In this protocol we provide insights into a small-scale and large-scale production technique of artificial circular RNA sponges relying on in vitro transcription and RNA ligation.Characterized by their covalently closed structure and thus an elevated stability compared to linear RNA molecules, circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Although the biological functions of naturally occurring circRNAs are largely unknown, they were reported to act as molecular sponges, sequestering microRNAs (miRNAs), resulting in a de-repression of target mRNAs. Taking these characteristics of naturally occurring circRNAs into account, artificial circRNAs could be a potential tool in molecular biology and medicine. Using the Hepatitis C virus (HCV) as a model system, this application of artificial circular RNAs was demonstrated. The virus requires cellular miRNA miR-122 for its life cycle, and circRNAs specifically engineered to efficiently sequester this miRNA impacted viral propagation. Since in this context the production of engineered circRNA remains the limiting factor, we present a method to produce and efficiently purify artificial circRNA sponges (ciRS)  Circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Naturally occurring circRNAs arise by an alternative splicing mechanism termed “backsplicing”  or “headin vivo”) by both a spliceosome-dependent exon circularization strategy [in vitro”) using recombinant phage RNA polymerase-mediated transcription and circularization by either employing genetically engineered autocatalytic group I introns  system) [The first evidence of the existence of circRNAs was reported in 1976, demonstrating plant viroid genomes are composed of single-stranded covalently closed RNA molecules ,9,10. Sistrategy ,13,14 anstrategy . Additio system) ,17,18 orAlthough the biological functions of endogenous circRNAs are still largely unknown, there are several lines of evidence showing that circRNAs have diverse important regulatory and developmental roles ,20,21,22in vitro and in vivo. When transfected into cells, these circRNAs are both present in the nucleus and the cytoplasm, and are more stable than their linear counterparts [Considering these functions and characteristics of naturally occurring circRNAs, artificial circRNAs could be a potential tool for molecular biology and medicine applied for the development of new strategies to combat human diseases such as viral infections or cancer. In this context, our laboratory utilized the Hepatitis C virus (HCV) as a model system in a proof of principle study. The single-stranded RNA genome of the hepatocyte-specific virus is bound by miR-122 at two distinct binding sites at its 5’-end (besides others), thereby protecting the viral RNA from exonucleolytic degradation and enhancing viral translation. Sequestration of miR-122 had already been shown to inhibit viral propagation in patients using the first locked nucleic acid (LNA)-containing antisense miRNA drug (Miravirsen). As a novel complementary approach, artificial circular RNA sponges (ciRS) produced as described in this protocol, were shown to efficiently bind and sequester the cellular miR-122 terparts .in vitro transcription and RNA ligation suitable for small and middle-sized RNAs. In this article, a small-scale as well as a large-scale production procedure will be described, typically yielding up to 20 µg and 350 µg of artificial circular RNA sponges, respectively.With the aim of targeting miRNAs misregulated within human diseases, artificial circular RNA sponges represent an efficiently functioning and inexpensive alternative to commercially available LNA-containing anti-miRNA drugs. Furthermore, circular RNA sponges facilitate functional analyses of cellular miRNAs by sequestration comparable to siRNA-based approaches to decrease the target mRNA level. However, since the production of engineered circRNA remains the limiting factor, this protocol provides insights into an efficient production technique of artificial circular RNA sponges relying on HiScribe T7 High Yield RNA Synthesis Kit Ribonuclease R RNaseOUT Recombinant Ribonuclease Inhibitor RQ1 RNase-free DNase T4 RNA Ligase Adenosine triphosphate Dithiothreitol Ethidium bromide solution 1% Guanosine 5′-monophosphate disodium salt hydrate Lipofectamine 2000 Transfection Reagent Polyethylenimine SYBR Gold Nucleic Acid Gel Stain w/v) in 1 × Tris-Borate-EDTA (TBE))Urea solution (50% urea (w/v))20% denaturing polyacrylamide solution (20% polyacrylamide (19:1 acrylamide/bisacrylamide) in 1× TBE containing 50% urea w/v), 0.05% xylene cyanol (w/v))Formamide loading buffer  Ensure used materials are RNase-free prior to use.Bunsen burnerCorning Costar Spin-X Plastic Centrifuge Tube Filters Eppendorf Tubes 5.0 mL Snap cap mini Quick Spin RNA Columns Phase Lock Gel Heavy 2 mL Tubes Pre-coated TLC plates CEL 400-10 UV254 Sterican Gr. 18, G 26 × 1""/ø 0,45 × 25 mm Design of microRNA binding sites. The initial design of the miRNA binding sites present in the circular RNA sponges, or the corresponding control sequences, is crucial to the procedure. Therefore, several features and limitations have to be considered. The goal is to design an array of miRNA binding sites that is specific for binding to the miRNA of interest, has sufficient thermodynamic stability to be able to compete with the endogenous miRNA targets, and is able to harbour the maximum number of binding sites without impairing in vitro circularization.Number of miRNA binding sites: The natural prototype of a circular miRNA sponge, ciRS-7/CDR1as, has ~70 miR-7 binding sites [in vitro ligation. Generally, shorter transcripts tend to circularize more efficiently compared to longer RNAs.ng sites ,13. Thisng sites  Another Binding site spacing: Besides the sequence composition and thus degree of complementarity to the target miRNA, the optimal spacing of miRNA binding sites is considered here. Based on analyses of natural miRNA:mRNA interactions , a spacBinding site types and specificity: The configuration of the miRNA binding site itself is also an important feature ( feature B. In seqBinding site repetitiveness for longer miRNA binding site arrays (OPTIONAL STEP): In case longer stretches of miRNA binding site arrays are desired, the perfectly repetitive units of the binding sites and spacer sequences . Note that seed mutant binding sites retain a certain non-canonical miRNA-binding activity [in vitro circularization efficiency.activity . OptimalTemplate generation as starting point for ciRS production in vitro. Conventionally, templates contain the following elements: a T7 promotor sequence for in vitro transcription, several binding sites complementary to the miRNA of interest, and optionally a double-stranded stem-loop sequence at the respective 5′- and 3′-termini to enhance circularization efficiency . The GMP-initiated in vitro transcription aims to obtain mainly 5′-monophosphorylated linear RNA transcripts which can be directly used for RNA circularization. Since all T7 polymerase-created in vitro transcripts start with three guanosines, a stochastic subset of RNAs will be initiated with GMP instead of guanosintriphosphate (GTP)  of DNA template, and 1/10 vol. of T7 RNA Polymerase Mix (10×). Adjust to final volume with RNase-free water:small scale: prepare 20 µL total volume of the transcription reactionlarge scale: prepare 100 µL total volume of the transcription reactionIncubate 2 h up to overnight at 37 °C.te (GTP) A. TherebDNase digestion to remove the template DNA , vortex, transfer into a Phase Lock Gel tube, and centrifuge 5 min, 20,000× g at room temperature. Transfer the aqueous upper phase into a new tube.large scale: Distribute RQ1-digested transcripts to two 2 mL tubes. For extraction, use RNase-free water to increase the reaction volume to 800 µL each. Mix each sample with 1 volume Phenol/Chloroform/Isoamylalcohol, vortex, transfer into Phase Lock Gel tubes, and centrifuge 5 min, 20,000× g at room temperature. Transfer each of the aqueous upper phases into a new tube.Ethanol or isopropanol precipitation to remove residualchloroform and salts. PAUSESTEP: Precipitate samples with 0.1 volumes 3 M NaAc (pH 6.5) and 2.5 volumes 100% ethanol or 0.7 volumes isopropanol at −20 °C for at least 30 min to overnight. Centrifuge at 4 °C for at least 20 min at 20,000× g. Wash the pellet with 70% ethanol and centrifuge again for 5 min, 20,000× g. Discard the supernatant completely, air dry the pellet briefly, and dissolve in 40 µL RNase-free water . OPTIONAL STEP: Mix 1% of the transcript with formamide gel-loading buffer and verify RNA integrity on denaturing polyacrylamide gel.Size exclusion chromatography to remove excess nucleoside triphosphates ; the sample volume increases to ~80 µL per column. Finally, measure the transcript concentration.osphates A. Apply Annealing of transcript ends. Within the annealing reaction, the double-stranded stem-loop (if included) is formed , thereby bringing 5′- and 3′-ends into close proximity, promoting an efficient circularization. This, together with an increased ligation reaction volume used in the next step, additionally favours intramolecular over intermolecular ligation reactions. OPTIONAL STEP: In this step, addition of equimolar amounts of a splint oligonucleotide can be useful if no stem-loop structure was included in the construct. Add 10× annealing buffer to a final concentration of 1× to purified transcripts and allow the annealing of the double-stranded stem-loop regions (or splint oligonucleotide) by incubating the samples for 2 min at 95 °C and decreasing the temperature by 1 °C/10 sec to 25 °C within a thermocycler.small scale: Add 0.99 volume RNase-free water, 0.28 volume 10× reaction buffer for T4 RNA ligase , and 40 U RNaseOUT  to annealed transcripts. The volume should be 202.5 µL. Incubate for 10 min at 37 °C. Then, add 0.02 volume 10 mM ATP , 0.15 volume DMSO , and 50 U T4 RNA ligase  for a total volume of 250 µL.large scale: Scale up the above reaction two-fold to a total volume of 500 µL. Incubate at 16 °C overnight. Considering the activity of the T4 RNA ligase, there are three possible scenarios: (i) the transcript is not ligated and remains a linear monomer, (ii) the transcripts are ligated in an intermolecular way, resulting in the formation of dimerized (or multimerized) linear transcripts or (iii) the transcript is ligated in an intramolecular way, forming a covalently closed circular RNA molecule . Note that rare larger multimeric circular RNA ligation products and RNase R-resistant linear RNA molecules remain if gel purification is omitted.Phenol/Chloroform/Isoamylalcohol extraction and ethanol or isopropanol precipitation are performed to ensure the purity of the reaction as previously described in  The RNA pellet is dissolved in 50 µL formamide gel-loading buffer .Polyacrylamide-urea gel electrophoresis for quality control and circRNA purification. While the mobility of linear RNAs within polyacrylamide-urea gels is proportional to an RNA standard, circular RNAs show an aberrant mobility. Due to their circular conformation, the migration of the latter within polyacrylamide-urea gels is much slower compared to their linear counterparts. This aberrant migration can be compared between polyacrylamide gels of different acrylamide content and allows distinguishing linear di- or multimers from circular RNA—detectable as a size shift. Generally, the polyacrylamide content of the gels is dependent on the circRNA size. Referring to a circRNA size of ~200 nt, we recommend using 6%  and 7%  denaturing polyacrylamide gels, whereas circRNAs with a size of ~350 nt can be identified within 5%  and 6% denaturing polyacrylamide gels. The optimal acrylamide concentrations should be determined experimentally for different RNA lengths. In order to analyse the circularization efficiency, 1% of the RNA transcript is loaded onto analytic denaturing polyacrylamide urea gels, followed by ethidium bromide  plate coated with cellulose using a 254 nm fluorescent indicator. Irradiation with UV light at 254 nm excites the plate coating and results in a green fluorescence. The RNA on the contrary absorbs the UV light at this wavelength, casting a shadow against the fluorescent background. Note that the lower detection limit of this method is around 500 ng. Cut the circular RNA band  from the gel using a clean razor blade. CRITICAL STEP—In order to increase the RNA purification efficiency, the excised polyacrylamide-urea gel fragments are to be crushed, as described in the following:Small scale: Take a 500 µL safe-lock microcentrifuge tube. The latter is pierced in the centre of the bottom. A thin  syringe needle is heated on a Bunsen burner flame to penetrate the plastic easily. In order to avoid injuries, we recommend extreme caution and suggest externally fixing the microcentrifuge tube in a tube stand within the course of piercing the tube, so no hands can be injured in the piercing process. Transfer the excised polyacrylamide-urea gel fragment into the pierced 500 µL safe-lock microcentrifuge tube and insert the latter into a fresh 1.5 mL Eppendorf tube. Centrifuge for 1 min at 20,000× g to crush the gel. PAUSE STEP: Gel fragments can be stored at −80 °C overnight. Add 400–800 µL 1× PK-buffer to the crushed gel and incubate on a rotating wheel either overnight at room temperature or at 50 °C for 1 h. Freezing  and thawing may increase yields. Use Costar Spin-X centrifuge tube filters to remove gel pieces by centrifuging for 1 min at 20,000× g.large scale: Take a 15 mL Falcon tube, which is cleaved at the 3 mL sign using either strong scissors or a heated blade. In order to avoid injuries, we again recommend extreme caution and suggest externally fixing the tube within the course of cutting the tube, so no hands can be injured in the slicing process. The upper part of the tube is discarded, whereas the bottom is subjected to the piercing procedure as described above for the 500 µL tube. Transfer the excised polyacrylamide-urea gel fragment into the pierced lower 15 mL Falcon tube part and insert the latter into a fresh 5 mL Eppendorf tube. Centrifuge for 1 min at 20,000× g to crush the polyacrylamide-urea gel. PAUSE STEP: Gel fragments can be stored at −80 °C overnight. Add 8 mL 1× PK-buffer to crushed gel, transfer this to the 15 mL Falcon tube, and rotate on a wheel either overnight at room temperature or at 50 °C for 1.5 h. Freezing with dry ice or liquid nitrogen and thawing in between may increase yields. Spin for 10 min at 20,000× g. Transfer the liquid into a fresh 15 mL tube, thereby preventing gel particle transfer.Phenol/Chloroform/Isoamylalcohol extraction and ethanol or isopropanol precipitation to purify and concentrate the produced circular RNAs.small scale: Follow the protocol described in in vitro-produced artificial circular RNA—is dissolved in an appropriate amount of RNase-free water, typically 20 µL.large scale: Precipitate 8 mL supernatant with 0.1 volume 3 M NaAc (pH 6.5) and 0.7 volume isopropanol overnight at −20 °C. During the RNA precipitation procedure, residual SDS from the PK-buffer containing supernatant may not be soluble at very low temperatures. Incubate the solution on a wheel at 4 °C for 30 min. Afterwards, spin for 30 min at 20,000× g. Wash the pellet with 5 mL of 70% ethanol, spin for 5 min at 20,000× g, and take up RNA in 400 µL RNase-free water. Use Costar Spin-X centrifuge tube filters to remove remaining gel fragments by centrifuging for 1 min at 20,000× g. Resulting artificial circular RNA sponges regularly yield up to 350 µg, when preparation is conducted according to the large-scale protocol. Measure the ciRS concentration. Assess the final circRNA quality, as mentioned previously, by loading 1% of the preparation onto denaturing polyacrylamide gels of various polyacrylamide contents, followed by ethidium bromide or SYBR Gold staining  treatment. Despite the differential mobilities of circular RNAs within polyacrylamide-urea gels of different acrylamide contents, circularity of the produced sponges should also be proven by RNase R treatment. Incubate ciRS with 1 U/µg RNase R, 10 × RNase R Rxn Buffer to a final concentration of 1× and 40 U RNaseOUT, add to 40 µL with RNase-free water, and incubate for 20 min at 37 °C. Phenol/Chloroform/Isoamylalcohol extraction and ethanol or isopropanol precipitation are performed to purify the RNase R-treated circular RNAs. Assess circularity by loading 100 ng of the RNA onto a polyacrylamide-urea gel (urea gel .Transfection of circRNA sponges in cell culture.OPTIONAL STEP: CiRS can be transfected into any eukaryotic cell culture-based system. Typically, transfection is performed using lipid- or synthetic-nanoparticles such as Lipofectamine 2000 or polyethylenimine formulations as used for siRNA transfections following the manufacturer’s instructions. Additionally, electroporation of circRNA sponges is possible. This procedure should use established protocols for the model system used.


Pseudomonas aeruginosa, consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93–99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of  During the last decade, RNA sequencing (RNA-Seq) has increasingly become the method of choice in transcriptome studies. It replaced conventional methods such as microarrays to quantify gene-expression levels more accurately in both eukaryotic and prokaryotic organisms. RNA-seq has benefitted from decreasing sequencing costs . HoweverIn eukaryotes, polyadenylation of the mature mRNA facilitates mRNA enrichment, e.g. by the use of bead pulldown approaches . In bactPseudomonas aeruginosa up to 25 % of the reads can map to tmRNA as found in transcriptome data recorded earlier by our group and in other studies  [Escherichia coli [Commercially available kits, e.g. Ribo-Zero (Illumina), which capture hybridized probes by the use of magnetic beads, are known to efficiently reduce the rRNA fraction down below 1 % in a wide range of bacterial species . Howeverarticle) . First iia coli , this smia coli . Anotheria coli  and a brP. aeruginosa-specific DNA probes, which allowed the efficient removal of tmRNA fragments in addition to the depletion of P. aeruginosa 16S and 23S rRNA. Our protocol outperformed the commercially available Ribo-Zero (Illumina) and Ribo-off kit (Vazyme) with a significant increase in the fraction of P. aeruginosa mRNA-mapped reads to more than 95 % of the overall mapped reads.Here we describe an RNase-H-digestion-based, organism-specific and easily customizable method for removal of overrepresented RNA species that goes beyond rRNA removal. We designed P. aeruginosa strain PA14 and incubated overnight in lysogenic broth (LB) at 37 °C while shaking at 180 r.p.m. Subsequent main cultures were inoculated at an OD600 of 0.05 in LB and grown at 37 °C while shaking at 180 r.p.m. until they reached OD600=2.0±0.1  or for 12 h (stationary phase).Planktonic cultures were inoculated with single colonies of Overall, 1 ml of the main culture was mixed with 1 ml RNAprotect Cell Reagent  and incubated at room temperature (RT) for 10 min. After a centrifugation at 8000 r.p.m. at RT for 15 min, the supernatant was discarded and samples were stored at −80 °C until use.−1 lysozyme were added to the cells and mixed thoroughly by vortexing for 30 s. After an incubation for 10 min at RT, intermitted by occasional short vortexing, 350 µl of RLT buffer  containing 1 % mercaptoethanol were added to the sample. The mixture was inverted several times and incubated for 1 h at −80 °C. Samples were then thawed on ice and loaded into a QIAshredder column , followed by centrifugation for 2 min at 14 000 r.p.m. RNA was extracted from the supernatant with the RNeasy Mini Kit  according to the manufacturer’s instructions. Subsequently, remaining DNA was removed from the extracted RNA samples by treatment with the DNA-free DNA Removal kit  according to the manufacturer’s protocol.For cell lysis, thawed samples were centrifuged for 5 min at 8000 r.p.m. and the supernatant was removed. Then, 100 µl of TE buffer containing 800 µg mlP. aeruginosa PAO1 were downloaded from pseudomonas.com (Tables S1 and S2). Sequence differences between the rRNA genes of the P. aeruginosa reference strain PAO1 and PA14 were checked and considered negligible.Sequences of 16S rRNA (NC_002516.2:c5269259-5267724), 23S rRNA (NC_002516.2:c6042736-6039846) and tmRNA (NC_002516.2:c901520-901872) genes of The reverse complement sequences of the respective gene sequences were segmented into fragments with a size of 50±5 bp. DNA nucleotides were designed to exhibit similar binding affinities to the target RNA, so that they exhibited homogenous melting temperatures  rather blast analysis [Check for off-target hybridization was carried out by analysis  and showet al. [2) were added and the digestion was performed in a final volume of 10 µl for 30 min at 37 °C. RNAClean XP magnetic beads  were used in a 1 : 1 ratio to purify the RNA and 15 µl of the eluate were incubated with 4 U rDNase I in 1× DNAse I buffer from the DNA-free kit  with a final volume of 20 µl for 30 min at 37 °C. DNase reaction mix was purified with 1× RNAClean XP magnetic beads obtaining 17 µl eluate. Fragment size distribution was assessed by the use of the Bioanalyzer  and RNA concentration was determined by photometric measurement .The protocol for hybridization of the designed oligonucleotides to their target RNAs and the subsequent cleavage of the hybridized RNA by the RNase H was adapted from Adiconis et al. . In brieFor the comparison of the depletion efficiency of the mRNA enrichment protocol described above, the commercially available Ribo-off rRNA Depletion Kit (Bacteria)  was used. Total RNA samples were treated for rRNA depletion as indicated by the manufacturer’s instructions.Sequencing libraries of the depleted RNA samples were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit  accordingly to the manufacturer’s instructions and then sequenced with an Illumina NovaSeq 6000 Sequencing System .https://github.com/s-andrews/FastQC). For removal of potentially remaining adapter reads and clipping of the reads, Fastq-Mcf  was used. Mapping was carried out by bowtie2 [https://github.com/BenLangmead/bowtie2) based on genome data retrieved from NCBI (CP000438.1) and featureCounts [http://subread.sourceforge.net/) was used to determine the number of sequencing reads that can be assigned to each genomic feature. The calculation of the read fractions for the different RNA species, the correlation analysis and the generation of the corresponding figures were performed by a custom R script.A first evaluation of the sequencing data quality was performed by using FastQC , both of which are based on an RNase-H digestion of RNA–DNA hybrids. Therefore, total RNA from P. aeruginosa cells harvested in the exponential and stationary growth phase was prepared in two biological replicates. The two independent depletion methods were applied on the same RNA samples in two technical replicates. Both protocols were stable and high correlation of gene-expression profiles across biological and technical replicates were achieved  among all mapped reads across all samples was below 1 % after following our custom-made depletion protocol. In contrast, 4–7 % ncRNAs were found in the RNA samples, in which the Ribo-off depletion protocol was applied . Furthermore, the transfer RNA (tRNA) fraction was larger in the Ribo-off-treated samples (0.2–0.3 %) than in the samples treated with our protocol (0.09–0.12 %).In order to rule out that biases in gene-expression profiles are introduced by the application of the specific depletion protocols, we determined the reads per kilo base per million mapped reads (RPKM) for each gene and compared the values of our method to those of the Ribo-off kit. No systematic divergences in the abundance of reads mapping to coding sequences (CDS) could be observed . Furthermore, blast analysis revealed no off-target hybridization of any of the designed probes to the rnpB RNA.As expected, the detected abundance of rRNA and tmRNA was higher in all samples treated with the Ribo-off kit. Furthermore, transcript levels of the tRNA genes were higher. As we did not explicitly target the tRNAs with our DNA probe design, the more stringent exclusion of small RNAs with an extensive secondary structure could be the reason for their lower abundance in our custom-made protocol treated samples. In addition, more P. aeruginosa RNA. As a result, the fraction of mRNA increased to >93 %. No overall bias was introduced and the gene-expression profile compared well to that of the mRNA-profile of the Ribo-off-treated RNA controls.We have established an efficient protocol for a highly specific depletion of non-mRNA such as 16S and 23S rRNA and tmRNA species from preparations of total rnpB does not lead to a drawback of the depletion protocol.We found that depletion of RNAs with a shorter length and a more extensive secondary structure such as tRNAs and other selected ncRNAs was stronger, although those RNAs were not explicitly targeted by our established method. We assume that these RNAs were depleted during the purification steps as short length and distinct 3D structure presumably impair their binding to the surface of the magnetic beads used for purification. Given that as a consequence the relative sequence information of the mRNA fragments increase, the stronger depletion of tRNAs and P. aeruginosa RNA but also to target non-mRNAs from other bacterial species. Thus, the newly established mRNA-enrichment protocol is particularly suited for high-throughput transcriptome analyses with RNA samples from the same species and represents a cost-efficient alternative. Its use can boost the elucidating power of studies examining the transcriptomic landscape of pathogenic bacteria and, therefore, allows deeper insights into the process of bacterial pathogenesis, e.g. in host-pathogen transcriptome experiments .The easily expendable spectrum of target sequences allows not only to deplete any additional Click here for additional data file.Click here for additional data file.


Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction, the FSE-arch, that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses and will aid future efforts to develop antiviral strategies.The   •Comprehensive RNA-RNA networks of the SARS-CoV-2 genome and subgenomes inside cells•Long-range structures spanning thousands of bases resulting in dynamic topologies•Multiple site-specific interactions between host and virus RNAs•An arch around the ribosomal frameshifting element is under purifying selection Coronaviruses use RNA structure to regulate their function. Ziv et al. identified maps of RNA-RNA interactions along the SARS-CoV-2 genome and subgenomes inside cells, revealing long-range base-pairing between distal elements, alternative co-existing RNA topologies, and interactions between virus and host RNA and providing insights into the coronavirus modes of action. Coronaviridae), namely the severe acute respiratory syndrome coronavirus (SARS-CoV), the Middle East respiratory syndrome coronavirus (MERS-CoV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are of special concern. SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has spread to date to nearly every country in the world, resulting in millions of infections, more than 1 million deaths, and a massive global economic impact  are translated from the same start codon at the 5′ end, resulting in the polyproteins pp1a and pp1ab. Translation of the longer product pp1ab is made possible by a hairpin-type pseudoknot RNA structure known as the frameshifting element (FSE), which regulates a programmed -1 ribosomal frameshifting that overrides with about 50% efficiency the stop codon of ORF1a . Previouin vivo crosslinking, the viral RNA is selectively captured, fragmented, and subjected to a click-chemistry reaction to add a biotin tag to crosslinked fragments. Crosslinked RNA duplexes are then selectively captured using streptavidin affinity purification. Half of the resulting RNA is proximity ligated, following reversal of the crosslink to create chimeric RNA templates for high-throughput sequencing. The other half is used as a control, in which reversal of the crosslink precedes the proximity ligation and accurately represents the background level of non-specific ligation. The coupling of two biotin-streptavidin-mediated enrichment steps, first of viral RNA and second of crosslinked RNA duplexes, provides high structural depth for identification of both long- and short-lived conformations. COMRADES can therefore measure (1) the structural diversity of alternative RNA conformations that co-exist inside cells; (2) short-distance, as well as long-distance (over tens of thousands of nucleotides), base-pairing within the same RNA molecule; and (3) base-pairing between different RNA molecules, such as those of host and viral origin (We recently developed crosslinking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture in living cells . COMRADEl origin .Figure 1Here we apply COMRADES to study the structural diversity of the SARS-CoV-2 gRNA and sgmRNA inside cells. We discover networks of short- and long-range RNA-RNA interactions spanning the entirety of SARS-CoV-2 gRNA and sgmRNA. We reveal site-specific interactions with the host transcriptome. Finally, we uncover a conserved long-range structure encompassing the programmed ribosomal FSE. In order to make our data more accessible to the community, we have developed an interactive, user-friendly interface for exploring the structures identified in this study.in vivo crosslinked, SARS-CoV-2-inoculated Vero E6/TMPRSS2 cells  and to discover long-distance RNA-RNA interactions as they occur inside cells. Indeed, we observed a high prevalence of long-range RNA base-pairing along the SARS-CoV-2 genome, with ORF1a demonstrating more long-range connectivity than any other ORF  that are essential for genome replication and discontinuous transcription . The 3′ the UTRs A and S4Athe UTRs , and a pthe UTRs .Figure 5in vivo evidence for genome cyclization in SARS-CoV-2, mediated by long-range base-pairing between the 5′ and 3′ UTRs .In addition to their coding capacity, nucleic acids have evolved structural capabilities to sense metabolites , catalyzc genome . Great ec genome . HoweverIntramolecular base-pairing within the same viral genome is more likely to occur than intermolecular base-pairing between two different viral genomes. However, we cannot rule out the possibility that some of the long-range interactions reported here might stem from intermolecular interactions between different viral genomes. Also, snRNAs are known to be heavily modified. As certain chemical modifications may affect the base-pairing capacity of RNA, modeling the exact structure of snRNA-viral interactions B is parteric.miska@gurdon.cam.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Eric A. Miska  cells were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB No. JCRB0403) and cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in a humidified CO2 incubator. All cell lines were regularly examined to exclude mycoplasma contamination.Chlorocebus sabaeus (Green monkey) VeroE6  were purchased from American Type Culture Collection . VeroE6/TMPRSS2 cells ,  were kin32165541) were inoculated with SARS-CoV-2 strain München-1.2/2020/984    at MOI =Total cellular RNA was mixed with a tiling array of 50 biotinylated DNA probes, 20 nucleotides-long each (IDT), antisense to ORF1a and ORF1b of the viral genomic RNA , and wasViral enriched gRNA and sgmRNA fractions were fragmented by incubating at 37°C for 20 min with 0.1 units/μl RNase III (Ambion). Reactions were terminated by cleaning with RNA Clean & Concentrator (Zymo Research). Biotin was attached to cross-linked RNA duplexes by incubating with 150 μM Click-IT Biotin sDIBO Alkyne (Life technologies) under constant agitation at 37°C for 1.5 h. Residual Biotin sDIBO Alkyne was removed by RNA Clean & Concentrator (Zymo Research). Biotinylated RNA duplexes were enriched using Dynabeads MyOne Streptavidin C1 (Invitrogen) at the following reaction conditions: 100 mM Tris-Cl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20, 0.5 unit/μl Superase-In (Invitrogen). Beads were washed 5 times on a magnet with 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 3.5 M NaCl, 0.1% Tween-20. RNA was eluted by adding 95% Formamide, 10 mM EDTA solution preheated to 65oC and purified using RNA Clean & Concentrator (Zymo Research).Each RNA sample was divided in two: one half was used for proximity ligation and then crosslink reversal , while in the other half, crosslink reversal was done before proximity ligation . Prior to proximity ligation, RNA was denatured by briefly heating to 95°C. Proximity ligation was done under the following conditions: 1 unit/μl RNA ligase 1 (New England Biolabs), 1x RNA ligase buffer, 50 μM ATP, 1 unit/μl Superase-in (Invitrogen), final volume: 200 μl. Reactions were incubated for 16 h at 16°C and were terminated by cleaning with RNA Clean & Concentrator (Zymo Research). Crosslink reversal was done by irradiating the RNA on ice with 2.5 KJ/m2 254 nm UVC using a CL-1000 crosslinker (UVP).Library preparation was done as described in , using 6https://cme.h-its.org/exelixis/web/software/pear/doc.html). PCR duplicates were removed using unique molecular identifiers via collapse.py (https://gitlab.com/tdido/tstk). Chimeric reads were identified and annotated to the respective genome using hyb (Homo sapiens reference genoem (GRCh38) with the addition of MERS (NC_019843.3).Data preprocessing was performed according to . In briesing hyb . SARS-Coin vivo structure produces sequencing reads differing by a few nucleotides. This results in increased computation load of folding each chimeric read separately. To overcome this issue, and to gain better structure predictions, the reads were clustered into chimeric groups. Each chimeric read is composed of a left side (L) and right side (R), each originated from a different position along the gRNA or sgmRNA. Each chimeric read can therefore be described as (g): the genomic distance between L and R, and chimeric reads that originated from the same structure will have a similar g and can be clustered based on their g values. Clustering of chimeric reads that originated from the same structure was performed using a network based approach whereby an adjacency matrix is created for all chimeric reads based on the nucleotide difference between their g values (Deltagap).Deltagap = 0 for identically overlapping gaps, and increasing Deltagap values for chimeric reads that share less overlapping sites. The clustering was performed twice per sample, once for the chimeric reads that represent short stem structures (g ≤ 10 nt) and once for chimeric reads that represent long distance interactions (g > 10 nt). Short range interactions weights were calculated as:g as vertices and weights as edges: G =  using iGraphDue to crosslinking and fragmentation, the COMRADES datas can provide redundant structural information whereby the same http://www.interjournal.org/manuscript_abstract.php?361100992). To identify densely connected subgraphs (communities) with chimeric groups containing chimeric reads that originated from the same structure, we clustered the graph using random walks with the cluster_waltrap function (steps = 2) from the iGraph package. Chimeric groups containing less than 10 chimeric reads were discarded. Chimeric groups often contained a small amount of longer L or R sequences due to the random fragmentation in the COMRADES protocol. To avoid introducing biases in the folding results, clusters were trimmed to the region from L to R for which evidence in the cluster is higher than the mean evidence - 2 standard deviations. The trimmed clusters can be found in (Folding of the chimeric groups was performed using the vienna package . For shohttps://github.com/JLP-BioInf/SARS-Cov2-COMRADES)Heatmaps, viewpoints and all other downstream analysis was performed using custom R scripts, all scripts to reproduce the analysis as well as functions to analyze other COMRADES datasets are available on GitHub . Sequences containing unidentified nucleotides were discarded. Four sequence sets for MSA  were generated:(I)MSA-1-SARSrel-3515seq: includes all complete non-redundant Sarbecovirus sequences. MUSCLE , (defaul(II)MSA-2-SARSrel-3515seq: Includes all complete non-redundant Sarbecovirus sequences containing only the four canonical nucleotide identifiers. MUSCLE .(III)(I) were calculated using the mbed function in the kmer package (MSA-3-SARSrel-137seq: pairwise distances between the sequences in  package . The “se(IV)(II) were divided into seven smaller sequence sets (six 500-sequences sets and one 515-sequence set). The seed sequences (“seeds” attribute of the mbed function in the kmer package (https://cran.r-project.org/web/packages/kmer/index.html)) for all seven sets were combined in a new sequence set to be aligned to make up multiple sequence alignment (IV). This resulted in a sequence set with less sequences than (I), but more than (III) and representative variation as (I). MUSCLE (default parameters).MSA-4-SARSrel-559seq: The unaligned sequences used to generate (I)SARS-CoV-2 COMRADES cluster coordinates were taken from (II)The cluster alignments were analyzed with R-Scape  using de(iii)The R-Scape output for each candidate co-varying pair includes an E-value statistical score . The default significance level of 0.05 was kept, so only position pairs with E-values smaller than 0.05 were considered in the subsequent analysis.(iv)Results tables of R-scape output combined with the corresponding coordinates in the non-aligned SARS-CoV-2 reference sequence, as well as nucleotide combination frequencies at the two positions across the alignment. We defined secondary base-pairing frequency as the percentage of sequences in which the pair of nucleotides differed from the most common base-pair type at the position but could still form a base-pair. For example, an imaginary pair of nucleotides has the composition:In all cases the NCBI reference genome for SARS-CoV-2 (NC_045512.2) was used as reference. For each of the four sequence alignments, the following steps were taken:https://github.com/JLP-BioInf/SARS-Cov2-COMRADES/tree/master/covariation). The identified base pairs were included in For the covariation analysis of sgmRNA chimera clusters, cluster coordinates were taken from Genome conservation data analyzed with synplot2  was takehttps://github.com/JLP-BioInf/SARS-Cov2-COMRADES. Base-pairing prediction, structure prediction and clustering data are available for exploration as a web interface: https://github.com/JLP-BioInf/SARS-CoV2-COMRADES-APP. Additional data supporting the findings of this study are available from the corresponding authors upon request.Computer code has been deposited on GitHub:


These retroelements are expressed as abundant linear and circular RNAs of ∼170-400 nt in different animal tissues. Bioinformatic and in vitro analyses indicate an efficient self-cleavage of the HHRs harboured in most invertebrate retrozymes, whereas HHRs in retrozymes of vertebrates, such as the axolotl and other amphibians, require to act as dimeric motifs to reach higher self-cleavage rates. Ligation assays of retrozyme RNAs with a protein ligase versus HHR self-ligation indicate that, most likely, tRNA ligases and not the ribozymes are involved in the step of RNA circularization. Altogether, these results confirm the existence of a new and conserved pathway in animals and, likely, eukaryotes in general, for the efficient biosynthesis of RNA circles through small ribozymes, which opens the door for the development of new tools in the emerging field of study of circRNAs.Ribozymes are catalytic RNAs present in modern genomes but regarded as remnants of a prebiotic RNA world. The paradigmatic hammerhead ribozyme (HHR) is a small self-cleaving motif widespread from bacterial to human genomes. Here, we report that most of the classical type I HHRs frequently found in the genomes of animals are contained within a novel family of non-autonomous non-LTR retrotransposons of the  Our results are consistent with a conserved role for most type I HHRs in the life cycle of a novel family of constitutively expressed non-autonomous non-LTR retrolements, which spread throughout animal genomes by means of small circRNAs transposition intermediates.bona fide HHRs, and their occurrence within tandem repeats. Sequence homology searches through BLAT , following the manufacturer's instructions. Shortly, tissues were homogenised (1:10 v/v) in Trizol, and then 0.2 volumes of chloroform:isoamyl alcohol (24:1 v/v) were added to the samples. The mixture was centrifuged, and RNA in the supernatant was precipitated with 0.1 volumes of 3 M sodium acetate (pH 5.5) and 2.5 volumes of 100% ethanol. RNA was resuspended and quantified as explained above.Genomic retrozymes were amplified by PCR using pairs of adjacent and divergent primers designed to bind the most conserved motifs within a species . The hotAcropora millepora, 300–400 nt for the mussel Mytilus galloprovincialis, and 200–350 for the axolotl Ambystoma mexicanum). RNAs were reverse-transcribed with SuperScript II (Invitrogen), and PCR-amplified with adjacent divergent primers previously phosphorylated, using Prime Star HS DNA polymerase. PCR products were cloned into a linearised pBlueScript KS (Agilent) vector by blunt-end ligation, and were sequenced automatically with an ABI Prism DNA sequencer (Perkin-Elmer).For circular retrozyme RNA amplification and cloning by RT-PCR, RNA was fractionated by native 5% 1× TAE PAGE, and extracted from gel slices corresponding to the size of the retrozyme monomer  in 5% (retrozymes) or 15% gels (ribozymes) with 8 M urea.RNAs of the cloned retrozymes were synthesized by tF, was determined by quantitative scanning of the corresponding gel bands and fitted to the equation tF = F∞(1 − ekt–), where F∞ is the product fraction at the reaction endpoint, and k is the first-order rate constant of cleavage (kobs).Analyses of retrozyme self-cleavage under co-transcriptional conditions were performed as previously described . TranscrFor northern blot analysis, 5–50 μg of purified RNA from the different animal tissues were examined in 5% polyacrylamide gels under native (1× TAE) or denaturing  conditions. After ethidium bromide staining, RNAs were electroblotted onto nylon membranes  and UV-fixed with a crosslinker . Prehybridization, hybridization (at 68°C in 50% formamide) and washing was done following the instructions of the manufacturer . The riboprobes were obtained by run-off transcriptions of linearized pBlueScript plasmids containing the full retrozymes in the presence of DIG-UTP (Roche Diagnostic GmbH) .in vitro analysis of the tRNA ligation and self-ligation capabilities of RNA retrozymes, monomeric retrozyme RNAs that resulted from double self-cleavage after transcription (either in the presence or in the absence of [α-32P]ATP) of dimeric constructs were purified from 5% polyacrylamide gels under denaturing conditions . For the assays of RtcB ligation and self-ligation, 1–10 ng of gel-purified radiolabelled retrozyme RNAs, or 1–4 μg for non-radiolabelled RNAs, were firstly denatured at 95°C for 1 min, cooled down to 25°C , and then incubated either in the presence of RtcB ligase in its corresponding buffer for 1 h at 37°C (New England Biolabs) or in 50 mM Tris–HCl, pH 8 and 10 to 50 mM MgCl2 for 1 h at 25°C, respectively.For Previous studies have reported that many type I HHRs in animal genomes can be found within short tandem repeats of a few hundred base pairs (150–450 bp), widely but patchily distributed among most metazoan phyla . In contAcropora millepora  tandem repeats of the kind mentioned above, ranging from dimers and trimers, up to 62-mers. A genomic copy of a monomeric retrozyme was amplified with adjacent oligos, cloned and transcribed in vitro. The HHR contained in the genomic retrozyme showed an efficient RNA self-cleavage, with a co-transcriptional kobs of ∼0.8 min−1  from different coral samples run in polyacrylamide gels (PAGE) under native conditions showed the presence of a single band >200 nt size, which corresponds to the size of the repeats found in the genome  of newts (Ambystoma mexicanum)    showed txicanum) . The HHRxicanum) . The HHRxicanum) . A genom) Figure . On the ) Figure   Figure  and 4B. ) Figure , indicatnd newts , left. Wnd newts , right, nd newts , indicatnd newts . Finallynd newts  carryingnd newts .in vitro by a chloroplast tRNA ligase  compared with its circularization through RtcB tRNA ligase (∼30%)  . For bote (∼30%) . Moreovee (∼30%) . Altogetligation .in vivo could be much higher than their linear counterparts. The observed levels of linear RNAs may result from either HHR self-cleavage or spontaneous breakage of the circRNAs during their purification and manipulation. Moreover, with the exception of the predicted secondary structure of some of the mussel retrozyme circRNAs, RNA circles of most metazoan retrozymes are predicted to allow the HHR to freely adopt the catalytically competent structure of the ribozyme (Figures in vivo by some mechanism other than self-pairing to prevent ribozyme activity and circRNA linearization.The results reported in this work answer a longstanding question about the presence and role of the paradigmatic HHR in diverse animal genomes. The first HHR motif described in a metazoan genome was reported more than 30 years ago in the satellite DNA repeats of newts . Althoug Figures . This fe Figures ,46. Our As previously hypothesized , plant ain vivo in mammalian cells by using constructs with tandem ribozymes of the Twister class (in vivo and in vitro of circRNAs.The presence of a genome-encoded circRNA in animals was first described anecdotally in the 1990s , but theer class , which cIn summary, these minimal retroelements in metazoans confirm the existence of a new natural pathway for circRNA biosynthesis through autocatalytic RNAs conserved in animals and, most likely, eukaryotes in general ,46. FutuM. galloprovincialis have been deposited in the GenBank database under the accession codes MN642551-MN642576.All data are available from the corresponding author upon request. The circRNA sequences cloned from gkaa187_Supplemental_FileClick here for additional data file.


Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses – animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5′-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses  contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3′-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size. The replicative complex of coronaviruses consists of 16 nonstructural proteins produced by the processing of pp1a and pp1ab by the viral proteases (in trans by the main chymotrypsin-like proteinase (MP) , wh, wh28], ral mRNA . 2′-O-meral mRNA .Cytology of replication. After entering the cell, the coronavirus nucleocapsid is transported to the endosomes, where genomic (+)RNA is released from the N protein. Free genomic (+)RNA enters the cytoplasm and is directed to the ribosomes [ibosomes  for the ibosomes , 21. Coribosomes . Genomicibosomes .Closteroviridae family includes about 40 plant (+)RNA viruses [The  viruses , 34 charGenome structure and expression. The RNA genome of beet yellows virus (BYV) was the first closteroviral genome sequenced. It consists of 14,480 nt and contains the 5′-cap, but lacks the 3′-poly(A) tail [(A) tail , 33, 35.(A) tail , 33, 34.(A) tail , 36, as (A) tail . The BYVThe BYV PLP autocatalytically releases the leader protein by cleaving the Gly588/Gly589 bond . Genomesin vitro, but, unlike its cellular orthologs, is unable to interact with unfolded proteins [The major capsid protein (CP) coats ~95% genomic RNA, forming the “body” of the filamentous particle, while the minor capsid protein (CPm) forms a ‘tail’ that includes the 5′-terminal genome portion , 42 Fig. Formatiproteins . The BYVproteins , 49. Virproteins . The proproteins  Fig. 4)in vitro,Replication-associated proteins. Closteroviruses belong to the supergroup of alpha-like viruses that includes (+)RNA viruses of animals  and plants   . Despite fashion .cis-signal on the (+)RNA and produces the antigenomic (–)RNA to be further used as a template for the synthesis of progeny genomic and subgenomic RNA strands. No experimental evidence has yet been reported in favor of either of two options of closterovirus transcription, namely, transcription from the subgenomic promoters on the antigenomic (–)RNA template or transcription on the antisubgenomic RNA templates [Comparisons of viral (+)RNA reveals a simple rule: the larger the size of genomic RNA, the larger the replicase gene. In other words, replication of large genomes requires more complex RNA replicative complexes . Replicaemplates . Obviousemplates -58.Cytology of replication. Closteroviral infection is accompanied by the induction of DMVs (~100 nm in diameter) and MVCs in the cells [Nicotiana benthamiana plants [he cells . These uhe cells . The DMVhe cells , 62. In a plants , 63. A 1a plants , 63.. [Closteroviridae have icosahedral (bromoviruses) or rod-like virions (tobamoviruses). The size of RNA that can be packaged into these particles is strictly limited, and it is possible that the evolution of the superflexible closteroviral particles built of several proteins has made it possible to resolve the problem of packaging of larger RNA RNA genome size. Several evolutionary “inventions” have allowed these viruses to solve the problems of packaging and replication of large RNAs. As suggested by Godeny et al. , the ancto 19 kb .Mutations introduced by viral RNA polymerases  and, posThe copying of large RNA strands is mediated by more sophisticated replicases, and expansion of the RNA genome is due, for a large part, to the acquisitions in the replication-associated genes . CoronavComparison of the structure and expression of coronaviral and closteroviral genomes reveals a striking similarity between the two groups and 4. ThFigs. 1


Drosophila key nuclear piRNA silencing pathway protein Piwi of the Argonaute family has been classically studied as a factor controlling transposable elements and fertility. Piwi has been shown to concentrate in the nucleolus for reasons largely unknown. Ribosomal RNA is the main component of the nucleolus. In this work the effect of a piwi mutation on rRNA is described. This work led to three important conclusions: A mutation in piwi induces antisense 5S rRNA expression, a processing defect of 2S rRNA orthologous to the 3′-end of eukaryotic 5.8S rRNA, and accumulation of fragments of all five rRNAs in Drosophilamelanogaster ovaries. Hypotheses to explain these phenomena are proposed, possibly involving the interaction of the components of the piRNA pathway with the RNA surveillance machinery. Drosophila genome UTP by polynucleotide kinase in Buffer B according to the manufacturer’s instructions  with subsequent washing by 2xSSC/0.1% SDS twice and 1XSSC/0.1% SDS once. Visualization was performed using a Typhoon FLA9500  phosphoimager. Usually the same filter was hybridized consecutively with different probes and stripped by boiling 0.1% SDS. Before transfer, RNA in the gels was visualized by SYBR Green II  on Bio-Rad Gel Doc gel documentation system . In addition to equal amounts of RNA analyzed, miR-8 microRNA and ribosomal protein rp49 transcripts were used as loading controls. Northern blot for each genotype was performed at least twice with independently isolated RNAs, except for supplementary material experiments performed once. To design the probes, GenBank accession numbers of the following DNA sequences were used: rRNA–M20107.1, 5S rRNA–J01122.1. The following probes were used:Various quantities of total RNA in equal amounts for mutant and control ovaries were separated by electrophoresis. For long RNAs, electrophoresis of 1, 2, and 4 mkg total RNA was carried out in a denaturing formaldehyde 1% agarose gel in MOPS buffer in accordance with the guidelines . Blottinn.)5S end AGGCCAACAACACGCGGTGTTCCCAAG (27 n.)18S end TAATGATCCTTCCGCAGGTTCACCTAC (27 n.)5.8S end CAGCATGGACTGCGATATGCGTTCAAA (27 n.)2S TACAACCCTCAACCATATGTAGTCCAAGCA (30 n.)28S end TCGAATCATCAAGCAAAGGATAAGCTT (27 n.)5S start TGTATTCAGC3GTGGTATGGTCGTTGGC (27 n.)5S mid CGACGCTGCTTAATTTCGGTGATCGGACGAGA (32 n.)5S end AGGCCAACAACACGCGGTGTTCCCAAG (27 n.)5S end sense CTTGGGAACACCGCGTGTTGTTGGCCT (27 n.)5S 3′ AAAAAGTTGTGGACGAGGC (20 n.) [5.8S mid GTCGATGTTCATGTGTCCTGCAGTTCACACGATGACGCACAG (42 n.) miR-8 GACATCTTTACCTGACAGTATTA (23 n.)2/Ntpiwi genotype was used. Thermal cycling consisted of 5 min at 95 °C, followed by 45 cycles of denaturation , annealing , extension , and a final extension of 5 min at 72 °C. RT– reactions did not show significant PCR signals. One biological replica per genotype was analyzed, in three technical replicates of real-time PCR. Adh transcript was used as a loading control.After RNA isolation with Trizol reagent, RNA was reprecipitated in 3.3 M LiCl, centrifuged, and dissolved in water, DNAse-treated . 1 mkg of total RNA was used to synthesize cDNA in RT+ and RT– reactions for each genotype using random hexamers with Mint reverse transcriptase . Real-time PCR was performed with SYTO13 intercalating agent and HS Taq DNA polymerase . For calibration the HeT-A retrotransposon Het-s2 (CGCAAAGACATCTGGAGGACTACC) and Het-as2 (TGCCGACCTGCTTGGTATTG) [Adh AdhRI_s (GCCTGCGTACATAGCCGAGAT) and AdhRI_as (GCTCCGTTAGTTGTTGGTTTCC). The following primers were used: for GGTATTG) , for Adh


In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.The HSV-1 virion host shutoff ( vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes. A key ct manner –8. Later(VP22) 9–, with th(VP22) 9–. In addi genomes –15. This, UL48 VP and UL49genomes –. Finally4 h p.i. .vhs-mediated mRNA degradation and global inhibition of transcription substantially alter the host transcriptome during productive infection. Virus-induced alterations in total RNA levels can be a consequence of either of these two global phenomena or due to gene-specific changes in RNA stability or transcription. Their relative contributions, however, could so far not be distinguished. Recently, Pheasant et al. presented a genome-scale transcriptome sequencing (RNA-seq) study analyzing nuclear-cytoplasmic compartmentalization of viral and cellular transcripts during lytic HSV-1 infection  labeling, followed by sequencing (4sU-seq), to characterize de novo transcription and RNA processing in hourly intervals during the first 8 h of lytic HSV-1 infection of primary human foreskin fibroblasts (HFF) (Both nfection . These rts (HFF)  20, 21), 21vhs-mvhs-mediated RNA degradation and global loss in transcriptional activity during lytic HSV-1 infection on a genome-wide scale, we now performed total RNA-seq and 4sU-seq time course analysis on HFF infected with a vhs-null mutant virus in which vhs has been inactivated by replacement of amino acids 251 to 489 with LacZ (Δvhs) . Notably, this included the key vhs-sensitive genes reported by Pheasant et al. Accordingly, increased reduction of total mRNA levels for these genes is not due to increased susceptibility to vhs-mediated RNA decay of the respective transcripts but instead due to additional, vhs-cleavage-activity-dependent effects on their transcription. vhs-dependent downregulation of transcriptional activity resulted in reduced protein levels of many of the respective genes already at 8 h p.i. in WT infection but not in Δvhs infection, as confirmed by quantitative whole-proteome mass spectrometry.To dissect the effects of Z (Δvhs) . Here, wZ (Δvhs)  20). Fu. Fuvhs-mfections . Mathemavhs, global inhibition of Pol II activity and host gene-specific regulation during productive HSV-1 infection, we employed the same experimental set-up for Δvhs infection as for our previous transcriptome analyses on WT HSV-1 infection (vhs at a high multiplicity of infection (MOI) of 10 and performed 4sU-seq in hourly intervals and total RNA-seq every 2 h during the first 8 h of infection  for all genes. It does not affect the correlation between fold changes , which is scale invariant. Since the analyses here were all performed without such normalization, the fold changes discussed here always represent relative changes compared to other genes regarding their relative contribution to overall RNA levels. Accordingly, this means that genes identified here as nonregulated only exhibit the global reductions in RNA levels or transcription that equally affect all genes. This enables the identification of gene-specific changes in RNA abundance and transcription levels that differ from the majority of genes.DESeq2 normalization assumes that there are no global changes in RNA levels between conditions. This is not the case in HSV-1 infection due to pike-ins , 25, 26.vhs and WT infections when comparing the same time points, confirming the high degree of standardization between the two independent experiments (Sr] ≤ 0.3), when essentially no (n ≤ 2) cellular genes were differentially expressed in both WT and Δvhs infections . This was expected as fold changes were only very small (median |log2-fold change| ≤ 0.1) and dominated by experimental noise. The highest correlations between 4sU-seq fold changes in WT and Δvhs infections compared to mock infections were observed at 4–5 h and 5–6 h p.i. (Srvhs on cellular RNA levels late in infection (vhs infection (from 6–7 h p.i.) were better correlated with slightly earlier stages (4–5 h p.i. and 5–6 h p.i.) of WT infection, indicating a slightly slower progression of Δvhs infection.Gene expression fold changes in 4sU-RNA were highly correlated between Δeriments  to F. ThrS≈0.8,  and E. CrS≈0.8, , consistnfection . Notablyvhs infection were only poorly correlated  , consistent with mRNA cleavage and degradation by tegument-delivered vhs protein. The negative correlation to RNA half-lives was also confirmed for total RNA fold changes from the study of Pheasant et al. at 4 h p.i. (Sr = –0.36), while at 12 h p.i., a weaker, but still highly significant, negative correlation was observed (Sr = –0.15).In contrast to 4sU-RNA, fold changes in total RNA obtained from WT and ΔMethods) . Please vhs infection, however, total RNA fold changes and RNA half-lives were positively correlated from 4 h p.i. onward  model of HSV-1 infection that models (i) global changes in host transcriptional activity, (ii) global changes in vhs endonuclease activity, (iii) subcellular compartmentalization and nuclear export of transcripts, (iv) differences in basal mRNA half-lives between genes, and (v) gene-specific transcriptional regulation . Using this model, we estimated the extent of loss in transcriptional activity from our total RNA-seq time course data in Δvhs infection and the increase of vhs endonuclease activity during WT HSV-1 infection from our total RNA time course in WT infection (see Text S2). Our results indicate that by 8 h p.i. in Δvhs infection, transcriptional activity dropped down to 10 to 20% of the level in uninfected cells . Furthermore, if the loss of transcriptional activity in WT infection were indeed dramatically higher than in Δvhs infection, vhs-mediated degradation would have to increase even faster and to higher levels to achieve the observed negative correlations. In summary, our model explains the wide range of total RNA fold changes observed between genes in HSV-1 infection simply by differences in basal RNA half-lives between genes in uninfected cells and gene-specific transcriptional regulation. Although statistically significant correlations were also observed between 4sU-RNA fold changes and RNA half-lives, these were relatively small in both WT (Sr ≥ –0.15) and Δvhs (Sr ≤ 0.25) infections. Thus, changes in newly transcribed RNA obtained during 60 min of 4sU labeling are also influenced by vhs-mediated decay and loss of transcriptional activity but substantially less strongly than for total RNA. We conclude that the poor correlation in total RNA fold changes between WT and Δvhs infections is a direct consequence of global effects of vhs on RNA stability throughout the first 8 h of lytic infection.To estimate the kinetics of ed cells . Assuminradation . This ravhs-mediated decay and loss of transcriptional activity on 4sU-RNA, we analyzed subcellular RNA fractions  from mock-, WT-, and Δvhs-infected cells at 8 h p.i.  and Δvhs (Sr = 0.82) time courses. This also confirmed the poor correlation of total RNA fold changes between WT and Δvhs infections (Sr = 0.24). Thus, it does not result from experimental bias between two independently performed time course experiments. Furthermore, negative (Sr = –0.36) and positive (Sr = 0.34) correlations to RNA half-lives were again observed for WT and Δvhs infections, respectively.Since our analysis revealed some effect of . n = 2;  to obtaiublished . Known nublished , confirmublished . Note thublished , 21. Thivhs-mediated RNA cleavage and degradation. The absence of any significant correlation between fold changes in chromatin-associated RNA and RNA half-life for both WT and Δvhs infections (Srvhs infection) confirms this assumption and provides further evidence for the efficient separation of the chromatin-associated RNA fraction. We thus focused on changes in chromatin-associated RNA to assess the effects of HSV-1 infection and vhs on transcriptional regulation. Strikingly, comparison of chromatin-associated RNA fold changes revealed that changes in relative, gene-specific transcriptional activity at 8 h p.i. were extremely similar between WT and Δvhs infections  only in WT infection and not in Δvhs infection  in both WT and Δvhs infections compared to mock infection (blue in P ≤ 0.001) for genes downregulated by type II interferon.Differential gene expression analysis on chromatin-associated RNA identified 225 genes  that were significantly downregulated at the transcriptional level , we harvested chromatin-associated RNA at 8 h p.i. in WT and Δvhs infections  that showed no gene-specific regulation . Like Pheasant et al., we used 18S rRNA as an internal reference for the qRT-PCR since it is not translated and thus not targeted by vhs. As expected, ARF4 and CNBP both showed higher (∼3-fold) chromatin-associated RNA levels in Δvhs infection than in WT infection. This is consistent with slower progression of Δvhs infection compared to WT infection and thus reduced global reduction in host transcriptional activity. Nevertheless, the loss of COL6A2, MMP1, and MMP3 transcription in WT infection compared to Δvhs infection was considerably greater (8- to 15-fold), thereby confirming their vhs-dependent transcriptional downregulation.Interestingly, a set of 150 genes  was significantly downregulated , in particular “extracellular matrix (ECM) organization” . This included fibronectin (FN1), integrin beta 1 (ITGB1), a subunit of integrin complexes binding fibronectin, and several genes encoding collagen alpha chains. Enrichment was also observed for “focal adhesion,” i.e., the integrin-containing, multiprotein complexes that anchor the cell to the ECM and connect it to the actin cytoskeleton .Functional enrichment analysis of skeleton , 32. Of vhs-dependent manner (P ≤ 0.001). The highest enrichment was found for the integrin adhesome components identified in HFF . Furthermore, genes of the HFF adhesome  showed a systematic shift in regulation between WT and Δvhs infections in total RNA, 4sU-RNA, and all RNA fractions , the stsubunits .vhs-dependent downregulation required vhs endonuclease activity, we performed RNA-seq of chromatin-associated RNA at 8 h p.i. using a vhs single-amino acid mutant (D195N) that no longer exhibited the mRNA decay activity but still binds to the translation initiation factors eIF4H and eIF4B . This confirmed vhs-dependent transcriptional regulation in an independent experiment  endoribonuclease muSOX and subsequent Xrn1-mediated mRNA degradation leads to transcriptional repression for numerous genes  and identified 342 muSOX-dependent genes. Although they found no clear links to specific biological processes in their functional enrichment analysis, the KEGG pathway “focal adhesion” was significantly enriched among muSOX-dependent genes (19 of 342 genes). To investigate whether vhs-dependent transcriptional downregulation of the integrin adhesome and ECM components might be mediated by general RNA degradation or represent a vhs-specific response, we analyzed the overlap of muSOX-dependent genes to our list of vhs-dependent genes , it is nevertheless small. Although this provides some evidence that vhs-dependent transcriptional downregulation for most genes is distinct from general mRNA-decay-dependent transcriptional repression, more work is required to rule out effects of different cell, species or virus backgrounds.Gammaherpesviruses also encode an mRNA-targeting RNase, SOX, which is not homologous to us genes . The samnt genes . Only 14vhs-mediated RNA degradation of a key cellular transcriptional regulator. We thus performed a motif search in promoters of vhs-dependently downregulated genes but surprisingly found no significantly enriched known or novel transcription factor binding motifs in the proximal promoter regions . To recover more distal regulation, we also performed a motif search in open chromatin peaks from ATAC-seq (assay for transposase-accessible chromatin using sequencing) data in uninfected cells  that act as cofactors of serum response factor (SRF) (vhs-dependent genes (P = 3.9 × 10–5). Nevertheless, only 42 (28%) of vhs-dependent genes were SRF targets and 93% of SRF targets were not vhs dependent in our study; thus, other regulatory mechanisms have to be involved. Further work is required to clarify this issue.A different explanation for the concerted downregulation of a set of functionally related genes could be ed cells  within 1or (SRF) . SRF hasor (SRF) . TCF-depor (SRF) . Though vhs-induced loss in total RNA levels between different cellular genes at 12 h post-WT infection  or low  reduction in total RNA levels in WT infection. Since actinomycin D treatment showed a similar stability of corresponding mRNAs in uninfected cells, these authors concluded that these differences were due to differences in the susceptibility of the respective transcripts to vhs cleavage activity. By RNA-seq and PCR on chromatin-associated rather than total cellular RNA, we demonstrated that all three of their PCR-confirmed highly vhs-sensitive genes are actually transcriptionally downregulated in a vhs-dependent manner. Moreover, genes defined as efficiently depleted during WT infection by Pheasant et al.  were significantly enriched for ECM organization . We thus hypothesized that a significant fraction of highly vhs-sensitive genes identified by Pheasant et al. might actually be vhs-dependently transcriptionally downregulated. Indeed, both original and additional vhs-dependently transcriptionally regulated genes identified in our genome-wide analysis were strongly enriched among efficiently depleted genes determined by Pheasant et al.  and were among the most significantly downregulated genes in total RNA at 12 h p.i. in WT infections  detectable in total RNA at 6 h p.i. or earlier in both WT and Δvhs infections. Not surprisingly, several of these genes  have previously been identified as highly upregulated in HSV-1 infection by microarray analysis on total RNA . Notably, 50% of genes in the orange cluster were upregulated by type I interferons (>4.5-fold enrichment).Analysis of chromatin-associated RNA identified a set of 462 genes that were significantly upregulated in both WT and Δegulated  was upreotal RNA , 44, andotal RNA . UpregulP < 10–13). In total, 76  upregulated genes (16.5%) were poorly expressed in uninfected cells. HSV-1-induced upregulation of genes not normally expressed has previously been reported for human alpha-globin genes (HBA1 and HBA2), which are normally only expressed in erythroid cells  and several of its targets are highly upregulated by HSV-1 infection . We thusvhs-infected HFF at 0 and 8 h p.i. (n = 3 replicates). In total, 7,943 proteins were identified (see Data Set S8). No filtering based on read-in transcription was performed, since read-through transcripts are neither exported nor translated -based quantitative proteomic analysis of WT- and Δanslated , 21. Proroblasts ); thus, P ≤ 0.001) from the proteomics time course for 3, 6, 9, and 12 h p.i. HSV-1 infection from the study of Kulej et al. . Five of these were also upregulated at protein level, including four genes that were upregulated in chromatin-associated RNA . Thus, transcriptional upregulation can have a small but measurable effect on protein levels by 8 h p.i. Only a few genes showed a significant differential effect . Strikingly, the 24 proteins downregulated in a vhs-dependent manner were strongly enriched for HFF adhesome components (>12-fold) and ECM organization (>23-fold). Accordingly, 9 of 14 (64%) proteins downregulated only in WT infection and included in our RNA-seq analysis were downregulated in chromatin-associated RNA in a vhs-dependent manner. An analysis of protein fold changes for all vhs-dependently transcriptionally downregulated genes (including our extended set) demonstrated that a significant number of respective proteins were either less downregulated or (relatively) more upregulated in Δvhs infection than in WT infection , to achieve a maximum level of reproducibility. Indeed, all major results were confirmed in experiments which were performed in parallel for WT and Δvhs infections.HSV-1 infection drastically alters host RNA metabolism at all levels by impairing host mRNA synthesis, processing, export and stability. Here, we differentiate and quantify their individual contributions to the RNA expression profile by combining RNA-seq of total, newly transcribed (4sU-RNA) and subcellular RNA fractions in WT and Δvhs nuclease activity based on the correlations between RNA half-lives and total RNA fold changes during the first 8 h of infection. This showed a drop in transcriptional activity down to 10 to 20% of the original level by 8 h p.i. in Δvhs infection, consistent with the well-described general loss of Pol II from host chromatin , which we did not consider in our model. In particular, vhs-dependent transcriptional downregulation contributes substantially to the reduction in total RNA levels for the respective genes. Furthermore, a recent study identified a set of 74 genes that escape degradation by four herpesviral endonucleases, including vhs vhs infections for both up- and downregulated genes  were upregulated by type I interferons (IFNs). Moreover, DUX4 was confirmed as a major transcriptional regulator in both WT and Δvhs-dependent, transcriptional downregulation of proteins involved in the integrin adhesome and ECM organization, which required vhs nuclease activity. Suppression of ECM protein synthesis during HSV-1 infection has already been reported over 30 years ago for the canonical integrin ligand FN1, type IV procollagen, and thrombospondin  governing these genes. It is unclear, however, why only a single or a very small number of transcription factors would suffer so much more dramatically from vhs nuclease effects. It is indeed surprising that vhs-mediated mRNA degradation does not cause a similarly pronounced dysregulation downstream of short-lived transcription factors involved in other processes. However, the surprisingly high correlation between fold changes in WT and Δvhs infections observed in chromatin-associated RNA excludes gross global effects of mRNA degradation of cellular transcriptional factors. Furthermore, no enrichment of any known or novel transcription factor binding motif could be identified in both proximal promoter regions or more distal open chromatin regions identified by ATAC-seq. Promoter analysis applied to all expressed HFF adhesome genes identified only one significant motif which was only observed in <6% of genes, suggesting that there is no single key transcriptional regulator for the integrin adhesome. Nevertheless, vhs may still directly interact with or target a major cellular transcription factor that governs the expression of the integrin adhesome and ECM via distal enhancers. Notably, ELK3, a TCF cofactor of SRF, was downregulated in a vhs-dependent manner early on in infection. While TCF-dependent genes were not enriched among vhs-dependent genes, an ∼2-fold enrichment of SRF targets was observed. Since TCF-dependent genes were determined by triple knockouts of all three TCFs  and confounded by the pleotropic effects of vhs nuclease activity. Nevertheless, we could show that vhs-dependent transcriptional downregulation has a clear impact on protein levels already by 8 h p.i., as confirmed by quantitative whole-cell proteomics. Proteins with strong vhs-dependent reduction at 8 h p.i. include matrix metallopeptidases MMP1 to MMP3, which are involved in degradation of ECM proteins, their inhibitor TIMP1 as well as other MMP-upregulating or -interacting proteins .Untangling the molecular mechanisms underlying specific In summary, our analyses provide a comprehensive, quantitative picture of the molecular mechanisms that govern profound alterations in the host cell transcriptome and proteome during lytic HSV-1 infection.vhs-inactivated mutant (Δvhs)  cells (obtained from the American Type Culture Collection) as described previously (vhs D195N mutant), at 37°C using an MOI of 10. Subsequently, the inoculum was removed, and fresh medium was applied to the cells.Human fetal foreskin fibroblasts (HFF) were purchased from ECACC  and cultured in Dulbecco modified Eagle medium with 10% FBS Mycoplex and 1% penicillin-streptomycin. HFF were utilized from passages 11 to 17 for all high-throughput experiments. This study was performed using WT HSV-1 strain 17  andt (Δvhs) . In thiseviously . HFF wervhs D195N mutant virus was constructed via en passant mutagenesis (Escherichia coli strain GS1783 containing the pHSV(17+)Lox BAC (The agenesis . Mutagen)Lox BAC . BAC DNAvhs infection was performed as reported previously for WT HSV-1 . Subsequently, the medium was aspirated and the cells were lysed with TRIzol (Invitrogen). Total RNA and newly transcribed RNA fractions were isolated from the cells as described previously (vhs infections were prepared as previously described (n = 2 replicates)  was prepared.Sample preparation for 4sU-seq in ΔWT HSV-1 . In brieeviously . In an ilicates) . To asseSequencing libraries were prepared using the TruSeq stranded total RNA kit (Illumina). rRNA depletion was performed after DNase treatment for total RNA and all subcellular RNA fractions using Ribo-zero but not 4sU-RNA samples. Sequencing of 75 bp paired-end reads was performed on a NextSeq 500 (Illumina) at the Core Unit Systemmedizin (Würzburg).qC values for each gene were calibrated to 18S rRNA for that sample, and relative expression between samples was calculated using the ΔΔqC method.Chromatin-associated RNA was isolated in TRIzol as described above and then DNase treated and purified with the Direct-zol RNA Miniprep kit . cDNA was synthesized using the “optional procedure” of the BioScript All-in-One cDNA Synthesis SuperMix . Real-time PCR performed with SYBR green qPCR Master Mix  using the recommended three-step protocol and 1 μM concentrations of the following primer pairs for each gene: 18S rRNA (GCAATTATTCCCCATGAACG and GGGACTTAATCAACGCAAGC), ARF4 (CCTTCTGCTTCTGCCCATCA and CGCATCTGCTTCTTGCCAAA), CNBP (AAACTGGTCATGTAGCCATCAAC and AATTGTGCATTCCCGTGCAAG), COL6A2 (GCAACGACTACGCCACCAT and GACCTTGATGATGCGGTTGA), MMP1 (TAGTGGCCCAGTGGTTGAAA and GGGCTGCTTCATCACCTTCA), and MMP3 (AGTCTCTGTGAATTGAAATGTTCG and AGTTCCCTTGAGTGTGACTCG). cDNA samples were diluted 1:10 in water for protein-coding genes and 1:1,000 for 18S rRNA; they were then diluted 1:4 for the final reaction, which was performed on a LightCycler 96 (Roche). The full description of H3K4me3 ChIPmentation is included in Text S10 in the supplemental material.vhs HSV-1 for 8 h at an MOI of 10. Infections were conducted in triplicate, with four uninfected controls . Washed cells were snap-frozen in liquid nitrogen. Cells were lysed in by resuspending in 100 μl 2% sodium dodecyl sulfate (SDS) and 50 mM tetraethylammonium bromide , followed by a 10-min (30-s on/off duty cycle) sonication in a Bioruptor sonicator (Diagenode). Lysates were quantified by a BCA assay, and 50-μg portions of each sample were reduced and alkylated with 10 mM Tris(2-carboxyethyl)phosphine (TCEP) and 40 mM iodoacetamide for 20 min at room temperature in the dark. Samples were diluted to 500 μl with 8 M urea–50 mM TEAB, assessed using 30-kDa Vivacon centrifugal ultrafiltration devices (Sartorius), and concentrated according to the manufacturer’s instructions. Samples were resuspended and concentrated in 8 M urea a further three times to remove residual SDS. There were a further three washes with digestion buffer  before the samples were resuspended in approximately 50 μl of digestion buffer with 1 μg of trypsin . Filter units were then incubated in at 37°C overnight in a box partially filled with water to reduce evaporation. Peptides were recovered into a fresh tube by centrifugation and a further wash with 50 μl of digestion buffer. The SDC was removed from each sample by precipitation with the addition of formic acid and two-phase partitioning with ethyl acetate. Peptides were then dried under vacuum. For TMT labeling, the samples were resuspended in 42 μl of 100 mM TEAB, and then 0.4 mg of each TMT reagent in 18 μl of anhydrous acetonitrile was added, followed by vortexing to mix the sample and incubation at room temperature for 1 h. A small aliquot of each sample was analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm the labeling efficiency, and samples were pooled 1:1 according to the total TMT reporter intensity in these quality control runs. The pooled sample was then acidified and subjected to solid-phase extraction cleanup using 50-mg tC18 cartridges (Waters) before drying under a vacuum.HFF were infected with WT or ΔSamples were resuspended in 40 μl of 200 mM ammonium formate (pH 10) and transferred to a glass high-pressure liquid chromatography (HPLC) vial. BpH-RP fractionation was conducted on an Ultimate 3000 UHPLC system (Thermo Scientific) equipped with a Kinetex EVO column . Solvent A was 3% acetonitrile, solvent B was 100% acetonitrile, and solvent C was 200 mM ammonium formate (pH 10). Throughout the analysis, solvent C was kept at a constant 10%. The flow rate was 400 μl/min, and the UV absorbance was monitored at 280 nm. Samples were loaded in 90% solvent A for 10 min before a gradient elution of 0 to 10% solvent B over 10 min (curve 3), 10 to 34% solvent B over 21 min (curve 5), and 34 to 50% solvent B over 5 min (curve 5), followed by a 10-min wash with 90% solvent B. 15-s (100 μl) fractions were collected throughout the run. Fractions containing peptide (as determined by 280-nm light absorbance) were recombined across the gradient to preserve orthogonality with on-line low-pH RP separation. For example, fractions 1, 25, 49, 73, and 97 were combined, dried in a vacuum centrifuge, and stored at –20°C until LC-MS analysis.18 column . The gradient was 3 to 10% solvent B over 10 min, 10 to 35% solvent B over 155 min, 35 to 45% solvent B over 9 min, followed by a wash with 95% solvent B for 5 min and reequilibration with 3% solvent B. Eluted peptides were introduced by electrospray to the MS by applying 2.1 kV to a stainless-steel emitter . During the gradient elution, MS1 spectra were acquired in the Orbitrap, and the collision-induced dissociation (CID)-MS2 was acquired in the ion trap. Synchronous precursor selection-isolated MS2 fragment ions were further fragmented using higher-energy collisional dissociation to liberate reporter ions which were acquired in the Orbitrap (MS3).Samples were analyzed on an Orbitrap Fusion instrument on-line with an UltiMate 3000 RSLCnano UHPLC system (Thermo Fisher). Samples were resuspended in 10 μl of 5% dimethyl sulfoxide–1% trifluoroacetic acid (TFA), and 5 μl of each fraction was injected. The trapping solvent was 0.1% TFA, the analytical solvent A was 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid. Samples were loaded onto a trapping column  at 10 μl/min for 5 min. Samples were then separated on a PepMap Ct test in R. P values were adjusted for multiple testing by using the Benjamini-Hochberg method  from within Proteome Discoverer v2.1 (Thermo Fisher) against the UniProt human database with appended common contaminants and the UniProt HSV reference proteome. The peptide spectrum match false discovery rate (FDR) was controlled at 1% using Mascot Percolator. The reporter ion intensities of proteins with high (1%) and medium (5%) FDRs were taken and subjected to LIMMA g method . ProteinJN555585) using ContextMap v2.7.9 (Sequencing reads were mapped against (i) the human genome (GRCh37/hg19), (ii) human rRNA sequences, and (iii) the HSV-1 genome , not downregulated in Δvhs infection (log2-fold change > −1), and there was at least a 2-fold increase in fold changes in Δvhs infection compared to WT infection.The numbers of read fragments per gene were determined from the mapped 4sU-seq and RNA-seq reads in a strand-specific manner using featureCounts  and geneHochberg . AdditioHochberg . To idenN*(t), t = 60], and then calculated the RNA half-life for each gene as t1/2 = –tln2/ln(1 – N*(t)).RNA half-lives were measured as described previously  from 4sUvhs infections is described in Text S2 in the supplemental material.The ODE model of WT and Δ2-fold change  ≥ 1, FDR  ≤ 0.001; downregulated, log2-fold change ≤ −1, FDR  ≤ 0.001)  . TCF-dep≤ 0.001) . Odds ra≤ 0.001) , and P vHochberg . Human pHochberg  (v10.5) Hochberg  and visuHochberg . Only asGSE70481). Mouse and human gene symbols were mapped to their orthologues in the respective other species using the Mouse/Human orthology table from the Mouse Genome Informatics (MGI) database (2-fold change ≤ −1 and FDR  ≤ 0.1) but not in ΔHS infection (log2-fold change > −1 or FDR  > 0.1). vhs-dependent genes were defined according to our criteria described above.Fold changes for WT and ΔHS MHV68 infection were taken from the study of Abernathy et al.  and downdatabase . muSOX-dvhs-dependently downregulated genes was performed using HOMER in proximal promoter regions  (n = 2 replicates) (vhs-dependently downregulated genes.Promoter motif search for rt site) . Potentilicates) . ATAC-selicates) , and opelicates) . Blacklilicates) , and peaGSE59717; 4sU-seq and total RNA-seq data of Δvhs infection, GSE129715; RNA-seq of total, cytoplasmic, nucleoplasmic, and chromatin-associated RNA in WT and Δvhs infections, GSE129582; RNA-seq of chromatin-associated RNA in WT, Δvhs, vhs D195N, and WT-BAC infections, GSE140068; and H3K4me3 ChIPmentation, GSE132920.All sequencing data are available in the GEO database under the following IDs: 4sU-seq and total RNA-seq data of WT infection,


There is an increased global outbreak of diseases caused by coronaviruses affecting respiratory tracts of birds and mammals. Recent dangerous coronaviruses are MERS-CoV, SARS-CoV, and SARS-CoV-2, causing respiratory illness and even failure of several organs. However, profound impact of coronavirus on host cells remains elusive. In this study, we analyzed transcriptome of MERS-CoV, SARS-CoV, and SARS-CoV-2 infected human lung-derived cells, and observed that infection of these coronaviruses all induced increase of retrotransposon expression with upregulation of TET genes. Upregulation of retrotransposon was also observed in SARS-CoV-2 infected human intestinal organoids. Retrotransposon upregulation may lead to increased genome instability and enhanced expression of genes with readthrough from retrotransposons. Therefore, people with higher basal level of retrotransposon such as cancer patients and aged people may have increased risk of symptomatic infection. Additionally, we show evidence supporting long-term epigenetic inheritance of retrotransposon upregulation. We also observed chimeric transcripts of retrotransposon and SARS-CoV-2 RNA for potential human genome invasion of viral fragments, with the front and the rear part of SARS-CoV-2 genome being easier to form chimeric RNA. Thus, we suggest that primers and probes for nucleic acid detection should be designed in the middle of virus genome to identify live virus with higher probability. In summary, we propose our hypothesis that coronavirus invades human cells and interacts with retrotransposon, eliciting more severe symptoms in patients with underlying diseases. In the treatment of patients with coronavirus infection, it may be necessary to pay more attention to the potential harm contributed by retrotransposon dysregulation. Emerging coronaviruses often spread rapidly from person to person and there seems to be an increased global outbreak of related diseases. MERS-CoV and SARS-CoV are two identified rare coronavirus strains which cause not only severe lung infection but also serious complications . More reTransposable Elements (TEs) are mobile DNA elements in virtually all eukaryotes and comprise more than 40% of human genome . They caLINEs are common autonomous retrotransposons and comprise about 17% of human genome . Some LIIn our study, we analyzed publicly available transcriptome data of human cells infected with coronavirus MERS-CoV, SARS-CoV, and SARS-CoV-2, and observed enhanced expression of TEs including several retrotransposons, as well as inflammation, immunity, and apoptosis related genes. We further noticed potential fusion of SARS-CoV-2 RNA with retrotransposon transcripts especially LINEs and SINEs. Therefore, further examinations on genome and transcriptome of cells from patients and studying models will be valuable to evaluate potential crosstalk between coronavirus and retrotransposons.Cell types below are used in this study. Calu-3, human lung cancer cell; MRC5, human fetal lung strain; A549, human adenocarcinomic alveolar basal epithelial cell; NHBE, primary human bronchial epithelial cell. Each group above has three replicates. For human intestinal organoids, each group has two replicates.Raw reads were processed with cutadapt v1.16 to perform quality trimming with default parameters except for: quality-cutoff =20, pair-filter=both. To include as many non-uniquely mapped reads as possible, trimmed reads were firstly aligned to human/mouse genome (hg19/mm10) by STAR (v2.5.1b) with default settings including parameters “–outFilterMismatchNmax 10 –winAnchorMultimapmax 2000 –outFilterMultimapNmax 1000”. RSEM was used to calculate FPKM value of genes. The annotation and fasta sequences for consensus transposable element sequences were downloaded from Repbase (version 20.01) . TEtransRNA sequencing data of coronavirus-infected human lung-derived cells are from GSE122876  , GSE5619Coronaviral infection led to not only respiratory failure but also multiple organ dysfunction syndromes, indicating that coronavirus impacts a wide range of human cells . TranscrTo examine whether coronavirus infection disturbs expression of specific gene sets in human cells, we analyzed public available RNA-seq data of human lung-derived cells with infection of MERS-CoV, SARS-CoV, and SARS-CoV-2. Through comparison of transcriptomes before and after infection, we identified thousands of dysregulated genes  for each group  in sperm were reported to transmit abnormal epigenetic information into preimplantation embryo, and epigenetic abnormality was further inherited to adult tissue, causing metabolic disorders . Two kinCoronaviruses are RNA viruses and are not supposed to integrate into host genome by themselves. However, it was reported that several RNA viruses have capacity to recombine with retrotransposons to invade host genome . RegardiWe found that 0.23% of SARS-CoV-2 RNA formed chimeric transcripts with non-TE genes and 0.14% with TE . RNA sequencing data of MERSCoV-infected and SARS-CoV-infected MRC5 cells are obtained from NCBI Sequence Read Archive with BioProject ID:PRJNA233943 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA233943). RNA sequencing data of SARS-CoV-2 infected A549 cells, Calu-3 cells, and NHBE cells (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615032). RNA sequencing data of SARS-CoV-2-infected human intestinal organoids (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA628628/). RNA sequencing data of IRF1 knockout and control human hepatocytes infected with hepatitis A virus are are obtained from NCBI Sequence Read Archive with BioProject ID: PRJNA473130 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA473130/). RNA sequencing data of STAT1 knockout and control human HepG2 cells treated by IFN (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA384926/). RNA sequencing data of human tissues and cell types (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA324812/). RNA sequencing data of human early embryos and embryonic stem cells (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA153427/). RNA sequencing data of 8-cell mouse embryos and adult mouse islet developed from zygotes with injection of sperm tsRNAs from high-fat-diet males (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA304514/).RNA sequencing data of MERS-CoV-infected Calu-3 cells  are obtaBE cells  are obtarganoids  are obtad by IFN  are obtall types  are obtaem cells  are obtaet males  are obtaL-QZ and XH conceived and designed the project. YY analyzed the data and wrote the manuscript. X-ZL performed analysis on chimeric transcripts. L-QZ and XH revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Key R&D Program of China  and the National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.