the microarray study we found a corresponding decline in the synthesis of rRNAs under stationary growth conditions using primer extension analyses. Moreover, we present evidence that the remarkable reduction in translational components is directly related to an increased basal ppGpp level in the mutant strain, probably resulting as the consequence of an imbalance in growth rate adaptation in the absence of 6S RNA. The role of 6S RNA as part of the network responsible for adjusting growth rate changes is discussed.70-dependent promoters (rrn P1 or rpoD P3), promoters known to respond to both σ70- and σ38-RNA polymerase holoenzyme (osmY) and promoters preferentially transcribed by σ38-RNA polymerase . The analysis revealed that 6S RNA affects promoters independent of the characteristics of the respective promoter, indicating that there is no absolute σ factor specificity  and the early stationary phase (OD600 ~2.4)  was also enhanced. In addition, a remarkable number of genes involved in stress adaptation also showed higher expression levels (e.g. asr encoding an acid shock protein and dps encoding a DNA protection protein showed about 2.5-fold higher expression levels in the 6S deficient strain). Likewise, genes encoding the ribosome stabilizing proteins sra and yfiA as well as the cold shock and general stress protein genes cspA, uspG and uspF were also up-regulated in the absence of 6S RNA. Furthermore, a number of mRNAs for transcription factors involved in environmental adaptation as well as several known and predicted/hypothetical transporters and (inner) membrane proteins showed increased expression in the ssrS- strain , or for the genes encoding transcriptional regulators, such as lldR (0.62 and 0.57-fold) or torR, gcvA, glcC, putA, iscR, sdiA, hcaR . Note, that the expression of this operon is known to be down-regulated during transition into stationary phase thereby weakening expression differences. Enhanced expression was also observed for genes encoding the amino acid transport systems mtr (tryptophane) and sstT (serine/threonine). In addition, the smpB gene shows a higher expression level .In the DNA microarray analysis of the early stationary phase, in the absence of 6S RNA, 148 genes showed increased expression and 125 genes decreased expression, when using 1.5-fold RNA level change as cut-off , yfjA (rimM), important for 16S rRNA processing, as well as the genes encoding primase (priB) or a tRNA methyltransferase (trmD). It should be noted that in addition to genes co-transcribed with ribosomal constituents several transcriptionally independent genes, yet functionally related to the translation process, were also found to be reduced in the ssrS deficient strain during stationary phase. Those genes comprise yibK, a predicted rRNA methylase and lepA, a GTP-binding protein known as a ribosomal elongation factor EF-4 (LepA), counteracting mistranslocated ribosomes [metG, lysS or tgt) showed reduced mRNA levels.The most striking result of the microarray analysis, however, is the expression of a large number of genes encoding ribosomal proteins and proteins involved in transcription and translation elongation, which show uniformly the tendency of slightly decreased and up to 2-fold lower expression levels in the 6S deficient RNA strain  as well as for several ribosomal genes/operons, such as rpsL , rplJ , rplN, rplY, . In contrast, a more than twofold reduction in P2-directed transcripts, representing the predominant RNA species during stationary phase, can be observed at the early stationary phase  revealed 38% higher basal ppGpp concentration in the absence of 6S RNA. This increase in the effector nucleotide is fully consistent with the reduced rRNA synthesis in the ssrS- strain and also explains the reduction in the translational components detected in the microarray analyses. We conclude therefore that the observed down-regulation of translational components during stationary phase is likely the result of an increased basal level of the regulatory compound ppGpp in response to the lack of 6S RNA. Together the results support the view that ppGpp compensates the defect in growth adaptation normally mediated by 6S RNA (see Discussion).Regulation of ribosome synthesis and most of the translational components critically depends on the concentration of the regulatory effector molecule ppGpp , whose crelA+ or relA- strains deficient in 6S RNA were compared (data not shown).It should be noted that the increase in the basal ppGpp concentration is mediated by an altered activity of SpoT and does not depend on the presence or absence of RelA because the same result was obtained no matter if 70-dependent genes are inhibited by 6S RNA, when cells enter stationary phase, but also other types of promoters are under 6S control.Given the respectable number of differentially expressed genes during exponential growth one must put into question the view that 6S RNA is a strictly stationary phase-specific regulator. Our analysis clearly shows, that not only σ70-dependent with weak -35 element or σ70 extended -10 promoters [32, σ38, σ54 and σ70 promoters of different specificity  is also reduced during exponential growth, suggesting a gradual regulation of the r-protein transcription units, which already initiates before cells enter stationary phase (see below).Interestingly, the mRNA level for rimM, fusA rpoB and rpoC. Repression affects almost all r-protein operons and as such r-proteins constituting the small and large ribosomal subunits. Moreover, primer extension analysis of the rRNA expression level performed under the same conditions clearly demonstrated a similar reduction, which strongly supports the conclusion that ribosome biogenesis must indeed be inhibited in the 6S RNA deficient strain. In addition to the core components of the ribosome a number of auxiliary genes involved in maintaining an active translation apparatus also show reduced expression. Among those genes are yibK and rimM, encoding rRNA methylases and fusA and lepA, encoding translation factors, which further underlines the fact that depletion of 6S RNA results in a coordinated reduction of the components necessary for active translation.The most interesting result of this study is the striking coincidence of r-protein operon-encoded genes and genes involved in translation, which are coordinately reduced in expression during early stationary growth. Those genes comprise r-protein genes from most r-protein transcription units including a number of genes co-transcribed within r-protein operons, such as smpB, encoding the protein SmpB that binds to tmRNA and serves to rescue ribosomes translating defective mRNAs [rssB, is involved in the degradation of RpoS, the master regulator for stationary phase and stress. RssB is part of a two-component system and as such regulated by phosphorylation. Whether the observed increase in rssB mRNA affects RpoS concentration is therefore questionable. A possible functional link between RpoS and 6S RNA is nevertheless an attractive hypothesis but to verify a possible direct connection to this very complex network certainly requires a separate study. In addition, the expression of folD is enhanced. This gene encodes a bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methylene-tetrahydrofolate cyclohydrolase. As such it is involved in the one-carbon metabolism and may be considered as functionally related to the gene ygfA, a putative 5,10-methenyltetrahydrofolate synthetase, which is co-transcribed with E. coli 6S RNA and found downstream of many enterobacterial and g-proteobacterial ssrS transcription units [guaD and add are also enhanced one might speculate that this is the functional link between the two genes ssrS and ygfA.The expression of a number of genes of interest, whether directly or indirectly, is also enhanced during stationary phase. Those genes comprise numerous transcriptional regulators but also genes related to the translation process, such as ve mRNAs . Many geon units ,8. The fE. coli two different enzymes are responsible for the different activities. The ribosome associated RelA protein catalyzes the high ppGpp concentrations in response to the codon-directed binding of a non-aminoacylated tRNA to the ribosomal A-site. On the other hand, the basal ppGpp level mainly results from the balanced synthesis and hydrolysis activities of the bi-functional enzyme SpoT, which acts as growth rate sensor. Changes in the balanced ratio of synthesis and degradation of ppGpp are subject to a variety of cellular compounds and their concentrations, such as carbon sources, iron, phosphate, fatty acids or metabolic compounds linked to stress. All these compounds are potentially acting as growth rate sensors [In contrast to the very high ppGpp levels induced under amino acid deprivation and nutritional stress, which trigger the stringent response ,37 the b sensors . One couE. coli is SpoT [ssrS- strain e.g. hisL, hisH, thrL or several aromatic amino acid transporters  or early stationary phase (~2.4 A600). Cells were instantly cooled to 0°C and concentrated by centrifugation. Total RNA was extracted by the hot phenol lysis as described [Cells were grown in YT-media at 37°C to either log- . The array design includes 9308 longmer oligonucleotide (70 mer) probes, representing genomes of four E. coli strains and three plasmids . Some genes are represented by different gene-specific oligonucleotides (e.g. guaD).Cell growth, time points of analysis and preparation of total RNA samples were carried out as described above. Synthesis of Cy3- and Cy5-dUTP labelled cDNA samples were performed as described . The E. http://www.bioconductor.org. The processed and loess-normalized data as well as detailed experimental information according to MIAME [Hybridization and stringent washing of the microarrays were performed according to the instructions of the supplier. Hybridization was carried out for 16-18 h at 42°C using a MAUI hybridization system . After washing the microarrays were dried by centrifugation  and Cy3- and Cy5-fluorescence was determined with 10 μm/pixel resolution using an Axon GenePix 4000B laser scanner . Quantitative image analysis was carried out using GenePix image analysis software and results were saved as GPR-file . For data normalization, GPR-files were processed using the BioConductor/R-packages limma and marray to MIAME  were stoto MIAME .- E. coli ssrSmutant MM139 with the wild- type MC4100 were carried out from 2 independent cultures for the exponential time point (2 hybridizations) and from 3 independent cultures for the early stationary phase, which also included a colour-swap (4 hybridizations). To filter for reliable signal detection and differentially expressed genes the following quality filter was applied: (i) flags > = 0 (GenePix Pro 6.0), (ii) signal/noise > = 5 for Cy5  or Cy3 , (iii) >1.5-fold change in the comparison MM139 versus MC4100. For statistical analysis p values were calculated by Student's t-test .To assess effects of 6S deletion on global gene expression pattern DNA-microarray analyses of RNA levels of osmY , rpoD , bolA  and rrsA as a control  were designed based on gene sequences using web-based Primer3 http://frodo.wi.mit.edu/primer3/. Primer pairs were checked for PCR amplification and specificity using E. coli genomic DNA as template. To test for contaminations in RNA samples with genomic DNA, the integrity and purity of generated cDNA and NAC were checked by qualitative PCR before real-time quantification. The cDNA template used for DNA amplification was equivalent to 10 ng RNA per 50 μl qRT-PCR reaction. The qRT-PCR reactions were performed in duplicate for each gene together with 1 reaction of NTC and NAC. After the qRT-PCR reaction PCR products were further characterized by melting curve analysis. The threshold cycles (CT) were calculated by the instruments software. The relative gene expression levels were calculated by comparing the CT values using REST 2005 software [qRT-PCR analyses were carried out with the Mastercycler ep realplex system (Eppendorf) and its instrument software (version 1.0) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen). To generate single-stranded cDNA, 2 μg of isolated total RNA were reverse transcribed with Superscript II (Invitrogen), using random hexamer primers. To generate no amplification control (NAC) from total RNA, the enzyme Superscript II was omitted in separate reactions. The primer pairs for realtime PCR (170-176 bp product size) of software .32P-ATP  and T4 polynucleotide kinase (NEB). Samples were heated for 3 minutes to 70°C and hybridisation was performed by slow cooling to room temperature. Primer extension reaction was stopped after 30 minutes at 42°C adding 5 μl formamide loading buffer. The samples were then denatured at 100°C for 5 minutes and separated on 15% polyacrylamide gels.Quantitative primer extension was performed as described . For mRNin vivo was performed as described [32P neutralized phosphoric acid for 1 h and 40 μl of this cell suspension were directly mixed with 1N formic acid and frozen immediately in liquid nitrogen. Cells were disrupted by three cycles of freeze-thawing, followed by 10 minutes centrifugation at 13000 rpm. The supernatant subsequently used for thin layer chromatography. As positive control high ppGpp concentrations were induced by the stringent control. In this case MG1655 cells were grown in YT-media to mid-exponential phase (OD600 0.6), labelled for 15 minutes with 32P neutralized phosphoric acid, followed by the addition of 0.7 mg/ml serine hydroxamate [Determination of basal ppGpp concentrations escribed . Cells wroxamate . The supTN and RG carried out the molecular genetic studies. TP carried out the microarray analysis and data evaluation. RW conceived of the study, participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.Promoter specificities of genes analyzed by primer extension. The table indicates different promoter specificities of selected genes analyzed by primer extension.Click here for filessrS-(MC4100) strainsGrowth curve of wild-type (MM139) and . The figure shows the growth curves of wild-type and mutant strains and the time points of RNA extraction.Click here for fileDifferentially expressed genes during exponential growth. The table lists all genes >1.5-fold differentially expressed in the DNA microarray analysis comparing the ssrS- strain MM139 with the wild type MC4100 during exponential growth.Click here for fileDifferentially expressed genes during stationary growth. The table lists all genes >1.5-fold differentially expressed in the DNA microarray analysis comparing the ssrS- strain MM139 with the wild type MC4100 during stationary growth.Click here for fileComparison of selected miroarray data with qRT-PCR analysis. The table lists a comparison of DNA-microarray data and qRT-PCR analysis for selected genes in early stationary phase.Click here for file


The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of  The dis in 2003 . The mur in 2003 A; ORF1 e in 2003 ; ORF2 en in 2003 .3 plaque forming units/ml versus >5 × 104 TCID50 per 35 mm dish for the Pol-I and FPV-T7 based systems respectively.A reverse genetics approach for MNV described previously relies on the transfection of a full-length cDNA construct of MNV into cells infected previously with fowlpox virus expressing T7 RNA polymerase (FPV-T7) . This syin vitro synthesised MNV RNA was examined as a possible reverse genetics approach as this has proven effective for other members of the Caliciviridae; both feline calicivirus , as described previously (Human embryonic kidney (293T) and murine macrophage cells (RAW 264.7) were obtained from ATCC and maintained in DMEM containing 10% foetal calf serum (FCS) at 37 °C with 10% COeviously , were ob2.2pT7:MNV 3′Rz containing the wild type MNV-1 sequence under the control of a truncated T7 RNA polymerase promoter was described previously . A modif2.3in vitro transcription. RNA transcripts produced in this manner resulted in the inclusion of GCUAG at the 3′ end of the viral transcript due to the NheI overhang. RNA was capped using the ScriptCap system from Epicentre according to the manufacturer′s instructions. Briefly, up to 75 μg of RNA was denatured by heating to 65 °C for 10 min prior to rapid chilling on ice. The capping reaction was then set up by the addition of 10 μl of 10× capping buffer, 10 μl of 10 mM GTP, 0.5 μl of 20 mM S-adenosyl methionine, 2.5 μl of Scriptguard and 4 μl of ScriptCap enzyme mix in a total reaction volume of 100 μl. The reaction was incubated at 37 °C for 1 h then the RNA precipitated using lithium chloride. RNA was washed with 70% ethanol prior to resuspension in RNA storage solution (Ambion). RNA was stored at −20 °C until required.Typically, transcription reactions contained 200 mM Hepes pH 7.5, 32 mM magnesium acetate, 40 mM DTT, 2 mM spermidine, 7.5 mM of each NTP, 25 ng/μl of linearised DNA template and 50 μg/ml of T7 RNA polymerase. Reactions were incubated at 37 °C for 2–7 h, treated with DNase at a final concentration of 0.1 unit/μl (New England Biolabs) then precipitated using lithium chloride, final concentration 2.5 M. RNA was resuspended in RNA storage solution (Ambion) and stored at −20 °C until required. The cDNA clones pT7:MNV 3′RZ or pT7:MNV F/S 3′RZ were linearised with NheI prior to 5 and 5 × 106 copies of MNV genomic RNA as determined using quantitative real-time RT-PCR (data not shown).Viral VPg-linked RNA was prepared as described previously . Briefly2.4In vitro translation reactions were performed using the Flexi rabbit reticulocyte lysate system from Promega. Reactions were prepared according to the manufacturer's instructions as described for capped mRNAs and programmed with 1 μg of RNA per 25 μl reaction. Reactions were incubated at 30 °C for 90 min prior to the addition of an equal volume of SDS-PAGE sample buffer. Samples were resolved subsequently on a 15% SDS-PAGE gel before exposure to phosphoimager screen.2.55 cells were seeded into a 35 mm dish in antibiotic free DMEM containing 10% FCS. Cells were then typically transfected with 1 μg of RNA, or amounts detailed in the figure legends, complexed with 4 μl of Lipofectamine 2000 for 20 min at room temperature in 200 μl of Optimem (Invitrogen). The transfection mixture was added to cells drop-wise prior to incubation at 37 °C. For virus recovery, cells were incubated for 24 h at 37 °C prior to freezing at −80 °C and subsequent titration by TCID50 on RAW 264.7 cells. Samples were prepared for western blot in an identical manner except rather than freezing at −80 °C, cells were lysed in RIPA buffer (50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and analysed subsequently by SDS-PAGE (15% acrylamide) and western blot using antisera to the viral polymerase NS7 or the minor capsid protein VP2.293T and BSR-T7 cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, 24 h prior to transfection, 7.5 × 102.66 cells per transfection were harvested and washed once in PBS (Invitrogen). Cells were then resuspended in 100 μl of resuspension buffer and electroporated as detailed in In order to determine the optimum transfection conditions for the RAW 264.7 cell line highly permissive to MNV infection, cells were transfected with 1 μg of pGFPmax plasmid (Lonza) using the Neon transfection system. 6 × 1033.1NheI restriction site after a 27 nucleotide long poly-A tail, followed by a 3′ hepatitis delta virus ribozyme sequence  were >4 × 105 TCID50, over 10-fold higher than the T7 RNA polymerase driven DNA based recovery system . The optimal conditions for virus recovery were identified as 1725 V with a 25 ms pulse length and under these conditions typical yields were 3.4 × 107 TCID50 at 24 h post-transfection of 1 μg of viral VPg-linked RNA . To obtain high efficiency transfection of RAW 264.7 cells, the optimum conditions for the delivery of DNA were first examined by electroporation of pGFPmax using the Neon electroporation system (Invitrogen). A variety of conditions were examined and the percentage of GFP positive cells determined A. Optimunked RNA A.3.4in vitro transcribed enzymatically capped RNA required for virus recovery was examined using the conditions demonstrated previously to give maximum yields from viral VPg-linked RNA. The yield of virus obtained by transfection of various quantities of RNA after a single cycle of replication, namely 24 h post-transfection, was examined pppG pppG(pN)). However, at best capping efficiencies of 75–80% can be achieved, with the remaining 20–25% of RNA containing a triphosphorylated 5′ end (7G(pN)) and as such the RNA is translated inefficiently pppG , into RNd 5′ end . Anothericiently . Typicaliciently . Howeverin vitro synthesised viral RNA as a possible approach to reverse genetics is not only limited by the inability of some of the RNA to translate efficiently as a result of either the absence of a 5′ cap or an incorrectly incorporated 5′ cap, but also by the induction of interferon by those transcripts which are not capped. Given the reported sensitivity of murine norovirus to the interferon response  interferon induction achieved by transfection of RNA prepared in this manner.The current report examines the use of enzymatically capped RNA, generated by the addition of an authentic cap structure to the 5′ end of nsferase , commercnsferase . The 5′ ach 100% . RNA proach 100% . It is w5 per 35 mm dish (1.5 × 106 cells)  . Transfe6 cells) , howeverVaccinia virus capping enzyme with efficient transfection of the murine macrophage cell line RAW 264.7, it was possible to develop an efficient method of recovery of MNV in permissive cells. Optimized transfection conditions were determined using the Neon transfection system from Invitrogen (9 TCID50 72 h post-transfection (By combining RNA capped using the vitrogen . In contvitrogen . Using tvitrogen A. Transfvitrogen . The obssfection , comparaIn conclusion, a new method for norovirus reverse genetics was developed, which allows the rapid and robust recovery of genetically defined viruses in tissue culture. This method, when combined with mutational analyses, provides an additional tool with which to uncover the molecular mechanisms of norovirus translation, replication and ultimately virulence, aiding understanding of this group of significant pathogens.