E. coli, a model bacterial organism.Bacterial small regulatory RNAs (sRNAs) play important roles in sensing environment changes through sRNA-target mRNA interactions. However, the current strategy for detecting sRNA-mRNA interactions usually combines bioinformatics prediction and experimental verification, which is hampered by low prediction accuracy and low-throughput. Additionally, among the 4736 sequenced bacterial genomes, only about 2164 sRNAs from 319 strains have been described. Furthermore, target mRNAs of only 157 sRNAs have been uncovered. Obviously, highly efficient methods were required to detect sRNA-mRNA interactions in the sequenced genomes. This study aimed to apply a modified CLASH  method to detect RNA-RNA interactions in Statistically significant interactions were detected in 29 transcript pairs. To the best of our knowledge, 24 pairs were reported for the first time and were novel RNA interactions, including tRNA-tRNA, tRNA-ncRNA (non-coding RNA), tRNA-rRNA, rRNA-mRNA, rRNA-ncRNA, rRNA-rRNA, rRNA-IGT (intergenic transcript), and tRNA-IGT interactions.Discovery of novel RNA-RNA interactions in the present study demonstrates that RNA-RNA interactions might be far more complicated than ever expected. New methods may be required to help discover more novel RNA-RNA interactions. The present work describes a high-throughput protocol not only for discovering new RNA interactions, but also directly obtaining base-pairing sequences, which should be useful in assessing RNA structure and interactions.The online version of this article (doi:10.1186/s12864-017-3725-3) contains supplementary material, which is available to authorized users. RNA-RNA interactions (RRIs) play important roles in multiple biological processes. For example, mounting evidence suggests that miRNA-mRNA interactions in eukaryotes and sRNA-mRNA interactions in bacteria can exert posttranscriptional regulation of gene expression –3. In adThe current bioinformatics methods for predicting RRIs take RNA sequences as input, and can be divided into two classes . The firC. elegans [E. coli, it doesn’t matter if they are associated with other partner molecules such as proteins. To the best of our knowledge, CLASH or similar methods have not been applied to detect transcriptome-wide whole RRIs in prokaryotes.An early strategy is the high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation(HITS-CLIP), which was developed to decode miRNA-mRNA interactions in the mouse brain . At firs elegans . HoweverEscherichia coli K12 MG1655 cells were centrifuged and washed with PBS, resuspended with PBS at a density of 5 × 109 cells/ml and incubated on ice. AMT (Sigma) was added to treat the cells at a concentration of 0.3 mg/ml, on ice for 10 min. Then, the cells were kept on ice and subjected to UV irradiation at 365 nm with an intensity of 10 mW/cm2 six times (10 min each); cells were shaken well before irradiation.After cross-linking, cells were washed twice with PBS. Lysozyme solution (TIANGEN) and 10% SDS (Sigma) were added for cell lysis at 64 °C for 2 min. Lysates were cooled to 4 °C. RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform extraction method . DNA conRNAs were trimmed with RNase T1 (Invitrogen) for 1 h.20-mer oligo-deoxy-ribonucleotides and buffer were added. The mixture was heated to 90 °C for 2min and cooled to room temperature naturally. RNase H (Thermo Scientific) was added for RNA digestion in DNA/RNA duplexes for 1 h. After 3 repeats, the oligonucleotides were removed by DNase I (NEB).RNAs were resolved on 10% urea polyacrylamide gels. The bands corresponding to 40-100 nt were cut out and recovered using a ZR small-RNA PAGE recovery kit (Zymo research).The recovered RNAs were incubated in a dephosphorylation mixture containing 8 U FastAP thermosensitive alkaline phosphatase  and 40 U RNase inhibitors in polynucleotide kinase (PNK) buffer for 45 min at 20 °C.RNAs were subsequently phosphorylated with 10 U of T4 polynucleotide kinase in PNK buffer (TAKARA) for 30 min at 37 °C. Cross-linked RNA molecules were then ligated using 40 U of T4 RNA ligase 1 , 1 mM ATP, and 40 U RNase inhibitors in RNA ligase 1 buffer for 1 h at 15 °C, and kept for 16 h at 4 °C.2, followed by 200 mJ/cm2. RNAs were then precipitated overnight using isopropyl alcohol and washed twice with 75% alcohol.For cross-linking reversal, the ligated RNAs were irradiated at 254 nm UV with a fluence of 400 mJ/cmE. coli, five samples were prepared. The sample with full treatment was termed ‘TAN’. Compared with the ‘TAN’ sample, ‘AN’ had no T4 RNA ligase treatment, while ‘A’ had no T4 RNA ligase and photoreversal treatments; ‘B’ was the sample without AMT, T4 RNA ligase and photoreversal treatments, while ‘U’ had no AMT, UV irradiation, T4 RNA ligase and photoreversal treatments.Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations. Library preparation was carried out on an Illumina HiSeq 2000/2500 platform. To detect RRIs in Escherichia coli K12 MG1655 with BLAST [The adapter sequences were removed from the raw sequencing reads using the Flexbar  softwareth BLAST . Only BLth BLAST . Then, cth BLAST . The strAdditionally, we defined the reads meeting the following criteria as “single reads”: (1) the read could be mapped continuously to the genome, and otherwise divided into two parts directly adjacent or with up to 4-nt overlap, mapped respectively to two nonoverlapping sequences within the same gene; (2) the helical regions should contain at least one classical cross-linking site of AMT, i.e. 5’-UR or 5’-RU. Reads mapped to multiple genes were discarded. Each combination of helical regions with AMT sites was used as constraint to derive the secondary structure of the read by RNAfold [1. Pairs of windows X and Y ligated by chimeric reads were counted. Window pairs  that appeared in no less than ten chimeric reads were further analyzed. For a ligated window pair , observed frequency was calculated as the number of pairs  divided by the total number of chimeric reads. The expected frequency of random ligation was calculated as:x and Ny are counts of windows X and Y, respectively. In case of non-random ligation of windows X and Y, the observed frequency should be larger than the random frequency. Here, observed to expected frequency ratio for each window pair was calculated, and varied from 0.44 to 130.97. Then, window pairs with ratios > 2 were subjected to Fisher's exact test. Finally, chimeric reads of windows  with P < 0.01 were identified as inter-molecular interactions frozen by AMT with statistical significance.As the reads were composed of RNA sequences in different lengths corresponding to various genomic locations, the genome was divided evenly into non-overlapping windows, and these RNA sequences were named for the windows in which they appeared. Considering that different window sizes can affect the reads grouping accuracy and resolution, we tried several window sizes and checked the grouping results by drawing graphs. The window size of 100 demonstrated the most reasonable results. So we select 100 as the final window size for analysis. All single read- and chimeric partner- windows were counted as total RNAs, TThis study Fig.  employedAs expected, longer reads in the TAN sample emerged Fig. . In sampWith mismatch and gap disallowed, each read from the TAN sample was mapped individually to the genome using the BLAST program . Some reE. coli genome, between the minimum and maximum number of ligated fragments in the sequenced TAN reads. Energies of the random sequences were mostly between -0.36 ~ -0.1 kcal/mol/nt. Finally, the difference of folding free energies between the 42 ~ 100 nt TAN sample and those from the random samples was calculated using t-test in R. A P value between the two samples was 2.2e-16, confirming that the long reads in the TAN sample were not originated from random ligation.If a read is composed of two RNA sequences which are cross-linked by AMT, the latter should form stable structures and thus possess low free energy. To test this hypothesis, free energy of secondary structures of reads from samples TAN, U, B, A, and AN were calculated using the RNAfold  program,We selected reads mapped uniquely for further analysis. As AMT was used to freeze the RNA interactions and enrich them for sequencing, the chimera should contain certain site pairs in both RNA sequences which could be recognized and linked by AMT. As for single reads, site pairs should also exist. We used GUUGle  and homep < 0.01) were selected as putative interacting pairs. Finally, 32 interacting pairs from 29 pairs of transcripts were identified, which corresponded to 1082 chimeric reads , 73 reads in 58 mRNAs, 15 reads in transfer-messenger RNA (tmRNA) ssrA, and 1 read in pseudo mRNA yneO. For the remaining reads, 86 were located in intergenic regions, 472 crossed gene boundaries, 7 crossed 3 repeat region boundaries, and 3 were located in a repeat region named REP31. These reads may reflect new RNA transcripts, whose functional roles remain to be discovered.The 1082 over-represented chimeric reads originated from different types of inter-molecular interactions  sRNA-mRNA interactions are dependent on specific environmental changes. The experimental conditions employed in this study may not be consistent with those of known sRNA-mRNA interactions; (2) with the high abundance of rRNAs and tRNAs, the sequencing depth was not high enough, and known interactions of sRNA-mRNA could not be detected; (3) other reasons such as many experimental steps do not promote the detection of sRNA-mRNA interactions. In the next study, we will try to improve the protocol to detect low abundance of RNA-RNA interactions by simplifing the experimental processes, eliminating rRNAs and tRNAs, and increasing the sequencing depth. Moreover, we will apply it in the comparing studies of various conditions and strains to find the RNA-RNA interactions underlying various physiological and pathogenic phenomena.Initially, we aimed to detect more sRNA-mRNA interactions using a modified CLASH protocol. Interestingly, another unexpected aspect of the complicated RNA-RNA interactions was revealed, indicating the necessity for studying RNA interactions broadly. The protocol applied here would be useful in future research for finding more RNA interactions and determining their interaction sequences. In addition, if combined with other CLASH protocols which can detect RRIs associated with proteins, we will be able to study the relation between the RNA-RNA interactome and the protein world.E. coli. Methods for detecting various kinds of RNA-RNA interactions may be different. The present study provides a high-throughput protocol not only for discovering new RNA interactions, but also obtaining base-pairing sequences directly, which should be useful in assessing RNA structure and interactions.As demonstrated by the novel RNA-RNA interactions discovered in this study, there may still be multiple RNA-RNA interactions to be uncovered in Additional file 1:Document S1. PCR and sequencing results to demonstrate the existence of a novel transcript which was detected to interact with rRNA rrlC in this study. (PDF 1869 kb)Additional file 2:List S1. List of interacting gene pairs detected in both this study and Waters’s study, including information of all reads mapped to these gene pairs. (TXT 28 kb)


We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use PTRE-seq to dissect sequence preferences and interactions between miRNAs and RBPs. The binding sites for these effector molecules influenced different aspects of the RNA lifecycle: RNA stability, translation efficiency, and translation initiation. In some cases, post-transcriptional control is modular, with different factors acting independently of each other, while in other cases factors show specific epistatic interactions. The throughput, flexibility, and reproducibility of PTRE-seq make it a valuable tool to study post-transcriptional regulation by 3′UTR elements.RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3′ untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the  A large number of RNA binding proteins (RBPs) and miRNAs bind to the 3′ untranslated regions of mRNA, but methods to dissect their function and interactions are lacking. Here the authors introduce post-transcriptional regulatory element sequencing (PTRE-seq) to dissect sequence preferences, interactions and consequences of RBP and miRNA binding. These trans-acting factors often bind to cis elements within the mRNA. Two important classes of trans-acting factors are RNA binding proteins (RBPs) and microRNAs (miRNAs).Cellular factors post-transcriptionally regulate mRNA by altering its modification, localization, stability, and translation9. miRNAs recruit the Argonaute containing miRNA-induced silencing complex (miRISC) to specific mRNAs by base-pairing with complementary sequences within their 3′UTR2. Mammalian cells typically express many miRNAs, with the human genome currently thought to encode 2580 miRNAs10. Those miRNAs are predicted to target most human mRNAs11.miRNAs are short non-coding RNAs that mediate translational repression and destabilization of target mRNAstrans-acting factors that affect mRNAs through processes including: splicing, adenylation and deadenylation, degradation, localization, and translation1. Recent studies have sought to identify the complete set of RBPs in mammalian cells, and based on these studies the human genome contains >1000 RBPs, most of which have unknown functions15. Over 800 RBPs have been identified in cultured HeLa cells alone12. One well-characterized RBP is Pumilio, a member of the Puf family, which is conserved from yeast to humans and regulates translation and RNA-decay19. The RNA binding protein Smaug is also conserved from yeast (Vts1) to humans (SAMD4A and SAMD4B) and regulates both translation and RNA-decay21. Another well-known RBP family is the ELAVLs, homologs of the Drosophila embryonic lethal abnormal vision, elav22. These proteins bind AU-rich elements within mRNAs and either stabilize or destabilize mRNAs, as well as enhance or repress translation22. While the function and mechanism of action of some RBPs have been partially elucidated, for the majority of RBPs their functions remain unknown.RBPs are a second prominent class of 21. Given the large numbers of uncharacterized RBPs and miRNAs, we urgently need new approaches with higher throughput, which can be employed in diverse cell types and developmental stages.Most evidence for the function of RBPs, such as Pumilio and Smaug, has come from low-throughput experiments that study their targets during embryogenesis or from reporter experiments15. Many RBP or miRNA binding sites have been shown to occur near predicted miRNA binding sites. In many cases these binding sites are immediately adjacent or even overlap28. Some RBPs cooperate with miRNAs in regulating the expression of specific genes. For example, Pumilio facilitates miRNA-mediated repression in both humans and Drosophila30. HuR, a RBP that binds AU-rich elements, can also modulate miRNA-mediated repression32. Understanding how mRNA trans-acting factors modulate the activity of one another is a major challenge. A tractable high-throughput approach would help unravel the interactions between different effectors of RNA regulation.Interactions between the miRISC and RBPs have been of great interest recently. With >2500 human miRNAs, that are predicted to target most mRNAs, and >1000 RBPs it is likely that many mRNAs are co-regulated by these factors35. While these methods provide the throughput required to study the effects of miRNAs and RBPs across the genome, they do not provide the flexibility to construct and assay large numbers of reporters designed to dissect the effects of different combinations and affinities of RNA cis-regulatory elements.The widespread availability of high-throughput sequencing is powering the development of “omic” technologies to study miRNAs and RBPs. RNA-seq combined with ribosome profiling can reveal the effects of RBPs and miRNAs on target RNA expression and translation39. An analogous technology for assaying the activities of the cis-acting RNA sequences bound by RBPs and miRNAs would help unravel the network of interactions that underlies post-transcriptional regulation of mRNA. Such a system should provide the flexibility and throughput to dissect individual 3′ UTR elements, assay the effects of changes in the strength and number of cis-acting RNA elements, and detect interactions between different types of cis-acting sequences.In studies of transcriptional enhancers, Massively Parallel Reporter Gene Assays (MPRAs) are useful complements to technologies that quantify the activity of endogenous genomic elements43. These approaches generally rely on synthesizing or amplifying portions of 3′UTRs and fusing them to a reporter. While these techniques have identified 3′UTR motifs that have effects on RNA stability and protein amounts, none have been combined with polysome profiling to separate effects on RNA stability, translation efficiency, and translational initiation. In addition, naturally occurring 3′ UTRs contain many different types of elements, making it difficult to deconvolve the effects of individual sites. A synthetic approach, in which large numbers of reporters with specific combinations of elements are designed and assayed, would provide the power necessary to isolate the effects of individual binding sites, as well as the interactions between sites. Because high-throughput methods for studying synthetic elements have proven to have great utility in dissecting interactions among transcription factors, we have extended this approach to post-transcriptional regulation47.Recently, several labs have employed plasmid or mRNA libraries to study endogenous 3′UTR elementselement sequencing (PTRE-seq), an approach that uses a massively parallel reporter library to study the effects of synthetic 3′UTR elements on RNA stability, translation efficiency, and translation initiation. We use PTRE-seq to study the effects of known binding sites for RBPs and miRNAs, both individually and in combination. With this approach, we determine that the binding sites for these effector molecules influenced different aspects of the RNA lifecycle, including RNA stability, translation efficiency, and translation initiation. We observe trans-acting factors acting independently or in some cases epistatically. Finally, deploying PTRE-seq across multiple cell lines revealed the influence of the trans environment on post-transcriptional regulation by specific trans-acting factors. Altogether these results demonstrate the throughput, flexibility and reproducibility of PTRE-seq.Here we report post-transcriptional regulatory We developed PTRE-seq to quantify the individual and combined effects of RBP and miRNA binding sites in 3′UTRs. We created 642 unique synthetic 3′UTRs composed of combinations of a let-7 binding site, the Pumilio recognition element (PRE), the Smaug recognition element (SRE), AU-rich elements (ARE), and a control sequence (“blank”). Bioinformatic analyses indicate that nearly 300 human transcripts contain a PRE, miRNA-binding site and an ARE  and the 40S ribosome fractions (initiating ribosomes) Fig. . Messengcis-elements in our library had strong effects in the data which we captured by fitting linear regression models for both RNA expression and TE to our data. For both the RNA expression model and the TE model, parameters included the identity of the element at each of the four positions and all pairwise interactions between elements at each position. The regression models captured the relationship between 3′UTR composition and relative RNA expression  , we excluded synthetic 3′UTRs containing ARE elements from this analysis. This model provides a formal biophysical framework to capture saturation effects and cooperative interactions between cis-acting elements. Each 3′UTR is described as a collection of states, in which each state represents a particular configuration of bound and unbound elements on a 3′UTR. The model uses parameters that describe the free energies of interaction between RBP/miRNA–RBP/miRNA and RBP/miRNA–mRNA to compute the probability, or weight of each state. These interactions can be neighboring or non-neighboring, however, our implementation of the model does not explicitly model position of the RBPs. In each state bound factors either facilitate or inhibit the recruitment of mRNA decay machinery (or the ribosome for TE), and the weights of the different states are used to compute the probability that the mRNA decay machinery (or the ribosome for TE) is present at an mRNA. In the model, this probability is proportional to the output RNA expression or TE50.To gain mechanistic insights, we also fit a statistical thermodynamic model to our RNA expression and TE dataR = 0.92) and RNA expression well (R = 0.94). The good performance of these models suggests that let-7, PRE, and SRE function mostly independently on UTRs. In most cases adding interaction terms did not improve the fit of these models to the data. This observation suggests that some of the self-interaction terms in the linear regression models (described below) are likely due to saturation of binding on UTRs with high copy numbers of cis-acting sites. The thermodynamic model naturally accounts for saturation without the need for interaction terms and describes the situation when saturation causes additional sites to have little or no effect. In two cases, the thermodynamic model for TE did improve with the addition of interaction terms, one for interaction between adjacent let-7 sites and another for interaction between adjacent PRE and let-7 sites  behaved differently than the let-7 binding site. Like the let-7 binding site, PREs also decreased RNA expression, but had a much more modest effect on TE Fig. . Additio53. Nucleotides 2–7 of the miRNA constitute the “seed” sequence, and weak seed pairing reduces the effectiveness of miRNA-mediated repression53. In addition to the 625 combinations of regulatory elements described above, we included constructs in the library to study the effect of base-pairing between the miRNA and its target on repression. This included a series of constructs with one, two, or four binding sites for let-7 with either 6-mer, 7-mer-a1, 7-mer-m8 or 8-mer base-pairing in the seed region52, as well as binding sites for let-7 that have perfect base-pairing with the target. We observed a clear copy-number-dependent and seed-pairing dependent effect on RNA expression and TE for these reporters  reduced RNA expression to a greater extent  than base-pairing MFE (R = 0.56) or secondary structure MFE (R = 0.12) alone. Because this model was made with only a few data points it is only suggestive. This secondary structure of the 3′UTR could explain the observation that some binding sites, even with better thermodynamics, were not as well repressed.The efficiency of miRNA-mediated repression depends on the number and quality of binding sites in its targeters Fig. . In addiing Fig. , the seqent Fig. . We susp30. In our library, the combination of a let-7 binding site and a PRE resulted in a reduction of RNA expression that was slightly less than the product of their individual effects  and AUF1 (hnRNPD), are also ARE-binding proteins (ARE-BP)22. In our library AREs either enhanced or repressed RNA expression and TE depending on their position in the 3′UTR , can either stabilize or destabilize target mRNAs, and can enhance or repress translationUTR Fig. . In the UTR Fig. . These oUTR Fig. . Since t2. During recovery from stress HuR relieves miRNA-mediated repression of the catalase mRNA (Cat1)32. In contrast, HuR binding to the c-Myc 3′UTR activates miRNA-mediated repression31. We observed AREs in our library either enhancing or suppressing miRNA-mediated repression in a position dependent manner  did we see a substantial reduction in RNA expression , human neonatal dermal fibroblast (HDF), and a mouse neuroblastoma (N2A). We observed wide variation in the effect of each regulatory element tested across the four cell lines. It is possible that some of these changes could be caused by differences in transfection efficiency or transcription across the cell lines tested. The let-7 binding site caused robust reduction of RNA expression in HeLa cells, but this effect was smaller in magnitude in HEK293, HDF and N2A . This altered structure might affect which ARE-BP bind to the sequence. The varied effects of AREs across cell-lines are consistent with this hypothesis. While AREs both increased and decreased RNA expression in most cell lines tested, in HEK293 we only observed increased RNA expression. As the expression of ARE-BPs is known to vary across cell and tissue types, this finding suggests that the ARE-BPs with different effects are binding to the same reporters in different cell-linestrans-acting factors. An intriguing possibility is that the ARE may be bound by cytoplasmic polyadenylation element binding protein, CPEB. The consensus binding site for CPEB is UUUUUAU61, but CPEB also binds the sequence UUUUAU62, which appears once in the ARE used in our library. CPEB has been previously shown to work with Pumilio in regulating mRNA translation63.Our experiments revealed strong epistatic interactions between AREs and sites for let-7 and Pumilio. The AREs either enhanced or suppressed miRNA- and Pumilio-mediated repression, depending on their position in the 3′UTR. These effects were not dependent on the proximity in linear sequence space between the two binding sites. These results demonstrate that AREs can modulate repression by both miRISC and RBPs such as Pumilio. Alternatively, the presence of the PRE or miRNA binding site may modulate the effect of the ARE by changing the secondary structure of the mRNA. These observations warrant future studies into which ARE-BP are responsible for these epistatic interactions, and whether the effects are mediated through mRNA secondary structure or potentially through interactions between Drosophila Smaug in HeLa cells, we observed a reduction in the RNA levels of SRE containing reporters. This suggests that at least in the cell-lines we tested the mammalian Smaug homologs, SAMD4A and SAMD4B, are expressed at low levels or are not efficacious.While we observed robust effects on the RNA expression and translation of reporters containing let-7 binding sites, PREs and AREs, in our experiments, the SRE caused only modest changes in RNA expression and no change in TE or 40S association. When we overexpressed cis-acting elements, both individually and in combination, and for unraveling their effects on different aspects of RNA stability and translational control.Our results provide further evidence for the mechanisms of post-transcriptional regulation by miRNAs, Pumilio and AREs. PTRE-seq will serve as a valuable tool studying the effects of multiple NheI, EcoRV, and KpnI sites downstream of EGFP. The PCR product was ligated into pCDNA5/FRT/TO that had been previously cut with PmeI.To create the PTRE-seq library we first generated the plasmid pCDNA5/FRT/TO-EGFP-RE. EGFP was PCR amplified with EGFP-F and RE-R primers, Supplementary Table GCTAGC-132nt regulatory element-ATGCATcGATATCaCTCGAGxxxxxxxxxGGTACCCGACTACTACTACG – 3′. The restriction enzymes are underlined and are from 5′ to 3′: NheI, NsiI, EcoRV, XhoI, and KpnI.A pool of 6500 unique 200-mer oligonucleotides was ordered from Agilent Technologies™. Oligonucleotides were designed to contain all combinations of either a let-7 binding site, PRE, SRE, AU-rich element or a “blank” control sequence. The sequence for each of these elements is described in Supplementary Table NheI and KpnI. We prepared plasmid DNA from ~40,000 colonies to generate library RE_Array*. We then cloned a “spacer” sequence in between the regulatory elements and the barcode. This “spacer” is the reverse of a sequence within the BGH 3′UTR and is used for amplification of the barcodes from cDNA or plasmid. The “spacer” was ordered as a pair of oligonucleotides that were annealed to form a dsDNA oligonucleotide with overhangs compatible with DNA cleaved by NsiI and XhoI. The “spacer” was cloned into the RE_Array* using NsiI and XhoI. We collected plasmid DNA from ~250,000 colonies to generate the library RE_Array_1.The library was PCR amplified for four cycles using Phusion High-Fidelity polymerase (NEB) and primers Lib_F and Lib_R. We cloned the amplicon into pCDNA5/FRT/TO-EGFP-RE using To clone individual reporters from the library we sequenced 94 colonies from the RE_Array* library. We chose from those clones seven reporters of interest. For the control reporter and three reporters targeted by let-7 , we ordered oligonucleotides that were ligated into the vector pCDNA5/FRT/TO-EGFP-RE as described above. The “spacer” was ligated into the plasmids containing the reporters, as described above.TM-293 cells  were grown in DMEM (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 1×Penicillin streptomycin and glutamine (Gibco) and 1 × MEM Non-Essential Amino Acids (Gibco). Transfection was carried using the Neon Transfection System (Invitrogen) per manufacturer protocol. For each transfection, 2.5 × 106 cells were electroporated with 8 µg of RE_Array_1. For transfection of mCh-Smg, we electroporated 8 µg of pCDNA-D40-mCh-Smg along with 8 µg of RE_Array_1 into HeLa cells as described above. The mCh-Smg plasmid was made by PCR amplifying the Smaug coding sequence (CDS) from Drosophila S2 cell cDNA using the primers described in Supplementary Table HeLa , HDFn , N2A CCL-131, ATCC) and T-REx31, ATCC 64 per the manufacturers protocol. The cells were split 24 h later into two separate 12-well plates and a 96-well plate. Forty hours after transfection the fluorescence was measured using a Synergy H4 plate reader (BioTek), at the same time RNA and protein was isolated from the 12-well plates.For transfection of the individual reporters we used Effectene (Promega). The cells were transfected in a 12-well plate with 500 ng each of the EGFP reporter and pCDNA-mCherry−1 cycloheximide for 5 min prior to harvesting and counting. A total of 3 × 106 cells were lysed and the lysate was subjected to ribosome fractionation using 7 to 47% sucrose gradient (Teledyne ISCO) as described previously65. In brief, cells were treated with 10 µg/ml cycloheximide for 5 min prior to harvesting and counting. A total of 3 × 106 cells were lysed in 250 µL of polysome lysis buffer  on ice for 20 min prior to clearing at 8000 × g for 10 min at 4 °C. The lysate was layered over a 7–47% sucrose gradient and subjected to centrifugation at 160,000 × g for 3 h at 4 °C. The gradient was fractionated by upward displacement with constant measurement of absorbance at 254 nm using a Teledyne ISCO fractionator. RNA was isolated from 40S and polysome fractions using Ribozol (Amresco). We collected polysome fractions from four biological replicates and 40S fractions from two. Isolated RNA was treated with Turbo DNase (Ambion). For qPCR of rRNA from total, 40S and polysome fractions, first strand cDNA synthesis was carried out using Superscript IV reverse transcriptase (Invitrogen) with random hexamer priming. qPCR was performed with iQTM SYBR Green master mix with the 18S and 28S rRNA primers described in Supplementary Table Total RNA was isolated using Qiagen RNA mini-prep per manufacturer’s protocol. For HeLa cells we collected total RNA from four biological replicates, when testing the library in other cell types we collected one biological replicate. For polysome profiling, cells were treated with 10 µg mlFor qPCR of the individual reporters: RNA was isolated using the Qiagen RNA mini-prep per manufacturer protocol. Isolated RNA was treated with Turbo DNase (Ambion) prior to first strand cDNA synthesis using Superscript Vilo (Invitrogen). Quantitative PCR was performed with EGFP and mCherry primers, Supplementary Table XhoI and SpeI. The digestion product was purified as before and ligated to the Illumina adapters described in Supplementary Table First strand cDNA synthesis for ribosome associated RNA or total RNA was carried out using Superscript IV reverse transcriptase (Invitrogen) with random hexamer priming. The barcode was amplified from cDNA or plasmid using RE_Amp_F and RE_Amp_R primers with Phusion-HF MM (NEB): 98 °C for 1 min, 22 cycles: 98 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The amplicon was purified using Nucleospin Gel and PCR cleanup kit (Macherey Nagel) and subsequently digested with The Illumina library was multiplexed and run on four lanes of Illumina NextSeq machine. Barcodes counts were determined. Only barcodes with greater than >10 counts in the cDNA and plasmid pools were used for analysis.64. The following primary antibodies were used in western analysis at the given dilution: GFP, 1:2000 ; β-actin-HRP, 1:2000, ; Anti-mouse IgG HRP, 1:10,000 .Cells that were transfected with individual reporters were lysed with Lysis Buffer . Western blot analysis was performed, as described previouslyRelative RNA expression for each regulatory element was calculated as described below. In brief cDNA counts for each barcode were normalized by the plasmid counts for the same barcode. The normalized expression was set relative to the median normalized expression of the control, 4×Blank.67. Coefficients are obtained in reference to “blank” sequence at each position. For cross-validation, we randomly divide the data into five parts and use 80% of the data to train and tested on the remaining 20%. This procedure is repeated five times. The parameters for our linear regression model are shown below:For each synthetic 3′UTR, we calculate median fold change across all 10 barcodes. Median fold change values are fit to linear model with interacting terms for the let-7 binding site, PRE, SRE, AU-rich element or space-holding sequence, at four positions using the lm function in Rj = 1 to 4 and i ≠ j, * = Interactionswhere 68 and then later modified51. We modified this framework slightly to model post-transcriptional regulation of mRNA by RNA binding proteins and miRNAs as described below.To model reporter mRNA levels as a function of the factors bound to their synthetic 3′UTRs we implemented a modified version of a thermodynamic model of transcriptional regulation proposed previously70.In our model we specify that each 3′UTR has four binding sites for factors that bind RNA (RNA binding proteins and miRNAs). When a spacer sequence is present at one of the sites we assume that no factor is bound to the site. We also specify that the 3′UTR has one additional binding site for a rate-limiting factor that controls the regulation of the mRNA. RBPs and miRNAs bound on the 3′UTR either facilitate or inhibit the binding of this rate-limiting factor. The primary assumption of this model is that the equilibrium binding of RBPs and miRNAs determine the occupancy of the rate-limiting factor on the 3′UTR. The equilibrium assumption is justified because the timescales at which RBPs and miRNA bind and unbind their sites are much faster than the timescale at which regulation of mRNA levels occurN) total binding sites, four for RBPs and miRNAs, and one for the rate-limiting regulator. Each 3′UTR can exist in many possible states, where a state is a particular configuration of bound factors. A state of the 3′UTR is specified by giving the occupancy σi, either 0 or 1, of each site. The model calculates a Boltzmann weight for each state of the 3′UTR using:σi,j represents the number of instances of factor i and factor j bound to each other in that configuration (see below), qi represents the change in free energy of RNA binding factor i binding to its respective site plus the natural log of the concentration of that factor, and ωi,j is the change in free energy of factor i and factor j interacting, which is a measure of cooperativity between factor i and j. We assume that no factor binds to the spacer sequence and that all of the RNA-binding factors are present in the cell at roughly equal concentrations and bind 3′UTR with same affinity. We also assume that the rate limiting factor binds the 3′UTR with a particular affinity, set to two units for the PTRE-seq data. We model different factor-factor interactions by allowing cooperativity in particular configurations. In some cases, we allow cooperativity only when two factors are simultaneously bound to directly adjacent sites. In other cases, we allow cooperativity as long as the two factors are bound to the same 3′UTR at any two sites. The rate-limiting factor is allowed to interact with all factors bound to the 3′UTR. In our notation qFactor and ωFactor are equal, respectively, to ln(qFactor) and ln(ωFactor) described previously51. To calculate the probability that the rate-limiting factor is bound to the 3′UTR, and therefore the predicted level of the mRNA, we use the equation:δ(rate limiting factor) is a delta function that is zero when the rate limiting factor is not bound and one when it is bound. The summations are over all possible states of the 3′UTR. To fit the model to data we search for values of α, all q, and all ω that best fit the observed mRNA levels measured by PTRE-seq. The parameters are fit with custom Python scripts using SciPy to minimize the objective function using constrained minimization optimization algorithms L-BFGS-B and SLSQP in alternating fashion until the parameter values converge. The asymptotic normal distribution for the parameter estimate is used to calculate the 95% confidence intervals for the parameter values46. After fitting, any non-zero values of a particular ωi,j are interpreted as cooperativity between the two factors.In our model of synthetic 3′UTRs we specify that there are five (https://github.com/hemangichaudhari/Cottrell_PTRE-seq_scripts). Sequencing data is available at the Sequence Read Archive under accession code SRP127467.The authors declare that the data supporting the findings described here are available within the article, the Supplementary Information or Supplementary Data Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7


Transcriptional analysis  of rice flower organs demonstrates correlations of 24-nt phased small RNAs with anther maturation and with three small RNA-binding Argonautes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes  that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations.Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes.  Oryza sativa), as a major crop, has been widely used as a monocot model species to explore the genetic basis of flower development in higher plants  and Tapetum Degeneration Retardation (TDR)  ( anthers .APETALA2 (AP2) genes controlling inflorescence architecture and spikelet meristem identity (SQUAMOSA Promoter Binding Protein-Like (OsSPL) genes, including OsSPL14, are targeted by miR156; this pathway has a role in flowering time, panicle architecture, grain yield, and other developmental phenotypes (trans-acting siRNAs (tasiRNAs), dependent on the activities of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICER-LIKE 4 (DCL4), generated from non-coding transcripts such as TAS3, play roles in both vegetative and reproductive development in both rice and maize by targeting genes encoding auxin response factors (ARFs). For example, the rice mutant of SHOOTLESS2 (SHL2), the ortholog of Arabidopsis RDR6, displays a severe phenotype of misregulation of adaxial-abaxial polarity patterning in both the lemma and anther  is defective in tasiRNA biogenesis and shows a pleotropic phenotype, including sterile male inflorescences  were used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments. The cDNA was synthesized using the mRNA fragments as templates. Short fragments were purified for end repair and single nucleotide A (adenine) addition, ligated to adapters, and then the second strand was degraded using UNG (Uracil-N-Glycosylase). After agarose gel electrophoresis, the suitable fragments were selected for PCR amplification as templates. All the small RNA and RNA-seq libraries were sequenced on an Illumina HiSeq 2000 platform by BGI .http://rice.plantbiology.msu.edu, accessed 23 September 2016) using the program Bowtie (www.bioconductor.org) package ‘edgeR’ was used for small RNA differential analysis . Rice miRNA sequences were downloaded from miRBase   package ‘pheatmap’ was used to represent the average abundance of miRNAs from three biological replicates. PHAS locus identification was performed using the same method described previously by PHAS loci identification. A phasing score of 25 was used as a stringent cut-off, followed by a manual check to remove loci producing highly abundant small RNAs in other sizes, which are most likely degradation products from t/rRNAs. The overall phasiRNA abundance for each PHAS locus was calculated by summing up the normalized abundance of 21- or 24-nt small RNAs generated from each corresponding 21-PHAS and 24-PHAS locus.Small RNA sequencing data were preprocessed by removing adapters, and then mapped to the version 7.0 of the rice genome assembly from the Rice Genome Annotation Project Database (er 2016) . The R (Paired-end strand-specific RNA-seq reads (90bp × 2) were mapped to the rice genome sequences allowing no more than two mismatches using ‘Tophat’ . The RNA in situ hybridizations were carried out as described by OsAGO1d, 5′-GCAATACCACCCACAAGGAC-3′ and 5′-GGTTCCAATACTCCCACTTCC-3′; OsAGO18, 5′-CAGTAT AACAGTACGGAACGC-3′ and 5′-TGTCATTACAACAAGTAG GAGG-3′.Freshly collected samples were fixed in formalin-acetic acid-alcohol (FAA) and dehydrated in a series of graded ethanol concentrations; these samples were then infiltrated with Histo-clear II, embedded in Paraplast Plus, and subsequently processed into 6-μm thick sections using a Leica RM2245 rotary microtome. Templates for RNA probe synthesis were amplified by PCR from the cDNA. Probes were transcribed RNA-seq and small RNA data are available from Genbank, under GEO accession number GSE77300.msp1-4 (‘msp1’ hereafter) and ostdl1a . We performed three biological replicates for each genotype and stage. The lengths of rice spikelets correspond to different anther developmental stages , and the distribution of lengths in different stages was analyzed. The three replicates were nearly identical . The 24-A number of miRNAs have characterized roles in plant development, targeting several families of transcription factors or other development-related genes . miRNAs,Supplementary Tables S2 and S3). From stage 3 to stage 5, >60 miRNAs increased significantly, while only 18 miRNAs decreased during the same time period; from stage 5 to stage 7, the numbers of miRNAs with significantly different levels (up or down) were both fewer than 20  of differentially accumulating miRNAs were observed, suggesting that the male sterile phenotype of these mutants has a limited impact on miRNA levels, relative to the wild-type. This was further confirmed by the observation that very few miRNAs were identified as significantly different in their levels when comparing the two mutants with the wild-type .Genome-wide differential analysis of miRNA expression was performed in wild-type rice across different stages of spikelet development . Prior work using PARE/degradome data confirmed the targets of a number of miRNAs in rice ; these were the only two targets that showed an inverse relationship with miR164 abundance. Three miR172 targets showed an inverse correlation with miR172 levels . miR156 showed an interesting pattern across the developmental stages of rice spikelets: a 21-nt miR156 isoform increased gradually from stage 3 to stage 7, while the 20-nt miR156 firstly decreased from stage 3 to stage 5, and then increased at stage 7 . Among the miRNA targets validated from prior work, only a subset had the expected inverse correlation with miRNA levels; thus, other factors may impact target transcript levels, including both transcriptional (e.g. transcription factors and epigenetic regulation) and post-transcriptional (e.g. mRNA turnover or sequestration) factors. Taken together, these data indicate that miRNAs together with their target genes are dynamically modulated during early stages of rice spikelet development, and are largely independent of the OsTDL1A-MSP1 pathway.Among the miRNAs differentially expressed in wild-type rice across the developmental stages, the level of miR164 showed a ~7-fold increase from stage 3 to stage 7, whereas miR172 showed a dramatic decline of ~4-fold from stage 3 to stage 5, then remained relatively steady to stage 7  and stage 7 (~240 RP5M)  ; miR2275PHAS loci and 50 24-PHAS loci (see Supplementary Table S4). By summing phasiRNA abundances from each PHAS locus, we obtained the overall phasiRNA abundances for both 21- and 24-nt phasiRNAs. In wild-type rice spikelets, the overall abundance of 21-nt phasiRNAs was ~120 000 RP5M in stage 3, increasing by >10-fold in stage 5, and then decreasing by <30% in stage 7 .We calculated both 21- and 24-nt phasiRNA abundances to see how phasiRNAs change during rice spikelet development and whether they are affected in the two mutants. Phasing analysis resulted in 1843 21- stage 7 . Compare stage 7 . ConsistWe next calculated the proportion of 21- and 24-nt phasiRNAs in the entire genome-matched populations of the 21- and 24-nt small RNAs in rice spikelets . We founPHAS transcripts) in the RNA-seq data to assess the correlation of phasiRNA and PHAS mRNA levels. As many 21-PHAS loci are found in large clusters in the rice genome (Supplementary Table S4), we selected a representative cluster on chromosome 3 for this analysis. Our earlier observations in wild-type rice showed a stage 5 peak for 21-nt phasiRNAs, which reduced slightly at stage 7 . Consistent with the precursor molecules consisting of a polyadenylated mRNA paired with an RDR6-derived antisense strand, the strand-specific RNA-seq reads were mapped to only one strand at a given PHAS locus . However, unlike 21-PHAS loci, both 24-nt phasiRNAs and 24-PHAS mRNAs were absent in the RNA-seq data from msp1 and ostdl1a mutants . In summary, sequencing data revealed the developmental modulation of phasiRNA biogenesis in rice anthers, with 24-phasiRNAs and their precursors disrupted in the msp1 and ostdl1a mutants, reflecting an intimate association of these transcripts with anther development and microsporogenesis in rice.We next examined the phasiRNA precursors , perhaps because of the excessive number of sporocytes, where MEL1 is expressed, in mutants .Argonautes (AGOs) are core effector proteins in small RNA-mediated silencing pathways. Different small RNAs are preferentially recruited into specific AGOs, mainly determined by the 5′-terminal nucleotide of the small RNA .We speculated that AGOs that accumulate in stage 7 could function with 24-nt phasiRNAs. Gene expression analysis showed that only three  3 and 5 . Since 2 mutants , suggestin situ hybridizations or examined published images for selected rice AGO genes. Published images for OsAGO2b demonstrate its accumulation in sporocytes and wall layers of rice anthers at pre-meiotic stages, and in later meiosis stage the transcript is restricted predominantly to the tapetum layer and microspores . This suggests that these miRNA-involved gene silencing pathways are genetically independent or upstream of the OsTDL1A-MSP1 pathway.miRNAs play important roles in plant development. We identified miRNAs with differential accumulation patterns during rice spikelet development, including conserved miRNAs . Among these, the role of miR164 in development is not well studied, although it accumulates in spikelet and floral meristems   suggests a role in histone modifications; histone H3 lysine 9 dimethylation (H3K9me2) is decreased in the y linked . ConsideJXB online.Supplementary data are available at Figure S1. MSP1 and OsTDL1A transcript levels in stage 3 of rice spikelets in the wild-type cultivar 9522 and two mutants.Figure S2. miRNA and target transcript levels in different stages and backgrounds of rice spikelets.Figure S3. 21-nt phasiRNAs and precursor transcripts were unaffected in both msp1 and ostdl1a mutants at stage 5.Figure S4. 24-nt phasiRNAs are strongly impacted in stage 7 spikelets of the two rice mutants.Figure S5. mRNA levels of MEL1 and OsAGO1d in different stages and backgrounds of rice spikelets.Figure S6. Expression of AGOs in different tissues and developmental stages of rice anthers in public microarray datasets.Table S1. Summary information for small RNA and RNA-seq libraries prepared in this study.Table S2. miRNA levels in different libraries.Table S3. Differentially expressed miRNAs in different stages and backgrounds of rice spikelet development.Table S4. 21- and 24-nt phasiRNA abundance from each PHAS locus.


Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule’s necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.DOI:http://dx.doi.org/10.7554/eLife.25940.001 Positive-strand RNA viruses, whose virions contain mRNA-sense single stranded RNA, represent the largest genetic class of viruses , includiThe structural organization of viral RNAs and proteins at such membrane-associated sites of RNA replication has been largely unknown. For example, in various studies, viral RNA replication templates and replication proteins have contrastingly been suggested to reside on the cytoplasmic face of membranes or inside vesicular compartments .To address these questions, we turned to flock house virus (FHV), a well-studied, model positive-strand RNA virus in the nodavirus family. The FHV genome organization is shown in Drosophila cells induces ~50 nm diameter ‘spherule’ invaginations of the outer mitochondrial membranes (Previous transmission electron microscopy (TEM) and EM tomography by our group and others showed that FHV infection of embranes . These sembranes , and remembranes . Similarembranes . While iembranes . MoreoveDrosophila cells by cryo-electron microscope (cryo-EM) tomography. Cryo-EM presents samples in a much more native state, bypasses heavy metal staining, and images the sample’s intrinsic electron density, providing direct visualization of all components – lipid, RNA, and protein – of the RNA replication complexes  that are active in viral RNA synthesis with a specific activity approaching that in infected cells  tilt series of images of mitochondria from FHV-infected and uninfected cells and processed these to reconstruct three-dimensional views. Previous EM of chemically fixed, heavy metal-stained FHV spherules  and othe3 and 145,500 nm3, with an average spherule volume of 66,100 nm3 and a standard deviation of ±32,900 nm3. When plotted, the distribution of spherule volumes showed three to four peaks, suggesting the possibility of distinct size classes of spherules  and a defined FHV RNA replication template transcribed in vivo from a DNA plasmid. 3, the smaller spherule class around ~12,000 nm3. Similarly, To test such possible relationships between viral RNA templates and spherule size, spherule volumes were measured in mitochondria from Subtracting the RNA1- and RNA3-associated spherule peaks from the size distribution for full infection leaves a broad range of intermediate sizes possibly containing more than the single peak that might be expected for replication of the remaining 1.4 kb genomic RNA2 . RNA2 re3 , dsRNA2 (1.4 kbp), and dsRNA3 (0.4 kbp) would occupy ~53,000 nm3, ~24,000 nm3, and ~7000 nm3, respectively. Accordingly, even the smallest of the RNA1-associated ~80,000–130,000 nm3 spherules . At a rpherules  could acpherules  could acOur previous examination of chemically-fixed, plastic-embedded samples of FHV replication compartments in whole infected cells, using both two-dimensional transmission EM imaging  and threTo better visualize these striking new features, we performed iterative subtomogram averaging  of the rAt its highest dimension, the turret was raised ~14 nm above the outer mitochondrial membrane surface and in cross-section was seen to reside in a slight depression of the outer mitochondrial membrane . Within The presence of crown structures on FHV spherules and their absence on mitochondria from mock-infected cells  indicateTo test for the possible presence of protein A in crowns, we used a rabbit antiserum against protein A and gold-conjugated secondary antibodies under cryo-EM conditions. This treatment produced abundant gold labeling on the cytoplasmic side of outer mitochondrial membranes, but only above membrane regions where spherules were present, and not above areas where spherules were absent . The disElectron microscopy and electron tomography have been used to visualize the basic membrane architecture of RNA replication compartments for a number of positive-strand RNA viruses , althougWhen imaged previously by EM and electron tomography of chemically fixed samples, spherules appeared elongated parallel to the neck axis and with indistinct contents, often shrunken, possibly due to chemical crosslinking . More na3 and linked the replication of sgRNA3 (0.4 kb) and genomic RNA1 (3.1 kb) with the smallest and largest subsets, respectively , which replicates its genome in similar spherular replication compartments on the plasma membrane, also shows a correlation of spherule size and template length .Another unprecedented feature of these replication compartments revealed by cryo-EM was the striking crown structure on the cytoplasmic side of the spherule aperture. Elements visible in individual images and refined by sub-tomogram averaging of hundreds of independent crowns included an ~18 nm high x 19 nm diameter, 12-fold symmetrical, cup-like main body, a central density within the cup, and an outer ring of 12 smaller projections . ImmunogCystoviridae encode a hexameric portal protein ring that also translocates parental RNA genome templates into virions and releases nascent viral RNA products to the cytoplasm  cells, purchased from ATCC in the course of these experiments ( (ATCC CRL1963); RRID:CVCL_Z232), were grown at 28°C in Schneider’s medium and 8% fetal bovine serum or in serum-free Express-Five media, supplemented with penicillin, streptomycin, and L-glutamine (Gibco). Passages 2–4 of this ATCC stock were frozen and on a regular basis, portions of these early passages were thawed to seed new batches of cells, yielding results that were highly reproducible. Sucrose gradient-purified FHV stocks were produced as previously described  or two 30 ms pulses of 1200 V in 100 µL tips on a Neon electroporator (Bio-Rad) and were harvested 48–72 hr post-electroporation.Schneider’s escribed . DrosophDrosophila cells under control of the baculovirus immediate-early 1 (IE1) promoter and trans-activating hr5 enhancer have been described previously (Plasmid constructs to express FHV RNA1 (pIE1-FHVRNA1), FHV RNA3 (pIE1-FHVRNA3), or protein A (pIE1PtnA) in eviously . Plasmid7 S2 cells using the Q-proteome kit (Qiagen) according to the manufacturer’s protocol. Briefly, cells were pelleted and re-suspended in a series of lysis buffers before being physically disrupted by drawing the suspensions ten times through a 25-gauge syringe needle. The resulting cell lysates were fractionated using several rounds of differential centrifugation until a pellet of concentrated mitochondria was obtained.Mitochondria were extracted from 6 × 1032P]UTP , to a final volume of 25 µL. Reactions were incubated at room temperature and flash-frozen in liquid nitrogen. RNAs were extracted using RNaqueous spin columns (Thermo-Fisher) according to manufacturer specifications. Following elution, samples were ethanol-precipitated, and RNA pellets were washed with 70% ethanol, dried, and re-suspended in RNase-free water supplemented with formaldehyde loading dye. Samples were electrophoresed at 100 mV for 2.5 hr on a 1.0% Tris-borate-EDTA agarose gel. The gel was then dried at 60°C, and exposed to a phosphorimager screen for imaging.FHV mitochondria RNA synthesis reactions were assembled using a modification of a previously described protocol . BrieflyEM-visualization of protein A replicase on mitochondria used a modification of the protocol from ° to +60° using defocus values ranging from 4 to 6 µm. The total electron dose per tomogram averaged 130–150 electrons/Å2. The energy filter slit size during collection ranged between 10–25 eV on both microscopes.Mitochondrial pellets were re-suspended to a final volume of approximately 50 µL isotonic buffer and supplemented with 10 nm fiducial gold particles (EMS). Copper grids with a 3.5 µm hole-size and 1.0 µm spacing (Quantifoil) were glow-discharged for 30 s using a Pelco GD unit and loaded onto a Mark IV Vitrobot (FEI). 3 µL of the mitochondrial preparation was loaded onto a grid at approximately 95% humidity and blotted for 2 s with Whatman paper at a −2 force offset. Grids were plunge-frozen in liquid nitrogen-cooled liquid ethane and stored under liquid nitrogen prior to loading into the cryo-EM holder and the microscope. Cryo-tomograms were acquired using either a Titan Krios electron microscope at 300 keV (FEI) at Janelia Research Campus (HHMI) with a pixel size of 2.9 Å or 5.6 Å or on a TF-30 electron microscope at 300 keV (FEI) at UW Madison with a pixel size of 4.6 Å. Both microscopes were equipped with post-column energy filters and post-GIF K2 Summit direct electron detectors (Gatan). Low and medium magnification maps were acquired using either UCSF tomo  or SeriaThe FHV crown data set included 6 tomograms, each containing between one and four mitochondria. Mitochondrial membranes were segmented semi-automatically , and mod In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Wenhui Li (Senior Editor) and two reviewers, one of whom, Wesley I Sundquist (Reviewer #1), is a member of our Board of Reviewing Editors.Thank you for submitting your article "Cryo-electron tomography reveals novel features of a viral RNA replication compartment" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The authors report cryo-EM tomographic reconstructions of Flock house Virus viral replication "spherules" formed as invaginations in the outer mitochondrial membrane. The structure is a relatively low resolution  but nevertheless reveals a number of new features that had not been characterized, including a 12-fold symmetric "crown" structure that contains viral replicase protein A, coiled dsRNA in the spherule interior, an unappreciated range of spherule volumes and a correlation between transcript size and spherule volume , and what appear to be progeny (positive sense) RNA strands being released from the spherule through the crown.Despite the relatively modest resolution of the reconstruction, the spherule structures are remarkable and other strengths of the paper include: 1) the experiments are generally well conceived and controlled, 2) the data provide strong support for models for spherule biogenesis and activity (summarized in Significant issues that must be addressed:1) The paper is generally straightforward and well written, but details of the reconstruction methodology and statistics are sparse . Most importantly, the assumption of 12-fold symmetry is not sufficiently justified – for example by providing a power spectrum showing a clear peak corresponding to 12-fold symmetry. There is no reason to think this assumption is incorrect, but the symmetry and structure of the crown are key new features and the experimental underpinnings should therefore be presented in detail.2) Interpretations of spherule size and content are an important component of this paper, but require clarification. There are several issues:A) Please explain exactly what is meant by the sentence: "Consistent with estimates of the persistence length of dsRNA" – didn't understand this sentence.B) C) The interpretation of D) Please clarify further the calculations between template length and spherule volume were derived and what relationship they have to cytoplasmic polyhydrosis virus (CPV) dsRNA. Perhaps it would be useful to show E) Postulating that 1.5 – 2.5 copies of dsRNA1 fit inside each spherule is not particularly satisfying or intuitive. Since multiple (+) strands come from the (-) strand wouldn't it be more logical to frame this argument in the context of a single (-) strand and multiple nascent (+) strands?Other issues that should be addressed:Introduction• "Understanding and potential antiviral manipulation would be greatly assisted[…]" – clumsy sentence.• It would be valuable to provide a brief discussion of structural information on Protein A that would be relevant and helpful to readers who are not familiar with Nodavirus research.Results• • Subsection “Spherule replication compartment volume correlates with RNA template length”, first sentence is missing a period.Discussion• The composition of the crown is not adequately discussed. It is clear that Protein A is involved but does it make up the crown or not? Is it possible that a host protein is co-opted? Are there any predictions about the structure of protein A that would allow fitting the structure into the crown? From the data it appears that the crown is likely to be a motor akin to the phage packaging motors, which was indeed mentioned, but a possible role in assembly was never discussed. Significant issues that must be addressed:1) The paper is generally straightforward and well written, but details of the reconstruction methodology and statistics are sparse .In the Results subsection “FHV spherule necks are crowned by cupped structures”, second paragraph, we have added further description of the reconstruction methodology and statistics. This added section refers to new panel G in what is now Most importantly, the assumption of 12-fold symmetry is not sufficiently justified – for example by providing a power spectrum showing a clear peak corresponding to 12-fold symmetry. There is no reason to think this assumption is incorrect, but the symmetry and structure of the crown are key new features and the experimental underpinnings should therefore be presented in detail.The power spectrum in question is now shown in new panel H of 2) Interpretations of spherule size and content are an important component of this paper, but require clarification. There are several issues:A) Please explain exactly what is meant by the sentence: "Consistent with estimates of the persistence length of dsRNA" – didn't understand this sentence.At the end of the subsection “Coiled interior filaments fill FHV RNA replication vesicles”, we have provided further information and explanation to clarify that the presence of relatively straight, continuous segments of at least 40 nm is consistent with the prior estimate of dsRNA persistence length, i.e. the length of dsRNA required to show significant bending, as ~60- 64 nm.B) In the relevant Results section, we have added a new paragraph  noting that the breadth of spherule sizes between those assignable to RNA1 and RNA3 appears greater than expected for a single peak associated with RNA2. We also note that potential contributions to this complexity could include the dependence of RNA2 replication on trans-activation by RNA3, which could lead to some spherules containing both RNA2 and RNA3, and the possibility, noted in reviewer comment 2C immediately below, that spherule sizes might vary dynamically through ongoing RNA synthesis and export. Since further comments on these points necessarily involve alternatives that cannot be resolved from present data, readers then are referred to the Discussion, where a fuller consideration of factors possibly contributing to the breadth of spherule sizes has been added .C) The interpretation of Please see the response to comment 2B immediately above. As indicated there, the possibility that spherule sizes may vary dynamically is now noted in the subsection “Spherule replication compartment volume correlates with RNA template length”, third paragraph and discussed further in the subsection “Relationships among spherule shape, filamentous contents, size distributions, and RNA templates”, third paragraph.D) Please clarify further the calculations between template length and spherule volume were derived and what relationship they have to cytoplasmic polyhydrosis virus (CPV) dsRNA. Perhaps it would be useful to show In the last paragraph of the subsection “Spherule replication compartment volume correlates with RNA template length”, we have more explicitly explained that we used published measurements of the diameter and packaging density of cytoplasmic polyhydrosis virus (CPV) dsRNA in dsRNA virus virion particles as a reference to calculate packaging density of FHV dsRNA in spherules. We appreciate the suggestion of a table comparing the CPV and FHV compartment volumes, but after testing some alternatives concluded that the presentation was simpler and more straightforward without this addition.E) Postulating that 1.5 – 2.5 copies of dsRNA1 fit inside each spherule is not particularly satisfying or intuitive. Since multiple (+) strands come from the (-) strand wouldn't it be more logical to frame this argument in the context of a single (-) strand and multiple nascent (+) strands?We agree that discussing spherule volumes in units of fractional dsRNA equivalents is not mechanistically well-founded and would only confuse readers. The relevant section in the last paragraph of the subsection “Spherule replication compartment volume correlates with RNA template length”, has been replaced with more meaningful comments noting that (i) these calculations show that even the smallest spherules associated with RNA1 and RNA3  are largOther issues that should be addressed:Introduction• "Understanding and potential antiviral manipulation would be greatly assisted[…]" – clumsy sentence.The sentence in question was not essential and was simply deleted.• It would be valuable to provide a brief discussion of structural information on Protein A that would be relevant and helpful to readers who are not familiar with Nodavirus research.To provide such introduction to protein A and nodavirus biology, we added a new Results• As requested, we have added arrowheads in what is now B – D). Scale bars would allow a better idea of what magnification and scale is being shown.As suggested, we have added scale bars to what is now E) Unclear when referring to The plot showing the distribution of inter-filament distance in what was • Subsection “Spherule replication compartment volume correlates with RNA template length”, first sentence is missing a period.We have added the missing period.Discussion• The composition of the crown is not adequately discussed. It is clear that Protein A is involved but does it make up the crown or not? Is it possible that a host protein is co-opted? Are there any predictions about the structure of protein A that would allow fitting the structure into the crown?While the composition of the crown is a question of great importance, no further information is available at present. To address this, in the first paragraph of the subsection “Multimeric FHV protein A crowns the cytoplasmic side of spherule apertures”, we have further clarified how the immunogold labeling and additional results are consistent with protein A being a major component of the crown. We also added sentences noting that while protein volume calculations show that crowns are sufficiently large to accommodate 12 protein A molecules, no structural information is available for protein A to a level that would permit meaningful modeling into the crown, and that not only host proteins but additional viral proteins might be recruited into the crown with protein A.From the data it appears that the crown is likely to be a motor akin to the phage packaging motors, which was indeed mentioned, but a possible role in assembly was never discussed.The potential molecular motor-like role of the FHV crowns in driving RNA translocation into growing spherules, via the force of RNA polymerization, is discussed in the context of the proposed model for FHV replication complex assembly in the first paragraph of the subsection “New model for spherule formation and function”.


Peg-IFNα and peg-IFNλ reduced HDV viremia  and serum HBsAg levels . Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA  Nucleosid(t)e analogs , which potently inhibit the HBV reverse transcriptase and are approved antiviral drugs for the treatment of chronic HBV infection, do not show beneficial effects in chronically HBV/HDV co-infected patients2. To date, no HDV-specific therapy is available or has been approved3. Thus, treatment options for chronically HBV/HDV co-infected patients are limited to interferons and pegylated interferon alpha (peg-IFNα) is the only currently approved treatment. Unfortunately, therapy outcomes with peg-IFNα (after 48 weeks of treatment) are unsatisfactory, as evidenced by the low rates of sustained virological responses, HDV relapses after treatment cessation4 and the occurrence of complicating side effects. A few clinical studies suggest that prolonging the IFN treatment to several years might lead to higher response rates5. Although peg-IFNα is able to reduce HDV viremia during treatment, knowledge about its antiviral effects or those of other potential therapeutic cytokines on HDV in infected cells is still scant.Around 15 million people worldwide are chronically infected with hepatitis Delta virus (HDV). Persistent co-infection with hepatitis B virus (HBV) and HDV leads to the most severe form of viral hepatitis, which is associated with an accelerated course of liver fibrosis and cirrhosis6 and leads to the accumulation of three different HDV RNAs: the genomic RNA, antigenomic RNA and mRNA7. The antigenomic HDV RNA is the central template for the transcription of full length genomic RNA and the hepatitis delta antigen (HDAg) mRNA. Newly produced virions consist of genomic HDV RNA, which is associated with HDAg proteins and surrounded by surface antigens of the hepatitis B virus (HBsAg). The HDAg proteins exist in two different isoforms: a 24 kDa small isoform, which is needed for replication, and the 27 kDa isoform generated as a consequence of an adenosine deaminase acting on RNA (ADAR)-mediated RNA editing event, which inhibits HDV RNA replication and drives viral assembly9. The balance between genomic and antigenomic RNA appears to be crucial to allow persistence of HDV infection and is highly regulated by the two forms of HDAg, as well as through epigenetic modifications11.HDV is a circular, negative, single-stranded RNA virus with a genome of 1,679 nucleotides in length. HDV replication occurs in the nucleus of hepatocytes in a double rolling circle processDue to the limited availability of experimental HBV/HDV infection models, knowledge about the behavior and stability of genomic and antigenomic RNAs in HBV/HDV co-infection and upon therapy is limited, although it may be determined to improve current treatment regiments.12, on HDV replication in vivo, by using human liver chimeric mice13 and focussing on intrahepatic changes of the different HDV infection markers. The capacity of the two therapeutic cytokines to reduce HDV was compared to treatment with the nucleotide analogs entecavir (ETV). In particular, we investigated the effects of the different treatments in HBV/HDV co-infected human hepatocytes by establishing a new magnetic beads-based qRT-PCR assay enabling specific quantification of both genomic and antigenomic HDV RNA forms in chimeric mice and patient liver samples, as well as a RNA in situ hybridization technique allowing the analysis of HDV RNA expression at single cell level.The aim of this study was to investigate the impact of both pegylated interferon alpha and lambda, which were reported to have similar antiviral effects in chronic HBV infected patientsStably HBV/HDV co-infected human chimeric UPA/SCID/beige (USB) mice displaying comparable viremia levels received peg-IFNα , peg-IFNλ  or ETV  for four weeks or remained untreated as controls (n = 3 for each treatment group). Figure In line with virological parameters, immunofluorescence staining of liver specimens from mice sacrificed at the end of the experiment revealed that the amount of HDAg-positive human hepatocytes was clearly reduced by the interferon-based treatments but not in ETV-treated mice. 60.2% (range = 51.0–76.4%) of human hepatocytes appeared HDAg-positive in control animals Fig. , while o14. After reverse transcription, biotinylated cDNA was purified via magnetic beads exclusively binding the biotinylated cDNA products which were then quantified by qRT-PCR14. For the strand-specific quantification of genomic and antigenomic HDV RNAs, artificial full-length genomic and antigenomic HDV RNA standards were generated as described in Supplementary Material and Methods. The standard curves of genomic HDV RNA showed a slope of −3.351 and 98.8% efficiency  a median of 0.097 genomic HDV RNA and 0.004 antigenomic HDV RNA relative to human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) were detected  to visualize both antigenomic HDV RNA and HDV mRNA replicative intermediates in liver specimens at a single cell level  and peg-IFNλ  treated mice compared to uninfected  or ETV treated animals , suggesting that both IFNs are able to clear HDV in a proportion of infected hepatocytes.To evaluate changes of intrahepatic HDV replication induced by the different antiviral compounds, treated and untreated human chimeric USB mice were sacrificed at the end of treatment and genomic and antigenomic HDV RNA levels were analyzed. Figure vel Fig. . Interesvel Fig.  and pegFNλ Fig.  treatedted Fig.  or ETV als Fig. , sugges15, total HBV RNA levels remained unchanged in ETV-treated mice.Total HBV RNA levels relative to hGAPDH appeared reduced 15.0-fold with peg-IFNα and 8.6-fold upon treatment with peg-IFNλ in the setting of HBV/HDV co-infection , hMxA (0.6x), hISG15 (1.2x) and hCXCL10 (1.1x) when compared to untreated mice  , human myxovirus resistance A (hMxA) , human interferon stimulated gene 15 (hISG15)  and human C-X-C motif chemokine 10 (hCXCL10)  Fig. . As previn vivo by employing HBV/HDV co-infected human liver chimeric mice. Moreover, we established and validated innovative magnetic beads-based qRT-PCR and RNA in situ hybridization assays to specifically quantify and visualize the different HDV RNA forms at single cell level in infected livers. We show that treatment of mice lacking adaptive immune responses with either peg-IFNα or peg-IFNλ for four weeks significantly lowered HDV viremia and amounts of HDAg-positive human hepatocytes. Previous reports have shown that IFNα and - to some extent - also IFNλ can mediate HBV suppression in infected cells by acting through different mechanisms18. However, to our knowledge this is the first study demonstrating that both peg-IFNα and peg-IFNλ can exert potent serological and intrahepatic anti-HDV effects in vivo, without the involvement of adaptive immune responses. It should also be noted that due to impaired lymphoid development, the activity of important innate components, like natural killer (NK) cells, are also strongly reduced in these humanized mice. Clinical observations indicated that peg-IFNλ administration provoked fewer side-effects than peg-IFNα19 and might therefore represent an attractive therapeutic alternative to peg-IFNα in chronic HDV infection, especially if prolonged regimens extending treatment duration beyond 48 weeks shall be employed in the attempt to increase response rates5. While peg-IFNλ is already used in clinical development for chronically HDV infected patients , this study underlines the importance to investigate the intrahepatic activity of different therapeutics that are emerging from preclinical development. Four-week administration of ETV strongly reduced HBV viremia and intrahepatic HBV DNA levels, but had no impact on circulating HBsAg, HDV viremia and intrahepatic HDV loads. These results are in line with data from clinical trials showing that 1-year treatment with peg-IFN-α4 but not with nucleoside analogs, like ETV20, reduced HDV viremia in infected patients20.The knowledge of the intrahepatic virological changes induced by different antiviral treatments in HBV/HDV co-infected patients is still limited. Both the availability of robust experimental models able to recapitulate the entire life cycle of HBV and HDV in infected hepatocytes and the establishment of sensitive molecular assays are necessary to gain insights about the impact of distinct drugs on HDV replication. In this study, we comparatively analyzed the antiviral effects of peg-IFNα, peg-IFNλ and ETV 6 and leads to the accumulation of three different HDV RNAs: the genomic RNA, antigenomic RNA and mRNA7. To dissect the antiviral effects of these three different compounds on intrahepatic HDV replication, we established a novel magnetic beads-based qRT-PCR based assay allowing the specific determination of genomic and antigenomic HDV RNAs. In the past, the differentiation between the genomic and antigenomic HDV RNA forms was attainable only by northern blot21. However, northern blot analyses are time-consuming, have lower sensitivity and are only semi-quantitative. In 2008, Tseng et al. proposed to quantify genomic and antigenomic HDV RNA by performing reverse transcription with nucleotide tagged HDV strand-specific primers followed by RT-PCR22. However, such qRT-PCR procedures may also generate false-positive PCR products since genomic and antigenomic HDV RNA are exact complements of each other and therefore may lead to self-priming and self-annealing. Our novel BMB qRT-PCR assay includes an additional purification step based on the use of magnetic beads, which is essential to circumvent self-priming and self-annealing of genomic and antigenomic HDV RNA. Moreover, the sequence of the HDV-specific primers and probes used in the BMB assay have been shown to detect all HDV genotypes14, thus making the assay useful for clinical trials or cohort studies.Active HDV replication occurs in the nucleus of hepatocytes using a double rolling circle process21. Proof of concept analyses performed in liver biopsies derived from chronically HBV/HDV infected patients using this BMB qRT-PCR assay indicated that genomic HDV RNAs were 3 to 41-fold more abundant than the amounts of antigenomic HDV RNA. Notably, these ranges are comparable to ratios obtained in livers of humanized USB mice (8 to 73 more abundance of genomic to antigenomic HDV RNA) and are also in line with previous studies detecting ratios of genomic to antigenomic HDV RNA in woodchucks and chimpanzees (5–22:1) by northern blot analysis21. Furthermore, this new BMB qRT-PCR assay is able to specifically detect genomic to antigenomic HDV RNAs up to a ratio of 1:100. This range appears sufficient to cover the ratios existing in hepatocytes of humans, woodchucks and chimpanzees.To our knowledge, quantitative estimations of genomic and antigenomic HDV RNA in the liver of patients infected with HDV have not been available and could only be estimated in woodchucks and chimpanzeesin situ hybridization assay (RNAScope), which permitted specific visualization of the HDV replicative antigenomic intermediates at single cell level. It is worth noting that in interferon-treated animals a large proportion of the human hepatocytes appeared both HDAg- and HDV RNA- negative, thus suggesting that HDV infection could be purged at least in a proportion of human hepatocytes after four weeks of peg-IFN treatment.In this study, we observed that both IFNs, but not ETV, led to a clear and comparable decrease of both genomic and antigenomic HDV RNA levels, while ratios of genomic to antigenomic HDV RNA were maintained in all treated and control groups. The strong decrease of both HDV RNA forms could also be confirmed by northern blot and by applying a new highly sensitive RNA in vitro23 and another study even indicated that HDV can hinder IFN-induced nuclear STAT1 translocation24. Accordingly, we previously showed that HBV can limit nuclear STAT1 translocation and enhancement of human ISGs upon treatment with regular IFNα in vivo25. However, prolonged therapy with higher doses of peg-IFNα was able to break such restriction in HBV-infected humanized mice15. Therefore, it is plausible that four-week treatment of HBV/HDV co-infected mice with peg-IFNs could also overcome the observed blockades of IFN signaling, since both peg-IFNs led to a similar and clear induction of human genes, which are involved in innate immune signaling (e.g. classic interferon stimulated genes (ISGs) and chemokines), providing evidence that ISG induction and antiviral activity could be elicited even in the absence of an adaptive immune system15. Previous cell culture analyses already suggested that signaling through either the IFNα or IFNλ receptor complex results in the activation of the same janus kinase (JAK)-STAT signal transduction cascade26, and accordingly, the RNA expression of human STAT1 was similarly induced in human hepatocytes of mice that were either treated with peg-IFNα or peg-IFNλ, but not with ETV. Consequently, the biological activities induced by either type I or type III IFNs seem to be similar26. However, the molecular mechanisms of IFN-induced HDV suppression in vivo are still unclear and needs to be addressed in further studies.Interestingly, short-term treatment with IFNα had no antiviral effect on HDV replication 4, our study suggests that peg-IFN-based therapies may still play a supportive role in combination with new therapeutic approaches involving, for instance, HDV entry27, assembly28 or HBsAg release29 inhibitors. Finally, this study underlines the importance of investigating the intrahepatic effects provoked by new treatment approaches and provides new molecular tools enabling the quantification of genomic and antigenomic HDV RNA contents in future preclinical and clinical trials.In conclusion, this study demonstrates that the antigenomic HDV RNA, which acts as template of viral replication, could be significantly reduced by peg-IFN treatment. Although the rates of sustained virological responses (SVR) in chronic HDV infected patients treated with peg-IFNα are low and viral relapse is observed in a high percentage of patients13. Primary human hepatocytes were isolated from rejected explant livers using protocols approved by the Ethical Committee of the city and state of Hamburg (OB-042/06) and accorded to the principles of the Declaration of Helsinki. Mice were maintained under specific pathogen free conditions in accordance with institutional guidelines under approved protocols. Repopulation rates were estimated by human serum albumin (HSA) concentrations in mouse sera  and confirmed at sacrifice by determining human cell contents by histology and qRT-PCR using the beta-globin gene kit 13. Animals displaying high levels of human chimerism (>2 mg/ml HSA in serum) were used for the study. To establish an HBV/HDV co-infection, human chimeric USB mice received a single peritoneal injection of 10 µl HBV- and HDV-positive, patient derived serum . The inoculum corresponded to a MOI of approximately 0.2 for both viruses, by estimating an average of 3 × 107 human hepatocytes per mouse liver30. Blood samples were taken every other week as indicated in the results. Mice were sacrificed at the end of treatment. Liver specimens removed at sacrifice were cryo-conserved in chilled isopentane for further histological and molecular analyses. All animal experiments were conducted in accordance with the European Communities Council Directive (86/609/EEC) and were approved by the City of Hamburg, Germany.Human liver chimeric USB (urokinase-type plasminogen activator (uPA)/severe combined immunodeficiency (SCID)/beige) mice were generated by transplanting one million thawed cryo-preserved human hepatocytes obtained from two different human donors (both with intermediate interferon responsive C/T IL28 locus) into homozygous USB mice, as previously reported15. In humans, 180 µg of either peg-IFNα or peg-IFNλ are used for a single injection, which would be an equivalent of 50ng/20 g mouse. According to commonly used dose scaling to adjust human doses to mouse equivalent doses and in line with previous reports17 we used 500ng/20 g mouse (25ng/g body weight) in this study. For ETV treatment, mice received drinking water supplemented with 1 μg/ml Baraclude Solution 15. Control mice were infected in parallel, but left untreated (n = 3 for each treatment arm).Stably HBV/HDV co-infected (>10 weeks post virus inoculation) human-chimeric USB mice were either treated with peg-IFNα  (n = 3), peg-IFNλ  (n = 3) or with ETV (n = 4) for four weeks. Uninfected mice were treated once with peg-IFNα or peg-IFNλ. All animals (four-week dose or single dose) were sacrificed 24 hours after their last interferon injection for intrahepatic analyses. The IFNs were injected subcutaneously twice a week (each 25 ng/g body weight)Cryo-preserved human liver biopsies were obtained from three chronically HBV/HDV-infected patients. The biological samples were obtained for diagnostic purpose. The surplus material and patients characteristics were used in accordance to the principles of the Declaration of Helsinki, after informed consent of patients and protocols were approved by the ethical committee of the city and state of Hamburg (PV4081).14 on a ABI Viia7 . In detail, RNA extracted from 5 μl mouse serum or 1 μl liver derived RNA were denatured at 95 °C for 10 min, immediately cooled down to −20 °C and reverse transcribed at 50 °C for 5 min. After inactivation of the reverse transcriptase at 95 °C for 20 s, amplification was performed under the following conditions: initial step 95 °C 20 s, 40 cycles at 95 °C for 3 s and 60 °C for 30 s. HBV viremia was determined as reported30. Known amounts of HBV or HDV containing plasmids were used as standards for quantification and hGAPDH for expression normalization21. HBsAg quantification was performed using the Architect HBsAg assay .Viral DNA and RNA were extracted from serum samples using the QiAmp MinElute Virus Spin kit  and from liver tissues using the RNeasy RNA purification kit . HDV viremia and intrahepatic HDV RNA levels were determined by reverse transcription and qRT-PCR using the ABI Fast 1-Step Virus Master , HDV specific primers and probes14 and immediately cooled down to −20 °C. After addition of a ABI Fast 1-Step Virus Master  to a final volume of 20 µl, reverse transcription was performed at 50 °C for 5 min on an ABI Viia7  and enzymes were inactivated at 95 °C for 20 s. Biotinylated cDNA was purified with the MinElute PCR Purification Kit  and isolated with dynabeads specifically interacting with biotin  following the manufacturer’s instructions. In brief, 5 μl dynabeads were washed and re-suspended in Binding Buffer, incubated with 20 μl biotinylated cDNA for 3 hours, then washed twice with Washing Buffer and once with sterile, RNase free water. For qRT-PCR 1 μl of purified biotinylated cDNA bound to dynabeads, HDV specific primers and probes14 and an ABI Fast Advanced Master  were used under the following conditions: Initial step 95 °C 20 s; 40 cycles at 95 °C for 3 s and 60 °C for 30 s. hGAPDH  was used for normalization of intrahepatic HDV RNAs. The BMB assay was established and validated using a genomic and antigenomic HDV RNA standard .RNA extracted from 10 μl mouse serum or 1 μg liver derived RNA was denatured at 95 °C for 10 min with 0.5 μM of a biotinylated HDV specific forward primer (biotin-GCGCCGGCYGGGCAAC) for genomic HDV RNA quantification or a biotinylated HDV specific reverse primer (biotin-TTCCTCTTCGGGTCGGCATG) for antigenomic HDV RNA quantification31. Radioactive probes were prepared by using a random priming protocol utilizing full-length HDV cDNA or hGAPDH cDNA templates and deoxycytidine triphosphate labeled on the alpha phosphate group with 32 P.Total RNA was extracted from liver tissue specimens with the TRIzol reagent  by following the manufacturer’s instructions. Extracted RNA was further purified using the RNeasy spin columns . RNA concentration was measured using an ND-1000 spectrophotometer  at 260 nm. RNA quality and quantity were monitored by ethidium bromide staining and by UV absorbance. Northern blot analysis was performed following standard procedures as previously described(32 P α-dCTP) . In particular, HDV cDNA templates specific for genomic and antigenomic HDV RNAs were obtained by the use of Transcriptor First strand cDNA synthesis  and HDV specific primers. Monomers of genomic and antigenomic HDV RNAs were transcribed from pHDVII- a pGEM-T easy vector  with dual-opposed promoters containing a 980 bp HDV fragment (from nt 305 to nt 1285)- with T7 and SP6 MEGAscript kit  after linearization by HindII and XbaI digestion, respectively. Densitometric quantitation of signal bands for genomic (1.7 kb) and antigenomic HDV RNA (1.7 kb) from untreated and treated mice was performed by Quantity One 1-D Analysis Software ; data are from 3 independent experiments and values are expressed as mean ± SD.13. Briefly, sections were fixed with acetone and incubated with mouse anti-CK18  and human anti-Delta . Specific signals were visualized with Alexa 488- or 546-labeled secondary antibodies . Nuclear staining was achieved by Hoechst 33258 . Stained sections were then mounted with fluorescent mounting media  and analyzed with the fluorescence microscope BZ9000  using the same settings for the different experimental groups. The percentages of HDAg-positive human hepatocytes were estimated as previously described13 and by using 2 visual fields (displaying approximately 500 human hepatocytes each) from 3 different liver sections per mouse.Cryostat sections of chimeric mouse livers were stained as previously describedin situ hybridization was performed on paraformaldehyde-fixed, cryo-preserved liver sections using the RNAScope Fluorescent Multiplex Kit  according to the manufacturer’s instructions32. Briefly, liver sections were dehydrated and pretreated with Pretreat 4  for 30 min. Samples were incubated with specific target probes which specifically bind both antigenomic HDV RNA and HDV mRNA  for 2 h at 40 °C . DAPI staining was performed to visualize nuclei. Stained sections were analyzed by fluorescence microscopy  using a 60 × /1.40 NA oil objective. Merged z stack images were prepared using the same settings for all groups.RNA To determine expression levels of genes related to IFN signaling (ISGs and cytokines) in human hepatocytes repopulating the mouse liver, primers specifically recognizing human transcripts and not cross-reacting with murine genes were used . Analyzed were hSTAT1 (assay ID: Hs01013989_m1), hMxA (Hs00895608_m1), hISG15 (Hs00192713_m1) and hIP10/hCXCL10 (Hs00171042_m1). For determining the expression of ISGs and cytokines, RNA was extracted as described above, cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis Kit  using oligo-dT primer according to the manufacturer’s instructions and qRT-PCR was performed with the ABI Fast Advanced Master  in an ABI Viia7 . Expression levels were calculated relative to the median of hGAPDH and human 60 S ribosomal protein L30 (hRPL30) and are depicted as log change from median values obtained from untreated control mice.Supplementary Material