While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals. RNA of some viruses is protected from degradation by a 5′ triphosphate group. Here the authors identify nudix hydrolase 2 (NUDT2) as novel antiviral defense protein that dephosphorylates viral RNA and thereby enables its degradation. Genomic RNAs and RNA transcripts of many viruses, including Orthomyxo-, Arena-, Paramyxo- and Bunyaviruses, do not bear a 5′-cap but present a 5′-triphosphate (PPP-) group. This terminal PPP- moiety linked to double-stranded RNA serves as a pathogen-associated molecular pattern (PAMP) sensed by the cellular helicase RIG-I, which activates a MAVS-dependent innate immune signaling cascade. Eventually, this culminates in the accumulation of type-I interferon (IFN) and IFN-stimulated genes and is critical to prevent virus spread3. It is currently unclear why some viruses bear terminal triphosphates despite the selective pressure of the innate immune system. However, a possible explanation may be the increased stability of PPP-RNA that inhibits 5′-to-3′ degradation by XRN1. Interestingly, despite the stabilizing PPP-group on viral RNA, specific highly secondary structured viral RNA sequences expressed by Flavi-, Bunya-, and Arenaviruses impair the activity of XRN15. Moreover, the depletion of XRN1 increases the stability of viral RNA6. Collectively, this suggests that 5′-to-3′ RNA degradation processes are operative in the case of viral RNA. In analogy to the eukaryotic mRNA degradation system, however, the degradation of viral PPP-RNA would require a processing step that removes the PPP-group. Indeed, recently the triphosphatases RNA/RNP complex-1-interacting phosphatase (DUSP11) and Decapping and exoribonuclease protein  were shown to dephosphorylate PPP-RNA to P-RNA that can be further degraded by XRN19. However, the mechanism of PPP-RNA dephosphorylation and the substrate specificity of DUSP11 is not yet well understood.All living organisms use RNA as the messenger molecule to translate their DNA encoded genetic information into proteins. Besides trans-activating sequences encoded on messenger (m)RNA and the subcellular localization of the RNA molecule, the abundance of mRNA is the primary determinant to modulate gene expression. mRNA abundance is regulated based on transcription and the stability of the molecule. In eukaryotes, the most prominent and evolutionary conserved processes that modulate the stability of mRNA are 5′-to-3′ RNA degradation by the cellular exonuclease 1 (XRN1) and 3′-to-5′ degradation by the RNA exosome complex. Eukaryotic mRNAs are protected from 5′-to-3′ degradation by a 5′-m7G cap structure, co-transcriptionally added to the mRNA molecule. To proceed with the 5′-to-3′ degradation process, the mRNA cap needs to be removed by the decapping protein 2 (DCP2) to generate a monophosphorylated (P-)RNA substrate, which can then be degraded by XRN1 Fig. 1,2. Geno11. In bacteria, the 5′ phosphates can be removed by the RNA pyrophosphohydrolase (RppH) to prepare RNA for degradation by the bacterial 5′-to-3′ endoribonuclease RNase E or 5′ exonucleases , usually resulting in NMP (nucleoside monophosphate) and P-X as products18. Since Nudix hydrolases process mammalian and bacterial mRNAs, we tested whether mammalian Nudix hydrolases may have dephosphorylation activity on PPP-RNA to initiate further exonucleolytic processing. Understanding such viral RNA degradation mechanisms is essential to further study cellular antiviral principles that are in place to limit the spread and pathogenicity of infectious pathogens and their involvement in antiviral immunity.Similar to viral RNA, prokaryotic mRNA also bears a 5′-PPP or 5′-PP groupJ13 Fig. ). Intere17 . Automated live-cell microscopy of VSV-GFP infected cells showed a distinct increase of GFP signal in XRN1 targeted cells as compared to controls Fig. , indicatach Fig. . Notablyach Fig. . Depletiach Fig. . For NUDach Fig. . We usedach Fig. . While tach Fig. .E. coli. As controls, we mutated the metal-coordinating glutamic acid residue (E) in their conserved Nudix hydrolase domain motif 20. Successful purification of the recombinant proteins was confirmed by SDS-PAGE and mass spectrometry  and led to almost complete dephosphorylation of the substrate RNA  and the second position  of the RNA Fig. . NUDT2 p22  and tested how NUDT2 processes these substrates. Notably, NUDT2 sequentially released single phosphates resulting in a di-phosphorylated dinucleotide intermediate that is further converted into a mono-phosphorylated product Fig. . This co22 Fig. . Neither22 Fig. . Removal22 Fig. c, 4e, f.We next tested whether NUDT2 can prepare PPP-RNA for degradation by the canonical XRN1 dependent 5′-to-3′ RNA degradation machinery. As a substrate, we used Hepatitis C virus (HCV) encoding PPP-RNA. Incubation with NUDT2 or XRN1 did not affect the stability of the PPP-HCV RNA Fig. , indicatTo extend this in vitro model to physiological conditions, we evaluated the activity of NUDT2 mammalian cells. In human cells, NUDT2 localizes to both the nucleus and the cytoplasm , inflammatory cytokines , or U1 snRNA in heart, liver, lung, or spleen , a virus that is highly sensitive to antiviral activities of IFNs. Extracellular application of Ap4A or its delivery into HeLa cells through lipofection impaired virus replication in a comparable manner as the double-stranded RNA analog polyI:C  (VSV-M2). Notably, accumulation of infectious virus particles was 10- to 30-fold increased in supernatants of bone marrow-derived from Nudt2−/− mice compared to corresponding Nudt2+/+ littermate controls  from ols Fig. . SimilarRNA Fig. . The incEFs Fig. . ImportaEFs Fig. . SimilarEFs Fig. . We concNudt2+/+ and Nudt2−/− mice with VSV using an intranasal infection regime, causing encephalitis. Surprisingly, despite significant growth differences in cell culture experiments, the infection in vivo did not lead to significant differences in survival rates when comparing Nudt2+/+ and Nudt2−/− mice  were identified as proteins that convert PPP-RNA into P-RNA, which serves as a substrate for XRN128. Since RNA degradation is an evolutionary, highly conserved process, we asked whether proteins known to be involved in PPP-RNA degradation in bacteria are also functionally active in mammalian cells. Using a siRNA screen, phosphate release assays, automated live-cell microscopy, and RT-qPCR, we identified that the protein NUDT2 could release phosphates from 5′-PPP RNA substrates and contributes to control the degradation of viral PPP-RNA. We show that NUDT2 is active on a broad range of PPP-RNA substrates but has a preference for processing longer substrates. NUDT2 sequentially releases two phosphates, and a monophosphorylated RNA is generated that can then serve as a substrate for the cellular exonuclease XRN1. This suggests that NUDT2 could be active in the degradation process of RNAs derived from diverse viruses.RNA turnover is a highly conserved process and tightly controlled to regulate RNA abundance and to target erroneous RNAs for degradation. PPP-RNA, as present in viral genomes and to some extent in bacteria11. Although PPP-mRNA is present in bacteria, recent reports of Luciano et al. suggest that E. coli predominantly contain diphosphorylated mRNA, which appears to be the preferred substrate of RppH in E. coli10. The high structural similarity of RppH and mammalian NUDT2 indicates similar activities of both proteins, which is supported by the ability of NUDT2 and RppH to process phosphorylated RNA. Likely, NUDT2 has a preference for longer RNA constructs . RNA of some viruses like Picornaviruses carry a structural element named internal ribosome entry site (IRES) that could also present a barrier for exonucleases like XRN133. HCV employs an unconventional mechanism to protect its RNA from degradation as it recruits the liver-specific microRNA miR-122 to its 5′-end. Through complementary binding to the HCV RNA, miR-122 protects the RNA from degradation by XRN134 by sterically hindering XRN1 binding to the 5′-end. Although NUDT2 dephosphorylated all tested RNA substrates, it may be that the activity of NUDT2 is similarly inhibited by steric hindrance under very special conditions, as seen for XRN-I. Flaviviruses form membranous replication factories that cover viral RNA from cellular proteins35, which may also be an evasion strategy to escape NUDT2 activity. It is not yet clear if NUDT2 can access such intracellular sub-compartments or if the activity of NUDT2 is limited to free 5′-PPP RNAs in the cytoplasm. Screening the impact of NUDT2 on more viruses would greatly enhance our understanding of the impact of NUDT2 dependent viral restriction and RNA decay. Since we could not observe a significant difference in survival rates between VSV-infected Nudt2+/+ and Nudt2−/− mice, we cannot exclude that other cellular pyrophosphatases have redundant functions. Such redundancies have also been reported for the activity of pattern recognition receptors (PRRs) in vivo. Despite the requirement of certain PRRs to induce cytokines and reduce virus growth in vitro, in vivo phenotypes were often less convincing. The RNA sensor Toll-like receptor (TLR) 7, for instance, only reveals its in vivo phenotype in the absence of the RIG-I sensing pathway36. Similarly, the in vivo activity of TLR3 strongly depends on the infection route of the pathogen37. Moreover, in an infection model for the DNA virus mouse Cytomegalovirus, the DNA sensor TLR9 only shows an effect in livers but not in spleens, clearly indicating an organ-specific effect38. Similarly shown for PRRs, the in vivo phenotype of NUDT2 may only be visible when using sophisticated in vivo models or when specific infection models are used. On an organismal level, organ-specific expression differences or viral counter mechanisms could affect the impact of NUDT2.The importance of RNA degradation is highlighted by the fact that many viruses evolved mechanisms to escape RNA decay pathways and potentially hide from NUDT2 activity. Potential evasion strategies include capping of RNA by either employing the virus-derived capping machinery or seizing it from cellular mRNAs in a process called cap snatchingAntiviral responses are often governed by proteins that are regulated by type-I IFNs. The expression of NUDT2 protein, however, does not change after cytokine treatment or virus infection. This may suggest that NUDT2, in addition to its ability to dephosphorylate viral RNA, may also have additional housekeeping functions. Interestingly, RNA Polymerase III products, including 5S ribosomal RNA, non-coding, and in some cases coding RNAs, should contain 5′-PPP termini. It is conceivable that proteins, such as NUDT2, DOM3Z (DXO), and DUSP11, are processing such aberrant RNAs or other RNA molecules to avoid stimulation of the innate immune system in the absence of pathogen encounter. Since self-RNA recognition may have dramatic effects on the cell, NUDT2, DUSP11, and DOM3Z (DXO) may fulfill redundant functions under steady-state conditions. Indeed, we could not obtain cells that lack both NUDT2 and DUSP11, despite the successful depletion of either of the two proteins. The full activity of these proteins may be required in case of viral infections since the abundance of viral PPP-RNA may exceed the catalytic capacity of either of these proteins.39. Hep3B cells were obtained from Prof. R. Bartenschlager, University Hospital Heidelberg (Germany). Mouse embryonic fibroblasts (MEFs) were isolated from 13.5-day old embryos from heterozygous breeding pairs. Murine bone marrow was isolated from mouse femur, and tibia and cultured in DMEM supplemented with 10% fetal calf serum , 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, and 2 mM L-glutamine. Cell lines were maintained in DMEM (PAA Laboratories) containing 10% FCS and antibiotics . All viruses used are classified as BSL2 pathogens in Germany, and all experiments were carried out according to official regulations. Influenza A/SC35M-NS1-2A-renilla-2A-NEP (IAV-ren)40, VSV, VSV-M241, VSV-GFP42, VSV-AV3-GFP19 have been previously described. HSV-1 (F-strain) was from Soren Riis Paludan , VSV-Luc was a gift from Gert Zimmer , and SFV was a gift from Andres Merits . Recombinant XRN1, CIP, and RppH and restriction enzymes for in vitro transcriptions were obtained from NEB, single nucleotides for in vitro transcriptions and phosphate release assays were from Jena Bioscience, RNasin was from Promega, TURBO DNase was obtained from Thermo Fisher Scientific, and T7 RNA polymerase was produced by the Core Facility of the MPI of Biochemistry. The CellTiter-Glo assay kit and the luciferase substrate d-Luciferin (E1602) were purchased from Promega. siRNAs were purchased from Qiagen or were synthesized by the Core Facility of the MPI of Biochemistry (see Supplementary Table HeLa (ATCC CCL-2) and HEK293T (ATCC CRL-3216) cells were described previously and were authenticated by ATCC43. To test the sequence specificity of NUDT2, RNA substrates with varying starting nucleotides, including single-stranded and double-stranded 5′-ends, were used as described13. In vitro transcribed RNA was generated from linear, double-stranded DNA templates, hybridized of complementary oligodeoxynucleotides , 50 mM NaCl, and 1 mM EDTA, heated to 95 °C for 5 min and cooled down to 20 °C at a rate of 0.1 °C/s on a PCR cycler. For the preparation of renilla luciferase RNA, the pRL-SV40 plasmid was linearized by BamHI cleavage. To prepare firefly luciferase RNA, the RiboMAX Large Scale RNA Production Systems Kit (Promega) control plasmid was used. Transcription was carried out overnight at 37 °C according to the manufacturer’s protocol of the T7 RiboMAX Large Scale RNA Production Systems kit (Promega). After DNase treatment for 30 min at 37 °C using 1 U of RNase-free DNase per µg of DNA template, ivtRNA was purified using either the MinElute Cleanup kit (Qiagen) or the Monarch RNA Cleanup Kit (NEB).As RNA substrate, IVT4, a defined double-stranded triphosphorylated RIG-I ligand, was used as described45 was linearized by MluI cleavage. Transcription was carried out for 6–8 h at 37 °C in 5× transcription buffer , 2.5 mM ATP, 2.5 mM UTP, 2.5 mM CTP, 2.5 mM GTP, RNasin (Promega), and 1000 U T7 RNA polymerase (Core Facility of the MPI of Biochemistry), in 100 µL final volume. 2 µL TURBO DNase (Thermo Fisher Scientific) was added for 30 min at 37 °C. Reactions were purified with mini Quick Spin RNA Columns (Roche) according to the manufacturer’s protocol. For the 44-mer single-stranded RNA, the pGEX-6P-T7-44-mer-RNA46 plasmid linearized by EcoRI cleavage served as a template. RNA was synthesized by in vitro transcription according to the MEGAscript T7 Kit (Thermo Fisher Scientific). After incubation for 16 h at 37 °C and TURBO DNase (Thermo Fisher Scientific) treatment for 30 min at 37 °C, the RNA was purified according to the Direct-zol RNA extraction kit (Zymo Research). For the generation of double-stranded RNA, a 20-nt antisense RNA (Metabion) (5′-acucucucucucucucuccc-3′) was annealed to the in vitro-transcribed RNA. In vitro transcribed RNA was capped using the ScriptCap Cap 1 Capping System .For the preparation of HCV PPP-RNA, the pFK-JFH plasmid DNA+Buffer (Biontex) for targeted gene knockdowns. Transfection was performed according to the manufacturer’s instructions for HeLa cells. Briefly, we transfected 15 pmol of pooled siRNAs per 1e5 cells in a 24 well format. Cells were infected 48 h after siRNA knockdown. Duplex siRNAs were either purchased from Qiagen or synthesized by the Core Facility at the MPI of Biochemistry  for the Nudix hydrolase siRNA screen or Metafectine Pro transfection reagent with the SINUDT2 knockout on virus growth, MEFs and bone marrow were seeded the day before infection. Cells were infected with VSV wt at a multiplicity of infection (MOI) of 0.001, SFV at an MOI of 0.001, HSV-1 at an MOI of 2, VSV-Luc at an MOI of 0.001, IAV-renilla at an MOI of 0.1, and HSV-Luc at an MOI of 2. Bone marrow was infected with VSV wt and VSV M2 at an MOI of 1. Twenty-four hours post-infection or as indicated specifically in the figure legend, cells were harvested for quantitative RT-qPCR, Western Blot or and the supernatant was harvested to test for virus accumulation by 50% tissue culture-infective dose (TCID50) assays on Vero E6 cells. To quantify the effects of siRNA-mediated knockdown of Nudix hydrolases, cells were infected 48 h after the knockdown was performed with VSV-Luc at an MOI of 0.001 for 24 h, IAV-renilla at an MOI of 1 for 48 h, VSV-GFP at an MOI of 0.001 for 24 h, and VSV-M2-GFP at an MOI of 0.0001 for 24 h. GFP levels were determined fluorometrically. Z-scores were calculated from three independent experiments individually for every gene and virus, as previously described47.To determine the impact of NUDT2, XRN1, or NUDT2 and XRN1, were infected with VSV-GFP at an MOI of 0.01 in five technical replicates. For each technical replicate, two phase-contrast and green fluorescence images were acquired by a 10× objective. Fluorescence images were further processed to remove the background signal using a top-hat filter. The integrated intensity of GFP+ cells per image was then normalized to the total area of cells by division for each time point.For RT-qPCR analysis, RNA was isolated using the NucleoSpin RNA Plus kit (Macherey Nagel) according to the manufacturer’s protocol. For proteomic analysis, cell pellets were snap-frozen in liquid nitrogen before further processing. For western blot analysis, cells were lysed in Laemmli buffer, boiled for 10 min at 95 °C, and subjected to SDS-polyacrylamide gel electrophoresis and western blot analysis. For GFP-tagged viruses, virus growth was measured by determining GFP levels in a microplate reader (Tecan Infinite 200 Pro) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. For luciferase-tagged viruses, cells were lysed in Passive Lysis Buffer (Promega), and virus load was determined by measuring firefly or renilla-luciferase. VSV-GFP growth kinetics were monitored and analyzed using the IncuCyte S3 Live-cell imaging system and software (Essen Bioscience). Heb3b cells, transduced with a non-targeting control vector or with a CRISPR/Cas9-mediated depletion of either NUDT2, NUDT2 E58A, NUDT12, NUDT12 E369A, NUDT14, NUDT14 E121A, NUDT17, NUDT17 E124A were obtained as gBlocks from IDT containing overhangs suitable for SLIC cloning. All constructs were cloned into pCoofy4, providing an N-terminal His6-MBP tag. Protein expression and purification were performed by the Core Facility of the MPI of Biochemistry. Briefly, plasmids were transformed in BL21-AI pRARE bacteria, and protein expression was carried out in an autoinduction medium containing 0.2% arabinose. Cells were lysed using an AVESTIN high-pressure homogenizer, and cleared lysate was used for protein purification using a HisTrap HP column  and further purified by gel filtration using a Superose 6 PC 3.2/30  . Proteins were dialyzed with D-Tube Dialyser Midi (cutoff 6–8 kDa) in a buffer containing 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 10 mM MgCl2, and 1 mM DTT. The identity of recombinant proteins was confirmed by mass spectrometry.DNA sequences of To test phosphate release from mammalian expressed NUDT2 cells, N-terminally 6-myc-tagged NUDT2, NUDT2 E58A, and NS1 of Influenza A virus  of strain A/PR8/34) were transiently expressed from pCS2-N-6xMyc plasmids. For this, HEK293T cells were transfected using 1.3 µg of plasmid per 1e6 cells and a ratio of the transfection reagent PEI to plasmid DNA of 3:1. The cells were collected after two days, washed 3× with PBS, pelleted, and frozen. Subsequently, the cells were lysed with TAP lysis buffer ) on ice for 30 min, followed by sonication  and clearing of the lysate . The expressed, Myc-tagged proteins were then precipitated using 80 µL slurry of equilibrated α-myc agarose beads . Beads were washed three times with NEB 2 reaction buffer to equilibrate for the phosphate release assay.Validation of precipitation of myc-tagged proteins: Immunoprecipitated proteins were eluted in the presence of 1x SDS sample buffer  and by boiling the samples at 95 °C for 5 min. Eluted proteins were then separated on a 4–12% Bis-Tris acrylamide gel  at 100 V for 90 min and transferred onto a 0.2 µm nitrocellulose membrane by wet-blotting at 300 mA for 1 h. The membrane was then blocked with 5% milk, 3× washed with PBS with 0.25% Tween-20 (PBS-T) for 5 min each, and stained with primary antibodies in 5% milk followed by incubation with an appropriate secondary antibody.Primary antibodies used in this study were as follows: monoclonal mouse α-NUDT2 , monoclonal mouse α-β-Actin-HRP , monoclonal mouse α-myc-HRP , and secondary horseradish peroxidase (HRP)-coupled antibody rabbit α-mouse IgG .For time-resolved analysis, the phosphatase activities of Nudix hydrolases, CIP, or RppH were analyzed with the EnzChek Phosphate Assay  according to the manufacturer’s instructions. For optimal performance, the following adjustments were made: one replicate (200 µL reaction volume) included 600 nM NUDT protein or the corresponding mutant, 4.5 µg of RNA substrate, 0.5 U of PNP, 0.2 mM MESG, and NEB 2.0 buffer. This setting was validated to yield optimal assay performance , and ribonucleotide triphosphate (rNTPs). The malachite green assay was performed according to the manufacturer’s instructions  after incubating 900 ng IVT4 with NUDT2 or NUDT2 E58A in NEB2 buffer for 3 h at 37 °C. This setting was validated to yield optimal assay performance. In order to test NUDT2 activity on a panel of possible substrates, NUDT2 (600 nM) and NUDT2 E58A (600 nM) were incubated for 3 h with rATP, rGTP, rUTP, and rCTP (2.5 µM) for 3 h at 37 °C in NEB2 reaction buffer. CIP (5 U) (NEB) was used as a positive control for released inorganic phosphate in all experiments. For immunoprecipitated NUDT2, NUDT2 E58A or NS1 (IAV) derived from HEK cell lysate, 20 µL of beads were incubated in 40 µL NEB 2 reaction buffer, including 900 ng IVT4, for 3 h at 37 °C. Beads were separated , and the phosphate release assay was performed using the malachite green assay as described above.32P labeled RNA was synthesized by in vitro transcription according to the MEGAscript T7 Kit (Thermo Fisher Scientific) supplemented with 0.17 µM [γ-32P] GTP (Perkin Elmer). For the 44-mer single-stranded RNA, the pGEX-6P-T7-44mer-RNA46 plasmid linearized by EcoRI cleavage served as a template. For the dsRNA IVT4, two annealed primer pairs (Supplementary Table 43 were used as a template. After incubation for 16 h at 37 °C and DNase digestion with TURBO DNase (Thermo Fisher Scientific) for 30 min at 37 °C, the RNA was purified according to the Direct-zol RNA extraction kit (Zymo Research). For the generation of a double-stranded RNA of the 44-mer ssRNA, a 20-nt antisense RNA (Metabion) (5′-acucucucucucucucuccc-3′) was annealed to the in vitro transcribed RNA.γ-The labeled RNAs were incubated with purified NUDT2 (600 nM), NUDT12 (600 nM), NUDT14 (600 nM), NUDT17 (600 nM), and RppH (5 U) purchased from NEB in a solution (20 µL) containing NEB2 buffer at 37 °C. Reaction samples (10 µL) were taken at 0 and 90 min and quenched with 2.5 µL of EDTA , analyzed by TLC on PEI-cellulose (Merck Millipore), and developed with potassium phosphate buffer . Spot intensities were imaged by using a GE Typhoon FLA 9000 Imager. For time-course experiments, the labeled RNA was incubated with purified NUDT2 or NUDT2 E58A (600 nM) in a solution (50 µL) containing NEB2 Buffer for 0–90 min at 37 °C. Reaction samples (10 µL) were quenched at time intervals with 2.5 µL of EDTA  and analyzed by TLC on PEI-cellulose (Merck Millipore) developed with potassium phosphate buffer . Spot intensities were imaged by using a GE Typhoon FLA 9000 Imager.48, except that after deprotection of the dinucleotides, the reaction mixture was converted into the triethylammonium salt using Dowex Et3NH+ before reverse-phase chromatography on a Source RP column. DecNHpppApG and DecNHpppGpA RP-HPLC product peaks were converted into pppApG and pppGpA as described. The pure pppGpA and pppApG were isolated as triethylammonium salts by ion-exchange chromatography on a Hi Screen DEAE FF column using a gradient of 0.0–0.6 M Triethylammonium bicarbonate. The integrity of the pppApG and pppGpA dimers was verified by LC-MS analysis.Dinucleotides were synthesized using DMT-2′-O-TBDMS-rA(bz), DMT-2′-O-TBMDS-rG(ib) amidites, and rA(Bz) and rG(ibu) CPG (Chemgenes) on a 10 µM scale by using standard solid-phase oligoribonucleotide coupling techniques. The CPG-bound dimers were triphosphorylated using the cyclotriphosphate protocol of triphosphate synthesisA short RNA  was incubated for 2 h at 37 °C in a solution (15 µL) containing NEB2 buffer (1×) with recombinant NUDT2 (600 nM), NUDT2 E58A (600 nM), RppH (5 U) or left untreated. Reactions mixtures were quenched with 3.75 µL of EDTA  and diluted 1:20 with water. For time-course measurements, the two short RNAs  were incubated with purified recombinant NUDT2 (600 nM), NUDT14 (600 nM), and NUDT2 E58A (600 nM), respectively, in a solution (75 µL) containing NEB2 buffer (1×) at 37 °C. Reaction samples (15 µL) were taken after 0, 1, 3, 6, and 19 h, quenched with 3.75 µL of EDTA , and diluted 1:20 with water. Aliquots (80 µL) were analyzed by reversed-phase (RP)-HPLC on an AGILENT 1100 System using an XBridge C18 column . Chromatography was performed using a two-eluent buffer system. Buffer A consists of an aqueous solution of 100 mM Et3NHOAc, pH 7.8, and buffer B consists of an aqueous solution of 100 mM Et3NHOAc, pH 7.8, and 40% (v/v) MeCN. Chromatograms were recorded at a wavelength of 260 nm. HPLC was performed using the following gradient condition: 0% B over 4 min, to 15% B over 20 min, to 18% B over 16 min to 100% B over 10 min, held at 100% B for 15 min, to 0% B over 2 min, held at 0% B for 13 min with a flow rate of 0.3 mL/min. Quantification of eluted compounds was performed by integration of the corresponding peak areas.To assess RNA integrity after treatment with recombinant Nudix hydrolases, small reaction samples were analyzed by the Bioanalyzer (Agilent) using the Agilent Small RNA kit according to the manufacturer’s protocol. For quantitative RT-qPCR analysis, total RNA from cells was isolated using the NucleoSpin RNA Plus kit (Machery-Nagel). RNA from mouse cerebellum, heart, liver, lung, or spleen was extracted using Lysing Matrix M and the FastPrep-24  instrument. Organs were directly lysed in LBP buffer, and RNA isolation was continued using the NucleoSpin RNA Plus kit (Machery-Nagel). Next, 200–500 ng of RNA was reverse-transcribed with PrimeScript RT Master Mix (TAKARA) and after that quantified by RT-qPCR using the QuantiFast SYBR Green RT-PCR Kit (Qiagen) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Each cycle included 10 s at 95 °C and 30 s at 60 °C, followed by a melting curve analysis. Primer sequences are depicted in Supplementary Table NUDT2 gene and a non-targeting control were designed and cloned into pLentiCRISPRv2 (Addgene #52961) as previously described50. HEK 293T cells were co-transfected with the obtained plentiCRISPR v2 vector (6 μg), pxPAX2 (3 μg), and pMD2G (1.5 μg) in a 10 cm dish using PEI. 24 h post-transfection, the medium was replaced with 10 mL of fresh DMEM. Seventy-two hours post-transfection, the medium was collected, filtered through a 0.45 µm pore size membrane (Millipore), and HeLa cells were infected with the lentivirus. Twenty-four hours post-infection, the medium was replaced with DMEM containing puromycin (1 µg/mL) for four passages. Knockout efficiency was analyzed by western blotting.Double-stranded 30-nt guide sequences  using 1 µL METAFECTENE Pro or 250 nmol ApNudt2 allele was rendered unfunctional by introducing a poly(A) signal after exon 1 in a construct purchased from the European Conditional Mouse Mutagenesis consortium (EUCOMM). This genetically modified ES cell clone  was injected into C57BL/6 blastocyst donors. Male chimeras were bred with C57BL/6 females to produce heterozygous Nudt2+/tm1a mice (Nudt2+/−) containing a tm1a cassette encoding for the lacZ gene, a neomycin resistance, a splice acceptor, and a polyA site flanked by FRT sites. Heterozygous animals were then bred to generate homozygous Nudt2tm1a/tm1a mice (Nudt2−/−).The For genotyping NUDT2 knockout mice, ear punches were collected from 21 to 23-days old mice and genotyped by PCR using the following primers: Nudt2_fwd: 5′-ccagctttcttgtacaaagtgg-3′, Nudt2 wt_fwd: 5′-gaatttctgctgctgcaggc-3′, Nudt2_rev: 5′-cctagtgaagggacaaagcagc-3′ and using the following parameters: 94 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, 65 °C for 20 s, and 72 °C for 1 min. The wild-type allele was detected as a band at 492 bp, whereas the inserted tm1a cassette was detected as bands of 417 and 912 bp.All animal experiments were performed in compliance with the German Animal Welfare Law . The mice were housed and handled in accordance with good animal practice as defined by FELASA. All animal experiments were approved by the responsible state office , the University of Magdeburg, under permit number AZ 42502-2-1344. During intranasal infections (i.n), 8–12-week-old mice were first anesthetized by i.p. injection with a mixture of ketamine (100 mg/g body weight) and xylazine (5 mg/g body weight). They then were infected with 5e6 plaque-forming units (PFU) of VSV in 20 µL PBS unless otherwise indicated. Mice that lost more than 20% of their body weight were sacrificed.Nudt2 knockout bone marrow cells  were lysed in 300 µL of lysis buffer  supplemented with complete protease inhibitor (Roche) and boiled for 10 min at 95 °C. The protein concentration of cleared cell lysates was assessed using Pierce 660 nm Protein Assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. Protein concentrations for all cell lines and replicates were equalized to 35 µg protein content and alkylated with 55 mM IAA for 20 min in the dark. Proteins were precipitated with acetone and resuspended in 150 µL of denaturation buffer . Protein digestion was performed by adding 1:100 (protein:enzyme) trypsin and LysC overnight at room temperature. Peptides were purified on stage tips and analyzed by LC-MS/MS using the EASY-nLC 1200 system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptide mixtures were separated on a 50 cm C18-reversed-phase column  using a 180-minute linear gradient of 5−30% buffer B (0.1% formic acid and 80% ACN) with a flow rate of 250 nL/min. The mass spectrometer was set up to run a Top10 method acquiring full scans  at a target of 3e6 ions, followed by isolation of the ten most abundant ions, HCD fragmentation  and detection in the Orbitrap analyzer.For proteomic analysis, wild-type and Peptide identification and quantification were performed using MaxQuant (version 1.6.17.0). For all MaxQuant searches, typical default parameters were employed. Spectra were searched against forward and reverse sequences of the reviewed mouse proteome, including isoforms . Settings included carbamidomethylation of cysteine as fixed modification and oxidation of methionine and N-terminal protein acetylation as variable modifications. Trypsin/P was specified as the proteolytic enzyme. The minimal peptide length was defined as 7 amino acids, and match-between-run was disabled for LFQ determination.t-test (FDR 0.05) was performed, including proteins with at least three valid values  and imputed (width 0.3 and downshift 1.8).The protein groups were further analyzed with Perseus (1.6.14.0). The matrix included 6288 proteins and was filtered with the default settings: potential contaminant only identified by site and reverse. After log2 transformation, a two-sided students E. coli RppH were analyzed using MAFFT53 with standard settings. The resulting tree was displayed using Dendroscope54 and visually adapted using Adobe Illustrator CS6 (16.0.3).To establish the phylogenetic relationship between different human Nudix hydrolases and bacterial RppH, protein sequences from all human Nudix hydrolases and Homo sapiens, Danio rerio, Caenorhabditis elegans, Mus musculus, Xenopus laevis, Gallus gallus, and Drosophila melanogaster were collected and aligned using the Clustal Omega tool with standard settings. The multiple sequence alignment was visualized using ESPript 3.055.Protein sequences of NUDT2 from All data were analyzed either using GraphPad Prism (8.4.3) or R (4.0.2) using R Studio (1.3.1056). Data acquired with the Incucyte live-cell imaging platform were analyzed using IncuCyte Analysis Software (2019B Rev2).Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Coral reefs provide numerous ecosystem goods and services, but are threatened by multiple environmental and anthropogenic stressors. To identify management scenarios that will reverse or mitigate ecosystem degradation, managers can benefit from tools that can quantify projected changes in ecosystem services due to alternative management options. We used a spatially-explicit biophysical ecosystem model to evaluate socio-ecological trade-offs of land-based vs. marine-based management scenarios, and local-scale vs. global-scale stressors and their cumulative impacts. To increase the relevance of understanding ecological change for the public and decision-makers, we used four ecological production functions to translate the model outputs into the ecosystem services: “State of the Reef,” “Trophic Integrity,” “Fisheries Production,” and “Fisheries Landings.” For a case study of Maui Nui, Hawai’i, land-based management attenuated coral cover decline whereas fisheries management promoted higher total fish biomass. Placement of no-take marine protected areas (MPAs) across 30% of coral reef areas led to a reversal of the historical decline in predatory fish biomass, although this outcome depended on the spatial arrangement of MPAs. Coral cover declined less severely under strict sediment mitigation scenarios. However, the benefits of these local management scenarios were largely lost when accounting for climate-related impacts. Climate-related stressors indirectly increased herbivore biomass due to the shift from corals to algae and, hence, greater food availability. The two ecosystem services related to fish biomass increased under climate-related stressors but “Trophic Integrity” of the reef declined, indicating a less resilient reef. “State of the Reef” improved most and “Trophic Integrity” declined least under an optimistic global warming scenario and strict local management. This work provides insight into the relative influence of land-based vs. marine-based management and local vs. global stressors as drivers of changes in ecosystem dynamics while quantifying the tradeoffs between conservation- and extraction-oriented ecosystem services. Coral reef ecosystems provide valuable resources. They buﬀer coastal erosion, provide a cornucopia of food resources, attract tourism dollars, supply construction and pharmaceutical materials, and provide recreational opportunities for humans and essential habitat for threatened and endemic organisms . Further2) emissions are slowly causing the world’s oceans to become warmer and more acidic. Ocean acidification reduces calcification rates of all calcifying organisms including corals. Intense or prolonged ocean warming can result in the expulsion of the symbiotic algae that live in the coral tissue leaving them looking “bleached” and is hence called coral bleaching. Bleached corals have a higher change of mortality as they become more susceptible to pathogens  establishment] could improve the provision of coral reef ecosystem goods and services, an ecosystem model that simulates impacts of both land- and marine-based management was parameterized for Maui Nui, Hawai’i, i.e., the islands of Maui, Lāna’i, Moloka’i, and Kaho’olawe. These management approaches were also combined with two future severities of climate-related stressors. Reefs of Maui Nui served as a case study, but this tool can be used in other areas with similar local and global threats . AlthougHere, we ask two questions: (1) What is the relative importance of land- and marine-based management action? and (2) Can local management mitigate the eﬀects of climate-related stressors? We expect that a combination of proactive local actions (sediment mitigation and fisheries controls) will attenuate declines in coral reef ecosystem goods and services delivery, but without local management, reefs will continue to decline, a trend exacerbated with more extreme future climate conditions.2 of shallow coral reef habitat across the Hawaiian Islands of Maui, Molokai and Lāna’i, i.e., Maui Nui  are classified as MPAs, with just over 9 km2 of the protected areas being designated as “no-take” area. The model domain consists of the shallow (0–30 m) reef zone around Maui Nui and is spatially represented by a 500 × 500 m grid cell network.Our study area encompasses ∼325 kmHIReefSim (Hawai’i Reef dynamics Simulator) is based on the framework of the Coral Reef Scenario Evaluation Tool (CORSET) developed by Fung  and adapThe HIReefSim model defines two consumer functional species groups, herbivorous  and piscivorous (predatory) fishes, and five benthic functional groups: macroalgae, turf algae, crustose-coralline algae (CCA), and spawner and brooder corals. Boosted regression trees generated spatial predictive maps of these ecological variables based on observations from a compilation of underwater surveys and an extensive gridded predictor dataset  Stamoul. Unlike Coral reef ecosystems are extremely complex systems and influenced by a myriad of variables. HIReefSim only includes key ecological dynamics by using diﬀerential equations to estimate the interactions among the functional groups and their response to stressors in each grid cell . For exaSpatially explicit stressor functions included nutrification and sedimentation , fishing , and coral and macroalgal mortality resulting from wave action , that escape piscivore predation because of their size. However, if fishing is not restricted, these larger-bodied fishes are key targets for spearfishers and an increase in spearfishing is not accounted for in the model. Additionally, reef structure is likely to erode due to coral cover decline, preventing fish recruits and juveniles from hiding  = aC / [1 + (a/b)∗Cd], where C is coral cover, and a, b and d are fitted parameters ], where C is coral cover and H herbivore biomass and Fpred a fitted parameter  arameter  ; andEvaluate whether the model can reproduce historic broad-scale dynamics for the Maui Nui region over the past 30 years (1985–2015), given a timeline of known stressors . Historigt, gm) and the biomass accumulation from grazing , and for piscivores this was prey availability (iph), predation pressure (gp) and biomass accumulation from predation (rp). We therefore randomly selected 50 values between the estimated ranges , which are directly correlated with storm intensity, are increasing as well . Murakam2), and “Fisheries Landings”  to translate HIReefSim model output into values important for management applications : “State of the Reef ” (unitless), “Trophic Integrity of the Reef ” (unitless), “Fisheries Production”  and CCA] and fleshy algae [turf (T) and macroalgae (MA)] and the trophic level of the fish community with herbivores (H) having a trophic level of 2 and piscivores (P) a trophic level of 4:The trophic integrity was estimated by the ratio of calcifiers [corals  was calculated as a ratio of total fish biomass . This raThe model directly calculates fish catch as a function of available fish biomass (basemap input layer) maximum fishing eﬀort, and accessibility  and marine-based management scenarios , sediment mitigation strongly impacted benthic composition, whereas fisheries management impacted fish biomass . For exaMarine-based management strategies had minimal impact on the trajectories of coral cover compared to the Current Management scenario , 5.2. CCThe expansion of current MPA boundaries to include areas encompassing the top 10% (B3) and 20% (B4) of fish biomass and coral cover or the randomly placed 30% MPAs (B1 and B2) showed no clear pattern in the results. Striking is that the placement of the 30% no-take MPAs had opposite outcomes for fish catch which declined by up to 20% under B2 and increased to 32% under B1  for herb2 across Maui Nui from 2015 to 2050  to increasingly stringent local management of sediment and fishing stressors under severe climate-related stressors , especially under the scenarios including 30% MPAs . Due to the low piscivore biomass, an additional 20% reduced piscivore pressure (F) had almost no additional eﬀect in landings .Looking at the eﬀectiveness of local management scenarios by comparing the end states of the EPFs of each scenario relative to current management, both the Trophic Integrity of the reef as well as the State of the Reef can benefit from additional management especially under less severe climate change . FisheriSpatial variation in the results of management can provide insights into where to target local management. For example, the southern coastlines of Moloka’i and Maui showed declines for the State of the Reef and strict local management had little eﬀect . By contSpatial patterns in Fisheries Production showed improvement under all scenarios and along most coastlines but less so along the northern coastline of Moloka’i  is needed everywhere–not just across 30% of the nearshore area–to obtain the best results for reef state and trophic integrity under all climate change scenarios. Secondly, place matters. We found that spatial variation in the eﬀects of local management was particularly high for reef state and fisheries production, and moderate for trophic integrity and fisheries landings, suggesting that fine-tuning place-based management could improve outcomes.Yes, despite a decline in Trophic Integrity and State of the Reef EPFs, both these EPFs displayed a buﬀering eﬀect of strict management on the degradation of coral reefs, mostly under the less severe climate change scenarios. However, as a tradeoﬀ, Fisheries Landings decreased overall compared to today’s levels but the maximum decrease was just under 6% . AverageHerbivores appeared to fare well under climate-related stressors as coral cover declined and algal cover increased , 7. The Alternatively, the increase in piscivores could be viewed as an undesired outcome since more piscivores led to elevated predation pressure on herbivores, reducing their biomass and the overall Fisheries Production –7. EcoloWide-scale spatial variability across Maui Nui was projected for the EPFs, with declines of up to 100% across the northern shores of Maui and Moloka’i for Fisheries Production, and large declines in State of the Reef along southern and eastern shores (where the impacts of hurricanes were projected to be highest), highlighting the importance of identifying areas where management will likely be most successful.Eﬀectively managing the entire coral reef ecosystem requires the combination of both land-based and marine-based approaches. Land-based management was most beneficial for the benthic community, especially coral cover, whereas marine-based management not only decreased macroalgal cover, opening up substrate for reef calcifying CCA, but most notably decreased the downward trend in piscivore biomass . Of equaRandom placement of no-take MPAs may not benefit the overall benthic community . For exaOverall, in the face of global threats, local management had mixed results for the ecosystem goods and services the reef provides. It abated the decline in Trophic Integrity of the reef and improved State of the Reef especially when both land-based and marine based approaches were combined but reducing greenhouse gas emissions is essential to avoid catastrophic loss of ecosystem services. Management that was more stringent required trading oﬀ Fisheries Production and Landings. By including extraction and reef resilience objectives in ecosystem goods and services, we provide a generalizable tool to clearly evaluate the tradeoﬀ of these conflicting goals under various management and climate-related stressors. Based on the limitations of the model structure, a cautionary interpretation of the model’s predicted long-term trajectory should be taken. Future work may want to improve the resource fish ratio, which we assumed to stay constant over the entire period. This assumption is unrealistic because fishers are likely to target specific species , causing community structure to shift. Furthermore, the results from the small number of MPA scenarios call for more thorough analysis, particularly with respect to their interaction with land-based pollution control. The model could also be used for evaluating the relative eﬀectiveness of diﬀerent fisheries management strategies, e.g., targeting diﬀerent trophic groups. A similar endeavor could focus on compliance within those MPAs to test the eﬀectiveness of restriction levels. Results of these improvements could greatly benefit the ongoing discussions of how to manage 30% of the coastline eﬀectively.sup1


Negarnavaricota, realm Riboviria, kingdom Orthornavirae, genus Phlebovirus) comprise three genome segments of ribonucleic acid (RNA) and which encode an RNA-dependent RNA polymerase, which they use to transcribe the viral RNA genome into messenger RNA and to replicate the genome. At least some of these viruses cause mild 3-day fevers in humans but some also have been associated with more severe illnesses in humans. The 67 recognized phleboviruses are listed here in a table composed by the authors from International Committee on Taxonomy of Viruses reports as well as the scientific literature.Sandfly-borne phleboviruses (phylum  Phlebovirus  comprise three genome segments of ribonucleic acid (RNA) which encode an RNA-dependent RNA polymerase, which they use to transcribe the viral RNA genome into messenger RNA and to replicate the genome . The genus currently comprises 67 species, listed in Phlebovirus, despite the observations that most tick-borne viruses formerly included in the genus Phlebovirus are now included in the genus Uukuvirus. Some phleboviruses have been isolated from vertebrates, such as wild or sentinel rodents in the Americas, and in Africa, such as opossums or sloths. Other phleboviruses have been isolated from febrile patients in South America  and Phlebotomus and Sergentomyia (both found in the Old World), the former two genera contain the more relevant species able to transmit viral pathogens [Austrosimulium, a type of black fly (https://en.wikipedia.org/wiki/Sandfly accessed on 10 April 2021).“Sandfly” (or “sand fly”) is a colloquial name for members of any species or genus of flying, biting, blood-sucking dipteran encountered in sandy areas. In the United States, “sandfly” may refer to certain horse flies that are also known as “greenheads”, or to members of the family Ceratopogonidae. Outside the United States, “sandfly” may refer to members of the subfamily Phlebotominae within the Psychodidae. The three main genera are athogens . Biting Infections with many of these viruses cause mild 3-day fevers, also known as pappataci fevers or phlebotomus fevers . These iA diagnostically complicating feature of phlebovirus replication may be reassortant generation resulting from multiple simultaneous phlebovirus infections . As withTo assess possible reassortant phleboviruses, amino acid sequences of the RdRp and correspondent M segments have been retrieved from GenBank and aligned with MAFFT . The perAdana phlebovirus species, according to the RdRp threshold. The possibility of reassortment involving the M segment would be a relevant phenomenon in the evolution of this group, as similarly reported for orthobunyaviruses [The likely reassortant phleboviruses have different M segments . Reassortment events have been described for phleboviruses of the Candiru antigenic complex , among Maviruses . The M saviruses ,19.


However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.Retroviral RNA genome (gRNA) harbors   This isthe gRNA . Gag is the gRNA . Howeverequences . Such seequences . Howevercis-packaging mechanism had initially been suggested for human immunodeficiency virus type 2 (HIV-2), it is now generally accepted that packaging of retroviral gRNA takes place independently from Gag translation  which are retrovirus-like elements also known as endogenous retroviral elements . LTR retnslation . It has nslation ,28 and/onslation  co-existnslation . A recennslation . Similarnslation . On the pol are necessary for packaging its gRNA, whereas in Ty1, a 144 nucleotide-long region at the 5′ end has been identified as critical for Ty1 gRNA packaging  also critically contributes to Pr77Gag binding. Furthermore, our in cell packaging data also established that ssPurines and PBS are not redundant during the MMTV life cycle, as loss of either of these Pr77Gag binding sites ablates gRNA packaging. To the best of our knowledge, these results demonstrate for the first time a direct role of the PBS in retroviral gRNA encapsidation. Future studies will reveal if this feature is unique to MMTV or if it also exists in other retroviruses and/or LTR retrotransposons. Identifying the structural motifs that allow gRNA/Gag interactions is likely to offer opportunities to develop small molecule-based anti-retroviral therapeutic interventions specifically targeting virus assembly especially given the fact that retroviral Gag is the only gene necessary for virus particle formation and gRNA encapsidation for the perpetuation of the virus life cycle.Therefore, to establish whether ssPurines or other sequences within the packaging determinants of MMTV mediate gRNA packaging, possibly by functioning as a Gag binding site(s), we performed a combination of  Pr77Gag  and biolNucleotide numbers refer to nucleotide positions of HYB-MTV, a molecular clone created by Shackleford and Varmus .Gag-His6-tag protein) was expressed, purified and characterized as previously described  of the molecules in solution, via the Stokes-Einstein equation:k is the Boltzmann constant, T is the temperature and μ is the viscosity of the solvent. Before sample acquisition, buffer was filtered through 0.2 μm filters (Millex®) and the offset of the solvent was measured for subsequent sample data treatment.Pr77SpeI sites were cleaved and then cloned into the subgenomic transfer vector DA024 that was previously cleaved with the same restriction endonuclease. All clones were confirmed by sequencing.Desired mutations in the ssPurines and other regions were introduced through splice overlap extension (SOE) PCR using MMTV subgenomic transfer vector DA024  as the tin vitro transcription were created as previously described , and resuspended in 30 μl Milli-Q water. Transcription was performed using MEGAscript T7 transcription kit according to manufacturer's instructions (Thermo Fisher Scientific) and following DNase treatment, RNA was extracted, precipitated and resuspended in 500 μl Milli-Q water. The in vitro transcribed RNAs were then purified by gel filtration chromatography using a TSKgel G4000SW column (Tosoh Bioscience) with running buffer containing 200 mM sodium acetate and 1% methanol. The eluted fractions under a same peak were pooled, ethanol precipitated, and resuspended in 150 μl of Milli-Q water. The RNA integrity was checked using denaturing polyacrylamide gel electrophoresis (PAGE).For in vitro transcription in the presence of [α-32P]-ATP, as described earlier  and 0.4 μg of yeast tRNA at 90°C for 2 min followed by chilling on ice for 2 min. The denatured RNAs were next allowed to re-fold by incubation in 1× folding buffer  at 37°C for 30 min. Next, increasing concentrations of Pr77Gag were mixed with the refolded RNA . The mixture was then incubated at 37°C for 30 min for binding, followed by incubation on ice for 30 min. Samples were separated using 1% agarose gel with electrophoresis performed in TBM buffer  at 150 V for 4 h at 4°C. The gels were fixed in 10% trichloroacetic acid (TCA) for 10 min, dried under vacuum for 1 h and analyzed using a FLA 5000 (Fuji) scanner. Quantitative analysis of the bands was performed using ImageQuant software  in a volume of 10 μl. In the meantime, a 20 μM stock of Pr77Gag was diluted in a buffer containing 30 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2, 10 mM DTT and 0.02 mg/ml BSA in a volume of 5 μl. The refolded RNAs were then mixed with the diluted protein  and incubated at 37°C for 30 min for binding and then on ice for 30 min for stabilization. The reaction mixtures were analyzed on 1% agarose gels with electrophoresis performed in TBM buffer  at 150 V for 4 h at 4°C. The gels were then fixed and analyzed as described above.For band-shift competition assays, internally labeled WT RNA (25 000 cpm) and 10 nM unlabeled WT RNA  using a dot blot apparatus. Membranes were pre-wetted with Tris buffered saline solution  and pre-washed once with 100 ml of buffer . After sample filtration, wells were washed three times with 60 ml of cold buffer , the membranes were removed from the filtration apparatus, and dried on air. The filters were exposed with an Imaging Plate (Fujifilm), scanned with a FLA 5000 (Fuji) scanner and quantified with the ImageQuant software  were mixed and denatured at 90°C for 2 min followed by cooling on ice for 2 min. The RNAs were then refolded in a folding buffer  along with 5 units of RNasin and 2 μg total yeast tRNA in 10 μl reaction volume for 30 min at 37°C. The 20 μM Pr77Gag stock was diluted in the same folding buffer and mixed with the refolded RNA to a final concentration of 4 μM; RNA in the folding buffer without Gag was used as a control. The mixture was incubated at 37°C for 30 min to allow for protein binding and then stabilized by cooling on ice for 30 min. This was followed by modification of the RNA samples by 100 mM BzCN dissolved in anhydrous DMSO or DMSO alone as a control. The modified and control RNAs were extracted using Roti®Aqua, ethanol precipitated, and the pellets were resuspended in 7 μl Milli-Q water.High-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) was performed on the WT (SA35) MMTV RNA either in the presence (4 μM) or absence of Pr77escribed . BrieflyGag were used as pseudo-energy constraints to fold the RNA secondary structure of the MMTV packaging signal using the program RNAstructure version 6.1  and AK30  as described above except that only yeast tRNA was used as the competitor. For AK29 RNA, Splice_1_1 and Splice_3_1 oligos were used for reverse transcription and Splice_1_2 and Splice_3_2 primers were used for sequencing. Similarly, for AK30 RNA, Splice_1_1 and Splice_2_1 oligos were used for reverse transcription and Splice_1_2 and Splice_2_2 primers were used for sequencing  pseudotyped particles produced by JA10 and MD.G (hygromycin B phosphotransferase gene (hygr), which helps to monitor the effect of the mutations introduced into this transfer vector following transduction of the target cells by the viral RNA in a single round of replication assay  293T producer cells and purified as described before ,60. A frNuclear and cytoplasmic fractions were separated from transfected cells as described previously . Cellulat-test was performed for determination of statistically significant differences in RPE between the WT and the mutations introduced.Quantitation of the transfer vector RNAs expressed in the cytoplasm and packaged into the pelleted viral particles was accomplished using a Taqman assay and the relative packaging efficiency (RPE) for each mutation was determined as described previously ,59,60. OIn vitro dimerization assays were performed using RNAs that were in vitro transcribed using MEGAscript™ T7 transcription kit (Invitrogen). The in vitro transcribed RNAs were purified using MEGAclear™ kit (Invitrogen) and ethanol precipitated as described previously. Briefly, 300 nM RNAs were denatured at 90°C and incubated on ice for 2 min followed by refolding at 37°C for 30 min in either the dimer  or monomer  buffers. The samples were then electrophoresed in TBM buffer  at 4°C or TBE buffer  at 25°C using agarose gels prepared in respective buffers. Density of the dimeric and monomeric RNA species was quantitated using ImageQuant software, and dimerization efficiency was calculated by dividing intensity of dimeric RNA band divided by intensity of total RNA bands  and expressed as dimerization relative to the WT for each mutation introduced.Gag polyprotein containing His6 tag at the C-terminus expressed in Escherichia coli .We first analyzed binding of gRNA to increasing concentrations of Pr77g Figure . Shiftedy Figure . After qy Figure , and theGag since an almost complete shift of radiolabeled unspliced gRNA (<1 nM) was observed at this concentration .Next, band-shift competition experiments were carried out in the presence of 800 nM Pr77n Figure . To deten Figure . We obsen Figure . Howevers Figure ; lane 9,s Figure , both gRGag to unspliced genomic and spliced RNAs by a different technique, direct filter-binding assays, not involving competition between several RNA species, were performed using radiolabeled WT (SA35) or spliced (AK29 and AK30) RNAs in the presence of increasing concentrations of Pr77Gag. Compared to WT unspliced gRNA (SA35), the spliced (env and sag) RNAs showed a drastic reduction in their affinity for Pr77Gag, further stressing the existence of high affinity binding site exclusively on the unspliced gRNA  on Gag binding, a mutation (AK20) was created in which six of the nine purines were substituted with other purines  or ssPurine mutant RNAs  and AK50  revealed slightly reduced levels of binding to Gag compared to the WT gRNA  and AK45 (5′ CCUGAAGUC 3′) containing partial substitution of purines to pyrimidines showed a slightly reduced competition to WT gRNA  and AK63  exhibited a drastically reduced ability to bind to Pr77Gag . The results obtained revealed that all the three mutations maintained dimerization abilities similar to that of the WT (SA35) RNA (To ensure that the introduced mutations in ssPurines somehow did not compromise the ability of the mutant RNAs to dimerize (which in turn may result in poor affinity for Gag), A35) RNA .Gag and the results observed were not due to effects of the introduced mutations on gRNA dimerization. Notably, the 5′ GGAG and 3′ AG nucleotides are important for retaining high affinity binding  act as a high affinity binding site for Pr77 Figures  and 6A.env; AK29 and sag; AK30; Figure in vitro binding assays performed on these spliced RNAs showed poor competition efficiency/binding capacity to Pr77Gag compared to unspliced RNA  and sag (AK30) spliced RNAs to elucidate the secondary structure of these RNAs. Results of these experiments revealed that the ssPurines in both the env and sag spliced RNAs were present in a base-paired state with neighboring sequences and therefore unavailable for Pr77Gag binding  as well as spliced mRNAs  and WT ψ-containing RNA, both in the absence as well as presence of Pr77Gag. The nucleotides showing reduced SHAPE reactivity in the presence of the protein correspond to protein binding site(s), as Pr77Gag binding protects these nucleotides from modification by BzCN. To minimize non-specific binding of Pr77Gag to WT RNA, hSHAPE was performed in the presence of excess spliced env mRNA . Pr77Gag was used at a concentration of 4 μM  to ensure complete binding of RNA to Pr77Gag. Under the conditions (protein and competitor RNA concentrations) used for footprinting, the band-shift assay showed formation of only a single complex, indicating that we were looking only at the high affinity binding site in the SHAPE footprinting experiments  was similar to wild type levels  showed nearly 80% reduction in packageability of the mutant RNA  was found to be slightly increased when compared to AK45 containing flanking substitutions  showed packaging efficiency comparable to WT  resulted in a drastic reduction in the packaging efficiency  and AG at 3′ end of ssPurines . Contrary to our expectations, RNA transduction efficiency of AK20 RNA was observed to be only 50%  despite the fact that the RNA containing this mutation was packaged almost to the WT (DA024) levels  that appeared when viral particles containing these mutants RNAs were used to transduce target cells. For all the introduced mutations, relative transduction of the packaged RNA corroborated well with the relative packaging efficiency except for mutation AK20  is schematically shown in Figure After having confirmed that the ssPurines are required for MMTV gRNA packaging, it was important to determine the role of other nucleotides that have shown attenuation of SHAPE reactivity in our footprinting assays. It was particularly important to ascertain whether the reduced SHAPE reactivity was due to direct Pr77After performing the same necessary controls as described for the other mutations , the pacGag binding, AK73 and AK74 mutations were tested in band-shift competition assays. Both mutations (AK73 and AK74) showed significantly reduced affinity to Pr77Gag compared to the WT (SA35) in band-shift competition assays  between nt 48–318  also showed a 40% and 96% reduction in their packaging abilities respectively when compared to WT (DA024). To determine if these nucleotides played a true role in Pr77s Figure . In ordet 48–318 . Howevert 48–318 . These dGag binding, SHAPE footprinting experiments were performed using the mutation AK74 RNA  that might affect RNA dimerization and, indirectly, RNA packaging  with a heterologous palindromic sequence (CGACGTGGCCG). On the other hand, mutation AK81 had disrupted the palindromic nature of the PBS while maintaining the nucleotides that showed attenuation of SHAPE reactivities in the presence of Gag. In the mutation AK82, the overlapping palindromes were maintained but the four downstream nucleotides that showed attenuation of SHAPE reactivities in the presence of Gag were substituted. Finally, in mutation AK83, the palindromic sequences were substituted with non-palindromic sequences concomitantly with substitution of three out of seven nucleotides at the 5′ end of the PBS showing Gag footprints, thus restricting the mutations to only the palindromic regions within PBS.Briefly, starting with a DIS pal mutation (SA44), a double mutation was created in which the palindromic nature of DIS as well as that of PBS pal was lost , the PBS pal  or both palindromes (AK84) were mutated Figure . While RNext, we compared the RNA packaging ability of the various mutations introduced in the 7 nucleotides of the PBS region which showed Gag footprints Figure . In the Together, these results indicate that (i) the palindromic sequences in PBS are not required for RNA dimerization  for Gag protein recognition  constitute the DIS  used in our study, Pr77Gag is unable to bind to those RNAs with high affinity  mediate RNA–protein interaction. Since gRNA packaging during virus assembly is vital for the continuity of viral life cycle, these findings have ramifications towards the development of therapeutic interventions based on unique antiretroviral drugs that target virus assembly, especially given the fact that only retroviral Gag is required for virus particle formation. These drugs could then target specific structural elements (such as ssPurines and PBS) within the packaging signal RNA or its interaction domains within the Gag protein using novel small molecule approaches ,95,96. UAll data pertaining to this manuscript has been provided as supplementary data.gkab223_Supplemental_FilesClick here for additional data file.


HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE  technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome. Lys3 primer that anneals to the gRNA at some stage during viral assembly and budding. The chaperone activity of NC facilitates the annealing of tRNA to the primer binding site (PBS), and aids reverse transcription by destabilising the secondary structures that would cause the pausing or stalling of the enzyme [The HIV-1 genome (gRNA) is a single-stranded RNA molecule that encodes the essential structural polyproteins Gag, Gag-Pol and the envelope glycoprotein Env, together with a number of accessory factors that aid viral replication and immune evasion. gRNA thus serves as a template for the translation of the viral structural proteins found in Gag and the enzymes encoded by Pol, as well as being captured by Gag for packaging into virions. During packaging, the gRNA undergoes dimerisation, resulting in two copies of the genome being encapsidated into the budding virions. During or after budding, the Gag and Gag-Pol polyproteins are cleaved into their individual components—matrix (MA), capsid (CA) and nucleocapsid (NC)—as well as three smaller peptides [e enzyme ,5,6,7,8.gag initiation codon by forming the U5:AUG helix [A critical step in this complex viral replication cycle is the recognition and packaging of the viral gRNA. The gRNA packaging process is highly specific and represents a novel drug target . It has UG helix ,26,27,28gag to gRNA dimerisation is facilitated by an RNA structural switch. There have been two predominant models for this. Firstly, a switch from a ‘Long Distance Interaction’ (LDI) conformation to a ‘Branched Multiple Hairpin’ (BMH) [gag initiation codon being located within a less stable structure than within the BMH conformation, to facilitate translation. In the BMH conformation, the gag initiation codon is sequestered through base pairing with the U5 region (referred to as the U5:AUG interaction), releasing the DIS and allowing it to base pair with the DIS on a second gRNA [It has been previously proposed that the shift from the translation of n’ (BMH) ,27,29. Iond gRNA ,27,29,30ond gRNA ,31. In tond gRNA ,33,34.The initiation of gRNA encapsidation is generally accepted to involve a small number of Gag proteins binding to Ψ . The swiWe previously demonstrated an in-gel SHAPE  method that was able to resolve the structures of the monomeric and dimeric HIV-1 leader sequences, without the need for stabilising mutagenesis, that identified certain key structural changes involved in RNA dimerisation . SHAPE rHere, initial experiments on the well-established TAR–Tat interaction suggested that SHAPE reagents reliably report upon the structural flexibility of the backbone at each nucleotide without being strongly affected by the ‘footprint’ of the protein binding. However, the structural ensemble of monomeric TAR RNA in the absence of Tat differs from the structural ensemble of the unshifted TAR that was incubated in the presence of Tat. Effectively, the technique appears to reveal the sub population of structures within the ensemble to which the protein did not bind.We then used in-gel SHAPE to study the 5′ region of the HIV-1 gRNA from the transcription start to within the beginning of the Gag open reading frame that contains the major sequences required for gRNA encapsidation. We sought to identify changes that occur in the structural ensembles of the HIV-1 RNA monomer and dimer RNA species upon the addition of Gag or NC during the gRNA dimerisation process.We found that the monomeric ensemble in the absence of a ligand, as well as the structures with Gag bound, largely resemble the LDI model, with the DIS paired in a long-range interaction with the U5 region. The monomeric ensemble with NC bound was more heterogeneous but still contained many of the LDI features. Within the dimeric ensemble TAR, poly(A) and SL1 structures were frequently present, but the dimer in the absence of a ligand did not contain the U5:AUG helix; however, the shifted dimer did. Our results show the surprising diversity of the RNA structural ensembles that could potentially be formed, and how they differ when Gag or NC bind to the RNA. They also indicate the structures preferentially selected by Gag or NC, as well as how the proteins remodel the RNA.XL-SHAPE was able to identify the initial interaction sites of Gag with the gRNA and show that these differ from those of NC at the same molar ratio of protein:RNA. Gag first interacts with the TAR region, and in doing so has structural effects on the downstream Ψ region structure. The interaction with NC alone is more promiscuous and is more reflective of how Gag interacts with the RNA when Gag is in higher concentrations. Our results suggest a mechanism by which HIV controls the switch between translating and packaging its genome, and provide insights into the RNA structures occurring during viral maturation. Interference with these critical structural transitions may have therapeutic potential.RNA was transcribed in vitro from DNA templates encoding the HIV-1 genome and containing a T7 RNA polymerase promoter at the 5′ end. DNA templates were synthesised by PCR with 1× BioMix red , 800 ng of plasmid DNA (pSVC21) and 30 pmol of forward (5′-TAATACGACTCACTATAGGGTCTCTCTGGTTAGACCAGATCTG-3′) and reverse (5′-CTTTCCCCCTGGCCTTAACC-3′) primers, TAR forward, 5′TAATACGACTCACTATAGGCCTTCGGGC CAAGGTCTCTCTGGTTAGACC-3′; TAR reverse, 5′CACT ACTTGAAGCACTCAAGG-3′ using plasmid pSVC21 as a template, as per Kenyon et al. (2013).gag RNA), or with 3 U of DNase per 20 μL reaction at 37 °C for 45 min (TAR RNA). RNAs were purified with MEGAclear columns (Life technologies), eluted in RNase-free water and stored at −20 °C.PCR products were purified by gel extraction  according to the manufacturer’s instructions. In vitro RNA was transcribed using the MEGAscript T7 . Each 20 μL transcription reaction contained 7.5 mM ribonucleotide triphosphates (NTPs), 1× reaction buffer, 800 ng of PCR products and 2 μL of T7 RNA polymerase, and was incubated at 37 °C for 3 h. DNA was degraded with 6 U of DNase  per 20 μL reaction at 37 °C for 90 min  was resuspended in 160 μL of renaturation buffer , heated at 85 °C for 5 min and slowly cooled inside the metal tube-holder insert by removing it from the heat block onto the bench, until it reached 29 °C. The volume was adjusted to 200 μL and a final concentration of 40 mM Tris-HCl, pH 8; 4 mM MgCl2; 0.2 mM EDTA, followed by incubation at 37 °C for 30 min.TAR RNA (24 µg) was refolded by being resuspended in 80 μL of renaturation buffer, and being heated and slowly cooled as above. The volume was adjusted to 100 μL with a final concentration of 40 mM Tris-HCl, pH 8; 130 mM KCl; 4 mM MgClw/w) in a binding buffer containing 65 mM Tris-HCl (pH 8), 26 mM KCl, 6.5 mM dithiothreitol (DTT), 0.13% Triton X-100 and 0.13 mM ZnCl2. RNA controls in the absence of protein were incubated with RNasin, tRNA and a binding buffer.For RNA with NC samples, 15× molar excess of NC (corresponding to 1NC:27.6nt) was incubated with the RNA for 15 min at 20 °C along with 4U/μL ribonuclease inhibitor  and 100 × excess of tRNA ), hence the RNA-only controls were also incubated with an equal volume of this buffer.For RNA with Gag experiments, RNA and protein (15× Gag/1 Gag:27.6nt) were incubated as above, with the exception that Gag was stored and added to the RNA in a different buffer , an additional 7.33 mM Tris-borate, pH 7 and 0.04% orange G dye. RNAs and RNA–protein complexes were then separated by native polyacrylamide gel electrophoresis using gels prepared with 4% acrylamide and 1× Tris-borate magnesium (TBM) . Samples were electrophoresed at 120 V for 15 min followed by 4 h at 110 V. The first three lanes of the gel were excised, containing an RNA ladder , one RNA sample and one RNA with viral protein sample. The gel fragment was then stained with 1.3 μM of ethidium bromide in 1× TBM for 5 min and visualised. The stained gel piece was aligned with the rest of the unstained gel, and specific bands  were excised with a scalpel. Each excised gel piece was then divided equally into two pieces. One piece of each band was incubated with 10% DMSO in 1× TBM (3 mL), and the other piece was incubated with 10% 100 mM NMIA (in DMSO) in 1× TBM (3 mL) at 20 °C for 45 min. Gel pieces were then washed twice with 1× TBM and soaked in 3 mL of proteinase K buffer (600 μg proteinase K (Ambion); 50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1% sodium dodecyl sulfate (SDS); and 10 mM EDTA) at 55 °C for 1 h. After incubation, gel slices were diced into small pieces (maximum 1 mm3) and washed three times with 1× TBM, then once with 1× Tris-acetate EDTA . Alternatively, during optimisation experiments, after gel pieces were diced, 1 mL of acetonitrile was added and centrifuged at 375× g at 10 °C for 10 min twice, and dried in a speed vac for 5 min. The gel pieces were rehydrated with 1 mL of 1× TAE. RNA was electroeluted using an Elutrap  overnight at 100 V and 4 °C. RNA was purified using phenol-chloroform extraction  followed by ethanol precipitation using 300 mM sodium acetate, pH 5.5 , and 2.5 volumes of ice-cold ethanol.Samples were mixed with native loading dye at a final concentration of 4% glycerol  per lane, and corresponding molar ratios of Gag. Samples were electrophoresed as described for in-gel SHAPE experiments above, gels were dried onto filter paper and visualised by autoradiography.RNA was in vitro transcribed using TM or PET® .For in-gel SHAPE experiments, 200 ng of RNA of both the DMSO control (−) and NMIA-treated (+) samples was resuspended in 12 µL of 2.1 mM Tris-HCl (pH 8.0) and 42 μM EDTA. VIC-labelled (Applied Biosystems) and 6FAMTM-labelled  fluorescent primers were added to the (−) and (+) samples, respectively, with a final concentration of 5 nM. Primers were annealed at 85 °C for 1 min, 60 °C for 5 min and 35 °C for 5 min. Primer extension was initiated by the addition of 5 mM DTT, 0.5 mM dATP, dCTP, dUTP and 7-deaza-dGTP, 40 U of SuperScript III reverse transcriptase  and 1× SSIII buffer to each sample. Extension was continued at 55 °C for 60 min. The cDNAs of (−) and (+) reactions were combined and RNA was degraded using a final concentration of 200 mM sodium hydroxide (NaOH), followed by incubation at 95 °C for 3 min. This was cooled on ice and the NaOH was neutralised with a final concentration of 200 mM hydrochloric acid. Sequencing ladders were generated using the Thermo Sequenase Cycle Sequencing Kit  and primers with the same sequences as those used in reverse transcriptions, but labelled instead with NEDn >/= 4 all replicates were aligned in Excel and outliers at each nucleotide position were defined as datapoints that were greater than the third quartile value plus 1.5 times the interquartile range, or less than the first quartile value minus 1.5 times the interquartile range. Average nucleotide reactivity data were then used in RNA structure to generate models of the 20 lowest free-energy structures. To examine and visualise the variety of structures (‘ensemble’) the RNA forms under each condition, each group of 20 structural models was assessed for the structures adopted by nts-1–57 (TAR), 58–104 (poly(A)), 105–115 (the 5′ side of U5:AUG), 236–282 (SL1) and 283–343 (SL1–3 and the 3′ side of U5:AUG). Firstly, the number of different structures for each structural element was counted. Minor variations which differed by up to two structural elements  were classified as variants of that structure, with the exceptions of smaller modifications at the base of TAR, poly(A) and U5:AUG, which were classified and counted as separate structures, as minor variations in TAR and poly(A) have been suggested to be of biological importance and U5-AUG contains only one structural element. For each of these structural elements, the proportion of the ensemble present in each different classified structure/variant was scored out of 20.The cDNAs and sequencing ladders were precipitated with sodium acetate and ethanol, resuspended in water and initially titrated individually onto sequencing plates to verify an amount at which maximum signal intensity and bleed-through into other channels did not occur, then combined at this amount and separated on a 3730xl DNA Analyser (Applied Biosystems). The sequences were aligned using SHAPEfinder, and mobility shift controls were performed as described in . DiffereRNA was renatured and incubated with/without protein as above, with the inclusion of an additional sample using aprotinin as an RNase-free negative control protein of similar charge. Samples were aliquoted onto two 96-well round-bottomed plates: one was cross-linked for 2 min at standard power on ice in a stratalinker (XL-1500) UV cross-linker and the other was incubated for 2 min on ice without cross-linking. Proteinase K (1.5 µL of 20 mg/mL) and 3.75 μL of 20% SDS were added to each sample, followed by incubation at 55 °C for 60 min. RNA was purified by phenol-chloroform extraction and ethanol precipitation as above.TM or VIC®, before being combined  for capillary fractionation. The average of this background was then subtracted from the following experiments: RNA-only samples with/without cross-linking were labelled with VIC, and RNA–protein samples with/without cross-linking were labelled with 6FAM. For capillary fractionation, a 6FAM-labelled cross-linked RNA–protein sample was combined with a VIC-labelled cross-linked RNA-only sample, and a 6FAM-labelled non-cross-linked RNA–protein sample was combined with a VIC-labelled non-cross-linked RNA-only sample. The capillary fractionation gave the differences in the amounts of 6FAM and VIC cDNAs of each nucleotide length, the corresponding fluorophore control background described above was taken away from this. Following this background subtraction, the non-cross-linked RNA–protein reactivity (controlling for RNA–RNA cross-links) was subtracted from the cross-linked RNA–protein reactivity to give a final cross-linking value for each nucleotide. As described and calibrated in [p < 0.05 by t-test).RNA was then reverse-transcribed as above. However, the combinations of primers used was different (as described in ). Firstlrated in , true sit-test was performed, assuming that the two samples have unequal variance.In instances where a difference from a control is calculated, a two-tailed Native RNA–protein interactions often involve the binding of multiple copies of the same or different proteins to the RNA, making it especially hard to resolve the RNA structures of individual complexes from within such a heterogeneous mixture. SHAPE has been previously used to identify structural changes induced in TAR RNA upon binding by a Tat protein . In thesIn-gel SHAPE has been previously used to identify RNA structural changes upon dimerisation of the HIV-1 viral RNA leader sequence . Its abiRefolded RNA containing the TAR and poly(A) sequences of HIV was incubated with a Tat peptide and complexes were separated by polyacrylamide gel electrophoresis (PAGE) using a native Tris-borate magnesium (TBM) gel a. Using To identify the molar ratio at which initial contacts between Gag protein and the HIV packaging signal RNA are made in vitro, we performed an electrophoretic mobility shift assay (EMSA) . In the At a molar ratio of between 10–20, we observe a shift in the RNA that is plausibly an intermediate conformation different from the original monomeric structure (such as the BMH structure) or the original monomeric structure  with Gag bound. At the lowest concentration of Gag that detectably binds to the RNA, both monomers and dimers are shifted. At a molar ratio of 30, there are no observable monomers. After this, Gag binding and the reduction in migration of the complex continues until Gag binding plateaus towards saturation. These results suggest that at low RNA concentration, initial Gag binding to the RNA induces a transition from a flexible structural ensemble of mainly monomers (with some dimers) to a more stable single-dimeric conformation via a transient intermediate monomeric conformation. Moreover, the striking mobility shift observed during successive Gag molecules binding between molar ratios of 20 and 30 suggests lower affinity binding of Gag to the initial RNA structures as a prelude to cooperative binding of Gag once the RNA converts to a more stable conformation. It must be noted that the experimental conditions used for the EMSA were optimised for a low concentration of radiolabelled RNA, and hence individual species from these gels could not be excised for in-gel SHAPE. This is because increasing the concentration of RNA to that required for in-gel SHAPE also increases the dimerisation of the RNA. In order to be able to investigate the initial effects of Gag binding upon both the monomeric and dimeric RNA, we chose a molar ratio intermediate between 10 and 20× Gag for in-gel SHAPE as it is the lowest molar ratio at which both the monomer and the dimer are reliably shifted .In order to probe the conformational changes occurring within the RNA upon protein binding, 15× molar excess of Gag protein was incubated with folded in-vitro-transcribed 5′UTR RNA and resolved using native PAGE; monomeric or dimeric RNAs or RNA–protein complexes were isolated, probed and analysed, as for gag. The difference in behaviour of the monomeric and dimer RNA upon Gag binding is shown in The 414-nucleotide RNA design contained 3Gs at the transcriptional start site and 78 nts of Uncleaved Gag protein and its NC cleavage product serve different purposes during the formation and maturation of the viral particle as well as during the subsequent reverse transcription process. Thus, we compared the differences in RNA flexibility upon binding of uncleaved Gag protein and NC protein, as NC is only formed during viral maturation and is responsible for chaperoning later events, such as during reverse transcription. The binding of the same molar excess of NC to the RNA as Gag (15×) leads to higher NMIA reactivity in the dimer, and thus increased RNA flexibility in the TAR stem–loop, the PBS region, the SL2 stem–loop and the region between SL2 and SL3 a, suggesWe then went on to examine the structural ensemble of the RNA bands probed, focusing on the TAR , poly(A)The monomeric RNA that had not encountered Gag/NC predominantly contained a fully or partially disrupted TAR structure, poly(A) involved in an LRI with SL1, similar to the LDI model, and detectable but minimal U5:AUG formation, whereas there was significant heterogeneity in the SL2–4 ensemble: the predominant structure contained a slightly extended SL3 and an intact SL4 . The monThe dimeric ensemble was modelled as the ‘hemi dimer’, with the additional constraint of the GCGCGC DIS sequence being maintained as single-stranded to enable pairing with its counterpart in the second molecule. The ligand-naive dimer ensemble predominantly contained a stable TAR and poly(A) stem–loop, but not the U5:AUG helix, SL1, and the SL4 sequence formed long-range interactions. This ensemble conformed to the BMH model except for the U5:AUG interaction, which was instead an alternative long-range interaction. The dimeric ensemble that had bound Gag mostly formed a TAR that was not additionally stabilised at the base by the 5′G, the poly(A) stem–loop, the U5:AUG helix , SL1 and SL4 forming the same long-range interactions as in the BMH. Overall, the majority of the structural ensemble was in the BMH or close to the BMH form. The dimeric ensemble with NC bound had an almost entirely disrupted TAR stem–loop, with this sequence forming various long-range interactions, considerable heterogeneity in the poly(A) ensemble, the U5:AUG helix predominantly intact, and the majority of the SL2–4 ensemble containing the same long-range interactions as are found in the BMH model.To illustrate the narrowing of the structural ensemble into the BMH form when Gag binds to the dimer, the improved fit of the SHAPE data are shown mapped onto the BMH structural model when Gag is not/is bound to the dimer a,b. The Overall, the ensembles suggest that Gag either specifically selects structures that resemble the BMH model, or that it remodels the RNA into the BMH when it binds. It can bind to alternative structures, as evidenced from the shifted monomer population, but these do not then remodel into a dimerisation-competent structure within the timeframe of this experiment, at this relatively low concentration of Gag.SHAPE coupled with photo-cross-linking (XL-SHAPE) has been used to identify protein binding sites and the impact of protein binding on RNA structure remodelling . The advIn-gel SHAPE is a precise and versatile technique that has been used to successfully separate and analyse the structure of multiple RNA conformers within a mixed population . Here, wgag sequences compared with previous studies of the effects of transcriptional start site heterogeneity. These 3′ nucleotides appeared to widen the structural ensemble we observed, as they were able to pair with the base of TAR, destabilising it to some extent, and hence enabling the structures previously seen in the presence of the 2G transcript to form. Our observations once again highlight the potential multifunctionality of the TAR RNA and its role in the translation/packaging switch, which make it an attractive drug target.As Gag concentration increases and higher numbers of Gag proteins bind the viral genome, the RNA is shifted towards a new optimal dimeric conformation . Using EA specific SL3-binding drug has been shown to be an effective small-molecule inhibitor of HIV packaging, and multiple methods have identified the GGAG tetraloop at the apex of SL3 to be a binding site for NC and Gag ,64,65,66Our results suggest that SL3 is stabilised in the dimer in the presence of Gag, possibly by the formation of extended intermolecular interactions 5′ and 3′ of it. This can be seen in Gag binding has also been shown to fully unwind SL3 RNA, whereas NC is unable to do this , and it The BMH conformation found in the dimeric ensemble has a number of SHAPE reactivity differences from the monomeric ensemble structures, notably within and surrounding SL1 and the U5:AUG helix. We observed sites of stabilisation and destabilisation along the stem and bulges of SL1 upon Gag binding to the RNA, which presumably facilitate the transition to the BMH model . It has In the dimer, upon Gag binding, there are sites of destabilisation in the PBS: the loop region above the U5:AUG, the loop of SL2 and the AU-rich sequence 5′ of SL3 . These cThe proteolytic maturation of Gag by the viral protease during viral particle maturation is vital for gRNA dimerisation and stability. Genomic RNA extracted from immature virions shows reduced stability, similar to that of a loose dimer ,37,71,72Overall, our data suggest a mechanism by which HIV-1 effects and times its packaging process to ensure that packaging only begins once sufficient Gag has been translated, and highlight the important gRNA sites and structures that allow packaging to begin. Such insights may provide new drug targets against HIV.