Nonetheless, RNA origami as a method for nanofabrication is not yet fully explored and the majority of RNA nanostructures are based on natural pre-folded RNA. Here we describe a biologically inert and uniquely addressable RNA origami scaffold that self-assembles into a nanoribbon by seven staple strands. An algorithm is applied to generate a synthetic De Bruijn scaffold sequence that is characterized by the lack of biologically active sites and repetitions larger than a predetermined design parameter. This RNA scaffold and the complementary staples fold in a physiologically compatible isothermal condition. In order to monitor the folding, we designed a new split Broccoli aptamer system. The aptamer is divided into two nonfunctional sequences each of which is integrated into the 5′ or 3′ end of two staple strands complementary to the RNA scaffold. Using fluorescence measurements and in-gel imaging, we demonstrate that once RNA origami assembly occurs, the split aptamer sequences are brought into close proximity forming the aptamer and turning on the fluorescence. This light-up ‘bio-orthogonal’ RNA origami provides a prototype that can have potential for  The scaffolded DNA origami technique involves short oligonucleotide staple strands which direct the folding of a long scaffold strand sequence into a pre-programmed nanostructure6 with well-defined shape, dimension and functional properties10.A plethora of self-assembled DNA origami and hybrid RNA-DNA origami nanostructures have been synthesized using the basic principle of Watson-Crick base pairingin vivo, their expression in large quantities, the formation of stable interactions11 and the control of gene expression12. Despite RNA’s functional capacities, the synthesis of RNA nanostructures via scaffold and staple strands is underdeveloped and still lacking. While significant advances have been made in DNA origami synthesis, the design and realization of RNA origami has been reported only recently13. Indeed, most of the RNA nanostructures have been developed under different approaches based on the self-assembly of building blocks such as loops and motifs16. In this context, several RNA sequences such as packaging RNA (pRNA) from bacteriophage ϕ2920, transfer RNA (tRNA)21 and an engineered modular ribozyme22 were used to guide the self-assembly of RNA nanoparticles23.As an interesting alternative, nanostructures can be self-assembled from RNA instead of DNA. The advantages of RNA structures include their potential applications 24 introduced a shape-based computational approach to design RNA hexagonal nanoring from two building blocks using stable RNAIi/RNAIIi loop-loop noncovalent intermolecular interactions. In this regard, a computational-based approach promoted the in silico design of a three dimensional cubic RNA-based scaffold that was self-assembled in a one-pot process. Instead of prefolded RNA, the nanostructure required relatively short RNA sequences and allowed selective point modifications27.In contrast to the above mentioned strategies used for the construction of RNA nanoparticles, Yingling and Shapiro29. Taking into account the powerful origami technique, Geary et al.30 showed the cotrascriptional folding of an artificially designed single stranded RNA (ssRNA) into a RNA tile: at the end of the RNA tile synthesis, the tiles were released and assembled into hexagonal lattices through kissing loop interactions. In contrast to the traditional origami technique, this strategy used RNA modules such as kissing loops to replace the staple strand role30. In the same year, a 7-helix bundled RNA tile and a 6-helix bundled RNA origami tube were designed and prepared from single stranded RNA scaffold and multiple staple RNA strands by employing for the first time a direct extension of the DNA origami strategy13.Recently, RNA nanostructures were also obtained with different tools and techniques imported from DNA nanotechnology, such as the use of the double cross over (DX) motifs and the DNA origami design. For instance, a tile-based assembly approach was investigated to assemble micron-size RNA scaffolds and a DX DNA tile motif was adapted to design DX RNA tiles that formed lattices and tubular structures via programmed sticky end interactions8, a square DNA origami and a triangle RNA-DNA hybrid origami were synthesized using ‘bio-orthogonal’ and uniquely addressable De Bruijn scaffold sequences (DBS). Unlike biologically derived scaffolds like M13mp18 or pUC19, these DNA and RNA ‘bio-orthogonal’ scaffolds do not contain genetic information, restriction enzyme sites or ambiguity in the addressability, making them candidates for future in vivo applications8. In this work RNA staple strands promoted the folding of a short ‘bio-orthogonal’ RNA scaffold sequence into a nanoribbon shaped structure: after an initial denaturation step, the self-assembly occured at physiological temperature (37 °C) within minutes. RNA origami assembly was verified by gel assay, atomic force microscopy (AFM) and using a new split Broccoli aptamer system able to bind the specific fluorophore only after the folding process. The Broccoli aptamer31 was divided into two non-functional RNA sequences each of which were integrated into two distinct RNA staple strands complementary to the scaffold sequence. Once the RNA origami was assembled, the split aptamer sequences were reassociated to form the functional binding site and the fluorescence was clearly restored.Here we take into account the large gap between DNA origami and RNA origami development and inspired by a bottom-up origami toolkit, we present a light-up biologically inert  RNA origami able to fold at constant temperature after an initial denaturation step. In our recent workin vivo origami applications when genetically encoded and transcribed RNA are used. RNA can control many cellular functions and the creation of new tools that allow the programmable control of gene expression is a fundamental goal in synthetic biology. In this perspective, RNA origami can be used as organelle-like structure able to act as regulatory machine.Herein, we investigate and combine three different aspects: i) ‘bio-orthogonality’, ii) physiologically compatible folding at 37 °C and iii) assembly monitoring via a new split Broccoli RNA aptamer system. These characteristics are important from the perspective of RNA origami expression and folding in living cells. Our RNA origami nanoribbon opens the way to new potential platform for future 8. Here, we use a similar method to obtain candidate sequences that interface with the Broccoli RNA aptamer system. The first synthetic scaffold was constructed to act solely as a platform for the split aptamer. In this design, the 32 nt-long De Bruijn sequence forms a three-way junction with the complementary domains integrated with the split aptamer, allowing for its reconstruction  are used to ‘seal’ the folding Fig. . Here, t32. The Spinach technology has been used to monitor different small molecules in live cells38, transcription in vitro and in vivo40 and RNA assembly41, to image mRNA42, to design genetic circuits43 and to measure ribozyme self-cleavage activity44.The Spinach aptamer sequence, an RNA mimic of green fluorescent protein, opened the way to a novel class of RNA sequences able to emit fluorescence in the presence of a specifically provided fluorophore molecule31. A unique characteristic of this aptamer is its short size that can improve its versatility when used as a tag. Moreover Broccoli aptamer shows robust folding and green fluorescence in physiological conditions.Considering the usefulness of Spinach, more recently a new smallest light-up aptamer, called Broccoli, has been describedet al.45 reengineered the natural ϕ29 three-way junction motif to generate an alternative ‘bio-orthogonal’ structure (named F30) that enabled stable aptamer expression in cells. RNAs tagged with this structure showed substantially increased stability compared with RNAs tagged with tRNA scaffold that were targeted for endonucleolytic cleavage in bacterial cells. Here, we considered arm 1 of the engineered ‘bio-orthogonal’ F30-Broccoli sequence to be a suitable point for spliting-up the fused aptamer: 8 base pairs of F30 sequence were included in our split aptamer sequences , that were able to self-assemble and restore the fluorescence function when hybridizing. In contrast here, the hybridization between Split1 and Split2 without the presence of the complementary Split1/Split2 DBS was undesiderable. Thus, we went a step further, presenting a split system that was able to promote the fluorescence activation only in the presence of a target complementary sequence.Recently, Alam in vitro transcription: the transcribed products were analyzed by denaturing gel that confirmed the expected size . After washing steps, the gel was stained with DFHBI-1T which binds to Broccoli with no background fluorescence45. Fluorescent imaging of the gel revealed bright bands corresponding to Broccoli aptamer and Split1, Split2, complementary Split1/Split2 DBS assembly, thus demonstrating that the fluorescence was restored only after the assembly process of the new split aptamer system  was calculated based on the standard deviations of the blank (10 independent measurements). The fluorescence emission values of Split1, Split2, complementary Split1/Split2 DBS alone and Split1 plus Split2 were comparable to the background value, as reported in Fig. 2, Split1_Split2_complementary Split1/Split2 DBS showed comparable fluorescence emission intensity values . Detail of its design is provided as supplementary information  and a snap cooling step (−1 °C/0.42 sec) from 75 to 37 °C, RNA origami were folded in isothermal condition at 37 °C for 20 min. Quality control of RNA origami was done by gel electrophoresis and atomic force microscopy. The quality of the gel bands provides a preliminary check about the quality of the folding and if smeared bands appeared rather than distinct bands, nanostructures are not folded52, thus the estimated dimensions of our RNA origami were approx. 27 nm × 5 nm. AFM images confirmed the correct formation of the RNA origami: their average lenghts were 25.6 nm ± 2.7 nm and 5.3 nm ± 0.8 nm in accordance with the expected size . This AFM yield was determined as a percentage of identifiable shapes in an entire AFM field, as previously described55. Due to the 2H-AE construct dimension, AFM characterization was performed on larger lattices obtained from 2H-AE assembly30 and the synthesis of the apta-FRET nanostructures was demonstrated by recording FRET outputs based on spectrofluorometric and flow cytometry measurements55.Recently, an alternative approach to the RNA origami method was provided: a single-stranded RNA origami was designed to fold during RNA transcription and a RNA aptamer-based FRET (apta-FRET) system was introduced in the nanostructure. In detail, authors showed FRET between fluorescent RNA aptamers by placing Spinach and Mango in close proximity on a single-stranded RNA origami 2H-AE 2 helix construct55, our RNA origami utilized seven staple strands that hybridized with a ‘bio-orthogonal’ ssRNA scaffold folding it into a nanoribbon well characterized by AFM. The Broccoli split aptamer system was inserted in the ‘bio-orthogonal’ RNA origami nanoribbon . The normalized fluorescence value of the Sc 2St sample was 0.1 . No difference was recorded between Sc 4St sample and background. These data showed the fluorescence activation due to the incorporation of the split aptamer system and to the formation of the active Broccoli aptamer on the RNA origami. The RNA origami folding was succefully confirm by fluorescence measurements. Moreover considering the purified RNA origami distinct band on native PAGE and the high folding yield (74%), the fluorescence value could be mainly determined by well-folded nanostructures.et al.45 developed an in-gel staining system as a Northern blotting alternative for the detection of RNA cleavage products tagged with Broccoli aptamer. Different molecular weight bands were separated by native PAGE: RNAs that contained Broccoli tag were visualized based on specific binding of DFHBI-1T to the aptamer tag. In our work, in-gel staining confirmed the RNA origami folding: RNA nanostructures migrated as a distinct band and triggered the fluorescence emission in the presence of the specific fluorophore  without initial denaturation and at lower magnesium concentration, which could result in future 63. Compared to the single stranded origami strategy based on a self-folding55, we demonstrated the isothermal self assembly at 37 °C of a ‘bio-orthogonal’ scaffold using multiple staple strands. Finally, the development of a new split Broccoli aptamer system allowed us to confirm the self-assembly.To the best of our knowledge, we are the first to investigate and combine three important aspects: i) ‘bio-orthogonality’; ii) isothermal folding at 37 °C of a nanostructure by employing scaffolded origami strategy instead of a single stranded origami approach and iii) self-assembly monitoring using a new split Broccoli aptamer system. Both unimolecular folding and scaffolded origami represent different approach fo synthesizing nanostructuresin vivo applications without the introduction of the burden related to fluorescence protein complementation64 commonly used to detect RNA assembly11 or to label native RNA65in vivo. In the perspective of the RNA origami expression and folding in living cells, we take into account all the above mentioned important characteristics. Natural and engineered RNA can control many aspects of the gene expression inside the cells. These regulatory functions can be tuned by the programmable formation of specific RNA structure12. Towards this vision, RNA origami nanostructure can represent a useful platform to organize new genetic control system.Our approach can find new development for Z)-5[methylene]-3,5-dihydro-2-methyl-3--4H-imidazol-4-one (DFHBI-1T) was purchased from Tocris Bio-techne. A DFHBI-1T stock solution (20 mM) was prepared in DMSO and stored in the dark at −20 °C for a week. All RNA oligonucleotides were purchased from Eurogentec and resuspended in ultrapure water to give stock solutions of 100 μM and stored at −80 °C. All DNA oligonucleotides and gBlocks Gene Fragment were purchased from IDT. The DNA oligonucleotides were resuspended in ultrapure water to give stock solutions of 100 μM and stored at −20 °C. The gBlocks Gene Fragment was resuspended at a final concentration of 10 ng µL−1 and stored at −20 °C. EDTA 0.5 M pH 8.0, Ultra PureTM 10x TBE buffer, Ultra PureTM 1 M Tris HCl pH 7.5, 10x TAE buffer pH 8.5, Ultra PureTM distilled water, SYBR® Gold, 6% NovexTM TBE gel and 10% TBE-Urea gel were purchased from Invitrogen, Life Technologies. HEPES 1 M pH 7 Bioreagent, KCl 1 M BioUltra, MgCl2 1 M in water BioUltra, 3-aminopropyltriethoxysilane (APTES) and agarose were purchased from Sigma-Aldrich. were also considered  and visualized under UV illumination. The low range molecular weight DNA ladder (NEB) was used as molecular weight marker. The PCR products were purified using Monarch® PCR & DNA Cleanup kit (NEB) and DNA concentration was measured on a NanoDrop spectrophotometer. AmpliscribeTM T7-FlashTM Transcription kit (Epicentre) was used to transcribe single stranded RNA from DNA template: a total of 20 µL mixture was prepared and incubated at 37 °C for 1 h and 30 min. After DNase treatment at 37 °C for 15 min, the RNA transcripts were purifed using RNA Clean & ConcentratorTM (Zymo Research) and quantified using a NanoDrop spectrophotometer. After the addition of the RNA loading dye 2x (NEB) and the heat denaturation at 65 °C for 5 min, in vitro-transcribed RNAs were loaded and run on 10% TBE-Urea gel in 1x Tris borate EDTA (TBE) buffer at 200 V for 40 min. After staining with SYBR® Gold in 1x TBE for 8 min, the gels were visualized using Typhoon laser scanner and Image Quant TL software . The low range ssRNA ladder (NEB) was used as molecular weight marker.The dsDNA templates containing T7 promoter sequence were prepared by polymerase chain reaction with Broccoli ssDNA, Split1, Split2, . Then, the gels were washed three times with RNase free water, stained with SYBR® Gold in 1x TBE for 8 min, and visualized using Typhoon laser scanner. The low range ssRNA ladder (NEB) was used as molecular weight marker.Split 1 RNA, Split 2 RNA and complementary Split 1/Split 2 DBS were mixed in a 1:1:1 ratio and incubated together at 37 °C for 25 min. All the samples including negative controls (Split sequences without complementary DBS) were run on 6% NovexThe fluorescence was measured with ClarioStar Plate Reader (BMG Labtech) and samples were excited at 460 nm and the emission was recorded at 544 nm. Samples and blanks (35 µL each) were loaded on a 96-well RNAse free µClear® black microplate (Greiner Bio-One) according to the following specifications. Negative controls , Split1 RNA_Split2 RNA_complementary Split1/Split2 DBS were mixed in a 1:1:1 ratio  and incubated at 37 °C for 25 min, as reported above. After the incubation period at 37 °C, the aptamer buffer was added to reach a final volume of 34.55 µL. The fluorophore solution (0.45 µL from a 0.8 mM DFHBI-1T in DMSO stock solution) was added to all samples and fluorescence intensities were recorded.−1) and visualized under UV illumination. The low range molecular weight DNA ladder (NEB) was used as molecular weight marker.Double stranded gBlocks Gene Fragment containing T7 promoter . The noDBS1 and noDBS2 scaffold sequences were transcribed in vitro at 37 °C for 2 h. After DNase treatment at 37 °C for 15 min, the RNA transcript was purifed using RNA Clean & ConcentratorTM (Zymo Research) and quantified using a NanoDrop spectrophotometer. After the addition of the RNA loading dye 2x (NEB) and the heat denaturation at 65 °C for 5 min, in vitro-transcribed RNA was loaded and run on 10% TBE-Urea gel in 1x TBE buffer at 200 V for 40 min. After staining with SYBR® Gold in 1x TBE for 8 min, the gels were visualized using Typhoon laser scanner. The low range ssRNA ladder (NEB) was used as molecular weight marker.The PCR product was purified using Monarch® PCR & DNA Cleanup kit (NEB) and the DNA concentration was measured on a NanoDrop spectrophotometer. The purified template was transcribed 2, 20 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0)13. After an initial thermal denaturation step at 75 °C for 1 min and a snap cooling (−1 °C/0.42 sec), the mixture was subjected to a folding step at 37 °C for 20 min. To confirm that the folding occured isothermically at 37 °C, samples were incubated at 75 °C for 1 min or subjected to an initial thermal denaturation at 75 °C for 1 min followed by snap cooling (−1 °C/0.42 sec).The set of staple strands  Staple s1 and Staple s2 (sample Sc 2St) and ii) Staple s1, Staple s2, Staple l1, Staple r1 (sample Sc 4St). The partially folded and folded constructs were purified from staple strands excess using the Amicon Ultra 0.5 mL 50 kDa or 100 kDa centrifugal filters . Capped Amicon Ultra were rinsed with 400 µL of folding buffer and centrifuged at 10000 g for 6 min at 15 °C. After this preliminary washing step, the samples were added to the filter device. Between every centifugation step, the flowthrough was removed and the filter was refilled with 400 μL of folding buffer. To recover the sample, the filter was turned upside down and centrifuged once more at 300 g for 10 min at 15 °C.TM TBE gel in 1x TBE buffer at 100 V for 40 min at low temperature (below 10 °C). After staining with SYBR® Gold in 1x TBE for 8 min, the gels were visualized using Typhoon laser scanner. The low range ssRNA ladder (NEB) was used as molecular weight marker. To confirm the RNA origami self-assembly by incorporation of Split Broccoli aptamer system, in-gel imaging with fluorophore DFHBI-1T was done as described above.Samples were run on 6% NovexThe fluorescence was measured with ClarioStar Plate Reader (BMG Labtech): samples were excited at 460 nm and the emission was recorded at 544 nm. After the addition of 11.2 µL of KCl 1 M and 1.29 µL of fluorophore stock solution (0.8 mM DFHBI-1T in DMSO), purified RNA origami, purified partially folded samples and blanks  were loaded on a 96-well RNAse free µClear® black microplate (Greiner Bio-One) and the fluorescence intensities were recorded.69 showed variations of less than 10% between cantilevers. The sample temperature control set at 20 ± 0.1 °C for all the experiments. All the images were corrected for tilt (line or plane flattening) and lightly low-pass filtered to remove grainy noise using the WSxM software 70.Freshly cleaved mica was passivated for 5 min with 10 μL of 0.1% APTES in water to ensure the adhesion of the negative charged RNA origami structures on the negative mica surface. After three washing with folding buffer, purified RNA origami samples (8 μL) were added to the passivated mica surface and allowed to adsorb for 5 min in a chamber. AFM imaging was performed with a Bruker multimode 8 AFM in Scanasyst mode using Bruker Scanasyst-Fluid+ tip for lower resolution images and with a Cypher ES for higher resolution. All images where acquired with the AFM operated in liquid, in tapping mode and with a setpoint ratio between the free amplitude and imaging amplitude of ~0.7. The Cypher was equipped with laser excitation (blueDrive) and temperature control, enabling ultrastable operation and fully reproducible imaging parameters. Measurements were conducted using cantilevers from a same wafer for better comparability  with a nominal spring constant of 0.76 N/m. Systematic thermal calibration54.Folding yield analysis based on AFM imaging was done as previously describedLabeling of DBS, noDBS1 and noDBS2 scaffolds were performed using Ulysis® Nucleic Acid Labeling kit (Life Technologies) following manufacturer’s protocol. Labeled RNA solution was purified by using Micro Bio-Spin® P-30 spin column (BioRad) and eluted in 10 mM Tris-HCl pH 7.4.The Alexa 488 scaffold was run on 2% agarose gel at 100 V for 1 h: gel was imaged using Typhoon laser scanner . After ethidium bromide staining, gel was again imaged using Typhoon laser scanner.8. NEB 5-alpha electrocompetent E. coli cells (NEB) were diluted 1:1 with sterile milli-Q water in a prechilled tube: 20 μL of cells suspension were used for each electroporation experiment after the addition of 5 μL of labeled scaffold . The mixture of competent cells and labeled molecules was immediately transferred into a prechilled electroporation cuvette  and electroporated . The negative control was prepared incubating cells with the same volume of the same labeled scaffold solution: the cells were not electroporated and were washed as the electroporated samples. After electroporation, cells were allowed to recover for 25 min at 37 °C in pre-warm SOC. Then cells were harvested by centrifugation at 3300 g for 1 min at 4 °C, washed 5 times with 500 μL of 1x phosphate buffered saline solution pH 7.4  and resuspended in 50 μL of PBS62. 2.5 μL of proteinase K  and 50 μL of melted SeaKem Gold agarose (Lonza) in TE  were added to the cell suspension and mixed gently. The agarose-cell suspension mixture was dispensed into a well of plug mold (BioRad) and allowed to solidify at 4 °C for 20 min71.Electroporation and plug preparation has been done as previously described−1 of proteinase k) for 15 min at 54 °C in a water bath71. After lysis, the plugs were washed four times (10 min/wash) at room temperature (once with nucleases free water and three times with TE pH 8.0). After washing steps the plug slices were loaded into the wells of 1% TBE agarose gel: the electrophoresis was performed for 1 h at 100 V. The 1 kb DNA ladder (NEB) and the low range ssRNA ladder (NEB) were used as molecular weight markers. The gels were imaged using Typhoon laser scanner . After SYBR® Gold staining for 25 min, the gels were scanned again on Typhoon laser scanner.The plugs were incubated in 5 mL of cell lysis buffer .Supplementary Information


Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent , but possess the same overall polycistronic genome structure, which is distinct from all other characterized picorna-like viruses. Consequently, we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species) and Sopolycivirus (11 species), with members of the latter infecting ants in at least 3 different subfamilies.Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5′ third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3′ two-thirds of the genome. The polypeptide translation of this 3′ open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. An inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and  Hel, helicase; IRES, internal ribosome entry site; LneV-1, Lasius neglectus virus 1; LniV-1, Lasius niger virus 1; MsaV-1, Myrmica scabrinodis virus 1; ORF, open reading frame; Pro, protease; RdRp, RNA-dependent RNA polymerase; SINV-2, Solenopsis invicta virus 2; SINV-4, Solenopsis invicta virus 4; SNP, single nucleotide polymorphism; TSA, transcriptome shotgun assembly.Picornavirales currently comprises the families Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae and Secoviridae, and the unassigned genera Bacillarnavirus and Labyrnavirus. Members of the order are characterized by (i) a positive-sense RNA genome, usually with a 5′ covalently linked VPg  and a 3′ poly(A) tail, (ii) a polyprotein gene expression strategy with cleavage mainly mediated by viral protease(s), (iii) a structural protein module containing three jelly-roll capsid protein domains which form small non-enveloped icosahedral virions with pseudo T=3 symmetry and (iv) a non-structural protein module containing a superfamily III helicase (or NTPase), a 3C-like chymotrypsin-like protease and a superfamily I RNA-dependent RNA polymerase (RdRp), encoded sequentially in that order [Caliciviridae and Potyviridae (for example), although termed picorna-like  are excluded from the Picornavirales order for various reasons; for example, caliciviruses encode a single jelly-roll capsid protein and virions have true T=3 symmetry, while potyviruses have an unrelated capsid protein and filamentous virions, and encode a superfamily II instead of a superfamily III helicase.The order at order . In contSolenopsis invicta Buren, an invasive species in the southern USA [S. invicta [S. invicta [S. invicta queens, including immune response genes [Solenopsis invicta virus 2 (SINV-2) is a positive-sense single-stranded RNA virus that infects the red imported fire ant,  invicta . While f invicta , there a invicta . Coloniese genes . The monse genes . IsometrSolenopsis invicta and four UK ant species. This led to the identification of four additional viruses. During the course of this work, 1445 new viruses were identified via high-throughput sequencing [polycistronic picorna-like viruses). The characteristic complement of picornavirus-like protein domains suggests that the Polycipiviridae should be included within the Picornavirales order.By searching the NCBI transcriptome shotgun assembly (TSA) database, we identified six related sequences, of which five were derived from ant samples. To further explore the diversity and prevalence of this group of viruses, we performed high-throughput sequencing of RNA derived from quencing , of whicet al. [The SINV-2 genome was first sequenced by Valles Solenopsis invicta RNA samples revealed the presence of a new SINV-2-like virus, which we named Solenopsis invicta virus 4 (SINV-4). A complete genome was obtained by Sanger sequencing and has been deposited in GenBank with the accession number MF041808.1. SINV-4 has the same genome organization  were identified, and their longest ORF extracted, translated and queried against NCBI RefSeq virus genome sequences. Sequences whose reciprocal best match was SINV-2 were retained, while other sequences  were discarded. This resulted in five long sequences with reciprocal best matches to SINV-2. An additional long sequence was obtained by merging the partly overlapping fragments LH935078.1 and LH935077.1. Five of the long sequences derive from ant RNA-Seq libraries . The sixth sequence, KA182589.1, derives from a Chironomus riparius RNA-Seq library . Sequence attributes are recorded in Next we queried the NCBI Transcriptome Shotgun Assembly (TSA) database using Lasius flavus, L. niger, L. neglectus and Myrmica scabrinodis) collected in Cambridge, UK, and performed two rounds of high-throughput RNA sequencing. Contigs were assembled using Trinity [blastx.Given that SINV-2-like viruses appeared to be most prevalent in ants, we obtained samples from four different ant species  anti-viral defence pathway, and standard RNA-Seq. In this case, good contig assemblies were not obtained for the small-RNA samples and no further small-RNA sequencing was performed. For the long RNA-Seq of L. niger, but not L. flavus, we identified one SINV-2-like sequence. In the second round, we performed long RNA-Seq for RNA obtained from M. scabrinodis, L. neglectus and a second L. flavus sample, and identified SINV-2-like sequences for M. scabrinodis and L. neglectus. Morphological ant species identifications were confirmed by blastn of cytochrome C oxidase subunit I (COI) sequences obtained from the transcriptome assemblies against the NCBI non-redundant (nr) nucleotide database.In the first round, we sequenced The three SINV-2-like sequences were verified by Sanger sequencing and extended where possible by 5′ and 3′ RACE (see below). Sequence attributes are recorded in >30 nt) back to the assembled virus genomes to assess coverage and variation. LniV-1, LneV-1 and MsaV-1 had mean coverage values of 6.4, 457 and 1348, respectively. For the two viruses with high coverage  and a mixed insect sample including members of the orders Lepidoptera, Diptera, Neuroptera and Coleoptera (KX883910.1), with respectively 19, 21, 21 and 30 % amino acid sequence identity to SINV-2 in the ORF5 polypeptide. The sequences from spiders, Odonata/Diptera and crayfish have low abundance (<0.1 % of sample non-rRNA), leading the authors to suggest the possibility that they may derive from parasites or gut contents (which in these cases might include insects) instead of the target organisms. Sequence attributes are recorded in During the course of the above work, 1445 new RNA viruses were identified via high-throughput sequencing of RNA samples obtained from a variety of invertebrates . The autIn summary, we identified a total of 15 SINV-2-like sequences , comprisFormica exsecta TSA contains an additional short ORF (ORF3a) between ORFs 2 and 3. Hubei picorna-like virus 81  present, though, given the variability in UTR lengths, we assume that several were incomplete. The 5′ UTR sequences were frequently >200 nt and ranged up to 366 nt in length. Eight sequences extend to the poly(A) tail and may therefore be assumed to have been 3′-complete. For these sequences, the 3′ UTR lengths ranged from 385 to 479 nt, excluding the poly(A) sequence. The length of the intergenic region between ORFs 4 and 5 ranged from 336 to 768 nt. To assess 5′-completeness we compared 5′ nucleotide sequences between different species . A number of polycipivirus sequences exhibit a predicted stable simple stem–loop structure close to the 5′ end. In SINV-4, SINV-2 and the C. riparius TSA – ORF1 was predicted to encode a picornavirus-like capsid protein, but for ORF4 this was only predicted for Hubei picorna-like virus 81, and for ORF3 it was not predicted for any of the three. However, for ORFs 3 and 4, HHpred [C. riparius TSA and Hubei picorna-like virus 82, or the single Hubei picorna-like virus 81 sequence, suggesting that these ORFs encode homologous proteins across all 14 sequences. For all sequences, ORF5 was predicted to encode helicase (Hel), protease (Pro) and RNA-dependent RNA polymerase (RdRp) domains. ORFs 2 and 2b had no HHpred matches; however, a transmembrane domain was predicted using TMHMM [−8; alignment length 126 amino acids), but homology to ORF2 of the C. riparius TSA/Hubei picorna-like virus 82 clade was less certain . The Formica exsecta TSA ORF3a polypeptide also had no significant HHpred matches.For each ORF, functions were predicted with HHpred  using th, HHpred  found sing TMHMM  in the mC. riparius TSA and Hubei picorna-like virus 82), the usually very well-conserved GDD in motif VI  was replaced with ADD.Following Koonin and Dolja , we idenPicornavirales. To test for monophyly, we obtained amino acid sequences for the conserved ‘core’ region of the RdRp from viruses in the order Picornavirales from the supplementary material of [muscle [Based on the genome structure and the identified protein domains, SINV-2-like sequences may be classified within the order erial of , appende [muscle  and gene [muscle . The SINPicornavirales. Thus we propose that they should be classified into a new family, Polycipiviridae (polycistronic picorna-like viruses), within the order Picornavirales. The virion morphology  observed for SINV-2 [Dicistroviridae) and the bipartite cheraviruses, sadwaviruses and comoviruses (family Secoviridae), the polycipivirus coding sequences are split into separate non-structural and structural protein modules. In contrast to these other groups, the polycipivirus structural protein module is further split into separate 5′ ORFs rather than depending on polyprotein expression of multiple jelly-roll domains from a single ORF.We have identified and sequenced four new viruses from ant species. Together with SINV-2, 6 sequences recovered from TSA databases and 4 recent additions to GenBank, these 15 sequences form a distinct group of arthropod-infecting viruses with a characteristic genome organization. Members have a polyadenylated positive-sense RNA genome that encodes three related picornavirus-like jelly-roll capsid domains, and a non-structural polyprotein containing superfamily III helicase, 3C-like chymotrypsin-related protease and superfamily I RdRp domains, characteristic of members of the order Solenopsis invicta polycipivirus) to include SINV-2, SINV-4 and nine related sequences, all of which contain ORF2b; Hupolycivirus (from Hubei picorna-like virus 81 polycipivirus) to include the two Hubei picorna-like virus 81 sequences; and Chipolycivirus (from Chironomus riparius polycipivirus) to include the C. riparius TSA and Hubei picorna-like virus 82, both of which contain the GDD to ADD substitution in RdRp motif VI.Although polycipiviruses form a distinct clade, there is considerable diversity among the 15 available sequences (some of the ORF5 pairwise amino acid identities are as low as 17 %), indicating that the family should be split into a number of genera. We propose the following groupings of the currently available sequences see : SopolycPicornavirales 3C-like proteases have a cysteine residue at this location (cysteine protease). The only other currently known exceptions are the sole member of the Picornavirales family, Marnaviridae, and some members of genus Nepovirus in the family Secoviridae [The polycipivirus 3C-like protease is unusual in that it contains a serine residue at its active site (serine protease), whereas the majority of oviridae . The ‘pioviridae .Potyviridae), polio virus (family Picornaviridae) or hepatitis C virus (family Flaviviridae) results in an in vitro RdRp activity of 5 to 12 % of wild-type activity [Picornaviridae) the RdRp was inactive in vitro [Hypoviridae) have SDD at this site. On the other hand, non-segmented negative-sense RNA viruses generally have GDN, whereas segmented negative-sense RNA viruses have SDD, and the reverse transcriptases of retroviruses have MDD [Permutotetraviridae and the double-stranded RNA virus family Birnaviridae have a permuted polymerase domain, with motif VI occurring between motifs III and IV; the GDD sequence is still present in permutotetraviruses but is substituted with ADN in birnaviruses, and mutation of ADN to GDD results in an almost complete loss of activity [The most conserved protein of positive-sense RNA viruses is the RdRp. In polycipiviruses, we were able to identify all eight signature motifs of the superfamily I RdRp. Unusually, however, in two of the sequences the highly conserved GDD of motif VI was replaced with ADD. Motif VI is responsible for magnesium ion coordination. The first aspartate (D) is mainly responsible for the coordination and cannot be substituted. There is, however, potential for flexibility in the third position, and even more flexibility in the first position (reviewed in ). Indeedactivity , althougin vitro . Positivhave MDD . Unusualactivity . In summPicornavirales species have been found to harbour a VPg protein covalently linked to the 5′ end of their genome that primes RNA synthesis during genome replication. The Picornavirales VPg is typically <5 kDa and normally encoded between Hel and Pro [Where investigated,  and Pro . Due to Picornavirales species, gene expression in polycipiviruses likely depends on internal ribosome entry site (IRES)-mediated initiation. Consistent with this, the lengthy 5′ UTRs typically contained a number of AUG codons that would be expected to inhibit 5′ end-dependent scanning. Consequently, we suppose the 5′ UTR to contain an IRES to direct ribosome initiation at the ORF1 start codon. Similarly, we suppose the long intergenic region between ORFs 4 and 5 to contain a second IRES to direct ribosome initiation at the ORF5 start codon. A similar situation occurs in dicistroviruses (family Dicistroviridae), where the translation of structural and non-structural polyproteins is directed by separate IRESes . Although we attempted to define the potential IRES RNA structure in silico by means of RNA-folding algorithms and inter-species comparisons, the results to date have been inconclusive. The translation mechanism of the additional 5′ ORFs  remains uncertain; however, close spacing of the stop and start codons of consecutive ORFs suggests a ribosome reinitiation mechanism.As in other Additional work will be required to confirm the composition and structure of virus particles, the presence and sequence of a 5′-linked VPg protein, the non-structural polyprotein cleavage sites and products, and the gene expression mechanisms of this unusual family of arthropod-infecting picorna-like viruses.Solenopsis invicta, Monomorium pharaonis and Lasius neglectus) were found to play host to more than one divergent viruses in the group, suggesting a long evolutionary history between Sopolycivirus species and ants. Although we cannot rule out other hosts, and our own sampling has been biased towards discovering new ant-infecting members, it seems possible that genus Sopolycivirus may be an ant-specific clade.Ten of the 11 members of the proposed Polycipiviridae genus Sopolycivirus appear to infect ant species, while the eleventh member (Shuangao insect virus 8) is apparently an insect virus whose host has yet to be identified . These vSolenopsis invicta ants were collected from 46 nests in the eastern portion of the state of Formosa, Argentina, and returned to quarantine in Gainesville, Florida, United States. Total RNA was extracted from 10 to 15 live worker ants from each colony using Trizol (Invitrogen) and the PureLink purification kit (Ambion). Total RNA (10 µg per group) was submitted to GE Healthcare  for mRNA purification, library preparation and Illumina RNA sequencing (MiSeq). Sequences were aligned to the S. invicta genome (GenBank accession AEAQ01000001.1) and non-matching sequences were compared to the UniProt annotated Swiss-Prot protein sequence database (download date 14 November 2014). The unmatched sequences were assembled  and a unique sequence with significant similarity to SINV-2 was identified by blastx analysis. RT-PCR with gene-specific oligonucleotide primers revealed that this sequence was present in some colonies of S. invicta in the USA. Total RNA from USA S. invicta colonies containing the sequence was extracted and used as template for cDNA synthesis, subsequent PCR and RACE (5′ and 3′) to obtain the entire genome sequence of this new virus, SINV-4, by Sanger sequencing. For 5′ RACE, the 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0  was used. cDNA was first synthesized with oligonucleotide p1476 (5′-TGGAATTCCAGAATTTTCTAAGGTTCCCATATTAGT), followed by PCR with the gene-specific primer p1474 (5′-TGAATTCCAGGTAACGCTTGAACCATTGGT) and the abridged anchor primer (Invitrogen). For 3′ RACE, the GeneRacer kit (Invitrogen) was used. cDNA was synthesized with the GeneRacer Oligo dT primer. PCR was subsequently completed with the GeneRacer 3′ primer and the gene-specific primer, p1536 (5′-ATGGCTGTTGCTGACATGTTATG CATTATGTT).Adult worker L. niger, L. neglectus, M. scabrinodis and L. flavus ants were collected from individual nests in Cambridge, UK; all ants were workers except the sequencing round 1 L. flavus sample, for which queens were used. Total RNA was extracted from 10 to 20 adult workers or 5 queens using Trizol (Invitrogen) following the manufacturer’s instructions. RNA was treated with DNase . For standard RNA-Seq libraries, ribosomal RNA was depleted using the Ribo-Zero kit (Illumina), and the remaining RNA subjected to alkaline hydrolysis, followed by acrilamide gel purification of RNA bands within the size range 35–50 nt for L. niger and 70–85 for the other four species. For small-RNA sequencing, RNA bands within the size range 18–30 nt were acrylamide gel-purified from total RNA after DNase treatment. All library amplicons were constructed using a small RNA cloning strategy [http://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-5781. RNA-Seq reads were trimmed using the FASTX-Toolkit and assembled using the Trinity (v 2.3.2) and Velvet (v 1.2.10) de novo assemblers [blastx, the assembled contigs were compared to a database of polypeptide sequences derived from SINV-2, SINV-4 and the SINV-2-like sequences identified in the NCBI TSA database. Contigs that mapped to SINV-2-like proteins with E-value <10−6 and length >300 nt of coding sequence were retained, manually joined where possible, and used to design primers for Sanger sequencing.Adult strategy  and sequsemblers . Using bGaps were filled and the entire genomes sequenced by Sanger sequencing and terminal sequences obtained by 5′ and 3′ RACE, as follows. Two µg of total ant RNA was treated with proteinase K and then recovered by acid phenol/chloroform extraction and used for 5′- and 3′-RACE using the SMARTer RACE 5′/3′ kit (Clontech) according to the manufacturer's instructions. Gene-specific primers for 5′ and 3′ RACE were located within 1000 nt of the expected 5′ and 3′ ends of the virus genome. The resulting clones  in the pRACE vector were sequenced with M13 universal primers. The rest of the viral genomes were PCR-amplified as eight partially overlapping DNA fragments using specific primers and the same cDNA templates as were generated for the 5′/3′ RACE PCRs. These fragments were cloned into the pJET1.2 vector (Thermo Scientific) and sequenced with pJET1.2-specific primers.blast [muscle v 3.8.31 [muscle alignments. To calculate the coverage and polymorphism frequencies, Bowtie 2 , using Picornavirales. Sequences for SINV-4, SINV-2, LneV-1, MsaV-1 and LniV-1 have been submitted to NCBI with the accession numbers MF041808, MF041813, MF041809, MF041810 and MF041812. Voucher specimens of the S. invicta ant species from which SINV-4 was originally sequenced are retained at USDA-ARS, Gainesville, Florida, USA.We have submitted a proposal to the International Committee on Taxonomy of Viruses (ICTV) to name a new family Polycipiviridae containing the genera Sopolycivirus, Hupolycivirus and Chipolycivirus in the order


A majority of indoor residential microbes originate from humans, pets, and outdoor air and are not adapted to the built environment (BE). Consequently, a large portion of the microbes identified by DNA-based methods are either dead or metabolically inactive. Although many exceptions have been noted, the ribosomal RNA fraction of the sample is more likely to represent either viable or metabolically active cells. We examined methodological variations in sample processing using a defined, mock BE microbial community to better understand the scope of technique-based vs. biological-based differences in both ribosomal transcript (rRNA) and gene (DNA) sequence community analysis. Based on in vitro tests, a protocol was adopted for the analysis of the genetic and metabolic pool (DNA vs. rRNA) of air and surface microbiomes within a residential setting.Sphingomonadales order, were present in lower relative abundances in the viable community.We observed differences in DNA/RNA co-extraction efficiency for individual microbes, but overall, a greater recovery of rRNA using FastPrep (> 50%). Samples stored with various preservation methods at − 80°C experienced a rapid decline in nucleic acid recovery starting within the first week, although post-extraction rRNA had no significant degradation when treated with RNAStable. We recommend that co-extraction samples be processed as quickly as possible after collection. The in vivo analysis revealed significant differences in the two components (genetic and metabolic pool) in terms of taxonomy, community structure, and microbial association networks. Rare taxa present in the genetic pool showed higher metabolic potential (RNA:DNA ratio), whereas commonly detected taxa of outdoor origins based on DNA sequencing, especially taxa of the Although methodological variations in sample preparations are high, large differences between the DNA and RNA fractions of the total microbial community demonstrate that direct examination of rRNA isolated from a residential BE microbiome has the potential to identify the more likely viable or active portion of the microbial community. In an environment that has primarily dead and metabolically inactive cells, we suggest that the rRNA fraction of BE samples is capable of providing a more ecologically relevant insight into the factors that drive indoor microbial community dynamics.The online version of this article (10.1186/s40168-018-0453-0) contains supplementary material, which is available to authorized users. The majority of individuals from the developed world spend over 90% of their time indoors, or in other built environments (BEs) , which nWhile earlier culture-based investigations have provided information on the viable and cultivable components of the indoor microbiome, high-throughput sequencing (HTS) of the DNA of the 16S ribosomal RNA gene (rRNA gene) offered unparalleled insights into the breadth of the diversity and composition of the BE microbiome. Studies have pointed to the outdoor environment, occupancy, and building characteristics  as the main sources of the indoor microbial community –7. AlthoAlternatively, the direct examination of rRNA through RNA isolation has been found, in general, to be a more reliable indicator of cellular viability than rRNA gene targets –19. UndeWith limited biomass, the BE poses a special challenge in providing sufficient material for RNA isolation, storage and analysis. Selective pressures imposed by the BE, such as desiccation and UV irradiation, generate additional challenges in extracting intact nucleic acids. Creating a robust and reproducible method for nucleic acid extraction, especially the RNA fraction, is essential to more accurately infer the survival and adaptive potentials of indoor microbes, and the viability of pathogens that are potentially present in BEs. Moreover, this will empower BE scientists to determine how the building and occupant attributes potentially shape the viable components of the BE microbiome.Variations in methodologies, such as sample collection, storage, or commercial extraction kits, have been shown to have an impact on microbiome data interpretation –30. FollA mock community including pure bacterial and fungal strains were included to test the effects of different DNA/RNA co-extraction methods, swab and surfaces types, and sample and nucleic acid storage conditions, over the recovery rate of both components (DNA and RNA). In vitro recovery rate of nucleic acid material was based on the result of quantitative PCR (qPCR). Based on in vitro results, sequencing analysis was performed on air and surface samples collected from an unoccupied bedroom to compare the total genetic pool and the viable components of the microbial communities. The following sections describe in detail the different tests conducted.Pseudomonas syringae, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus nidulans. , 20% of yeast (S. cerevisiae), and 10% of fungi (A. nidulans). Microbial isolate proportions were based on recent indoor dust studies that quantified the bacterial and fungal community in BE samples [4 cells/mL was spiked onto the samples, and the samples were extracted along with an extraction blank and an aliquot of the dilutions in PBS buffer as a reference of the actual number and RNA content of the spiked cells.Four pure strains representing different types of cells were used:  samples –33. For Specific primers targeting the 16S and 18S rRNA genes were designed for each strain using primer-BLAST  . The cloned sequences were confirmed by Sanger sequencing at UC Berkeley DNA Sequencing Facility. Plasmids were extracted and purified , then linearized  followed by the quantification of the DNA concentration  and preservation at − 20 °C.The qPCR standards were constructed amplifying the rRNA genes from the pure strains using the designed primers and the same quantification protocol , and quantified on the same day. Following extraction by one of the three evaluated protocols, the RNA was digested with the TURBO DNA-free Kit . Reverse transcription of the isolated RNA into complementary DNA (cDNA) followed manufacturer’s recommendations  were evaluated. Also, three types of swabs  were compared  were used as recommended by the manufacturers  with a D-plate accelerator (collects particles with a diameter larger than 0.25 μm). All windows were closed, and the room was left unoccupied during sampling (except when required to change sampling filters and disinfect the apparatus). To minimize the effect of sampling time on differences in microbial community composition [°C until nucleic acid extraction.Indoor residential samples were collected during April 2017 in an approximately 10 mong Kong , 40. Bioong Kong , 40 usinposition , all theg 15 min and the LifeGuard discarded. The pellet was resuspended with the appropriate lysis buffer and proceed with the extraction. To assess the effect of sample preservation and storage time on environmental BE samples, DNA and RNA from air filter samples were extracted on six different time points spanning 6 weeks of storage.gDNA from the indoor surface and air samples were extracted using the DNeasy PowerSoil Kit with slight modifications as previously described . Based oRNA from environmental samples was processed as described above. gDNA and cDNA were subjected to bacterial PCR by primer pairs targeting the 16S rRNA V4  pipeline . The rawp values were adjusted for multiple comparisons using the false discovery rate (FDR).The results of the in vitro test are expressed as the proportion of the DNA (as 16S/18S rRNA gene copies) and the RNA (as 16S/18S rRNA copies) recovered from the spiked samples in comparison with the reference sample of each set of experiments. R software  was usedp < 0.05 considered statistically significant. Only OTUs with DeSeq2 log-fold changes of at least |2| were considered to be differentially abundant. Where indicated, p values were adjusted for multiple comparisons using the FDR, and Kendall’s τ ranked correlation was computed in R [ANOSIM Global R and PERMANOVA pseudo-F statistics were calculated for the indoor microbiome samples using QIIME, based on the default setting of 999 permutations. To identify differentially abundant OTUs between genetic and metabolic pool, DeSeq2 was performed with an adjusted ted in R .Molecular-based methods have greatly increased our understanding of the diversity of ecological interactions observed among the members of the BE microbial community when compared to culture-based methods. However, discrepancies in the conclusions based on microbial community composition and inferred metabolic activity among different studies have highlighted the need to better understand how the various sample preparation methods influence an individual study’s results. In this study, we compared the efficiencies of several common DNA/RNA sample co-extraction methods, materials for surface sampling on different surfaces, and sample and nucleic acid storage methods.P. syringae, B. subtilis, S. cerevisiae, and A. nidulans with final cell densities of ca. 104 cells/mL were used to evaluate the DNA/RNA co-extraction efficiencies of the DNeasy PowerSoil Kit and PowerMicrobiome RNA Isolation Kit (Power), the AllPrep DNA/RNA Mini Kit (AllPrep) and the FastDNA and FastRNA SPIN Kit for Yeast (FastPrep), all with minor modifications as stated in Additional file p < 7×10− 5) although no statistically significant differences were found among the three methods in DNA extraction. The Power Kit appeared to have slightly less variation in efficiency between microorganism type for DNA and one of the lower overall extraction efficiencies for RNA compared to the other kits.Log-phase cultures of S. cerevisiae was among the highest in recovery of DNA and the lowest for RNA (p < 0.1) when using the FastPrep Kit. Conversely, the filamentous fungi A. nidulans had one of the lowest efficiencies of recovery for DNA from the AllPrep Kit, but one of the highest efficiencies for RNA. We believe that this is due to the very large method variation that was observed, rather than in intrinsic differences in RNA copy number characteristic of each species. This observed variability in nucleic acid extraction efficiency indicates that it is important to understand the range of technical variation in setting a threshold for what is a significant difference in inferring metabolic activity/viability using RNA:DNA ratios.Within each method, differences in extraction efficiency were observed for individual microbes when comparing DNA to RNA. For example, the yeast p > 0.07), showing a similar DNA and RNA extraction efficiency to the obtained for the spiked swabs extracted with the same protocol, FastPrep (MW p = 0.67).Cellulose nitrate filters from the air sampler were spiked with the BE mock community, followed by nucleic acid co-extraction, to evaluate the potential interference with chemicals accumulated on the filters during the air sampling. No significant differences between the clean and used filters spiked with microorganisms were detected . None of the preservation methods outperformed the others in the storage of RNA. This high level of intraspecies variability should be of concern when setting up an experimental design for a BE study so that, whenever possible, immediate nucleic acid extraction should be attempted.BE microbiome studies typically require an extensive sampling regime over multiple locations and/or time points. To avoid extensive degradation of the nucleic acids prior to analysis, samples are stored in a way to optimize their integrity. We evaluated six different methods for sample storage preserving the DNA and RNA and an additional method specifically for post-extracted RNA. The two most striking findings were the very high level of variability in replicate performance of the nucleic acid preservation within each method Fig. , and theAn additional commercially available solution that preserves extracted RNA, RNAStable, was also evaluated. Tests with the BE mock community demonstrated post-extraction RNA preservation during the 3-month storage with no significant degradation Fig. . The RNAAlthough microbes that are found in indoor air and surfaces commonly originate from external sources, their ability to persist is dependent on their ability to survive environmental stresses, such as low humidity, UV light, and lack of nutrients. Identification of the indoor microbial community composition and abundance through DNA-based methods does not provide the ability to distinguish the majority of the microbial community that is either metabolically inactive or non-viable from the minority that remains viable. Even with the previously mentioned caveats of using the more labile RNA to identify potentially viable or active microbes, it may still provide more ecologically relevant information than DNA when a majority of the microbes are dead or dormant. With multiple replicates, we identified very high levels of variation in all steps of process of BE samples for analysis. In many cases, we found that the variation in response among different organisms was greater than the different commonly used methods, leading us to conclude that standardization of methods among BE researchers may not produce the desired clarity that is hoped by its proponents. A practical solution may be to use this information to attempt to minimize variation in sample preparation and storage wherever possible, and to require that the differences among samples be greater than the observed differences found within the methods for a biologically meaningful conclusion. As a test case, we examined the rRNA and DNA nucleic acid fractions of surface and air samples in a residential unit. By examining differences in the potentially active/viable subset in comparison with the total BE microbial community, we were interested in determining if the rRNA fraction would add value to the more commonly used, DNA-based, microbial analysis.p = 0.001) and community composition . Environmental genera drove the differentiation of the genetic pool, whereas host-associated genera drove the differentiation of the viable population . The differences in the taxonomic profiles of the DNA and RNA populations were also consistent with the community composition data  and RNA  components of the indoor air microbiome. To our understanding, this is the first account involving DNA and RNA fractions of the of indoor air and surface microbiomes. Similar to a previous work of outdoor air , the genion Fig. . Within ata Fig. . Specifinditions . Therefonditions .Fig. 4DiSphingomonadales order. While members of Sphingomonas and Sphingopyxis have been identified in air and on surfaces of different BEs [Deinococcus is the most differentially abundant in the viable population, which is consistent with this genus’ ability to be resistant to radiation and desiccation, and survive in harsh indoor environments [Streptococcus, Corynebacterium, Staphylococcus) were also significantly and differentially abundant in the viable populations. Given that some of these genera include potentially pathogenic species, species- and strain-level analyses of the RNA population within indoor air may be warranted, as potentially live and pathogenic microbes may be transmitted between indoor individuals [DeSeq2 was performed to determine differential abundance of specific OTUs between the genetic and the metabolic pool . OTUs with high RNA:DNA ratios include members of Microlunatus, a genus previously postulated to be a rare but active in cleanroom surface microbiome [Brachybacterium had an RNA:DNA ratio at over 170. Different species of Brahchybacterium collected over multiple seasons from bioaerosols of Chinese residences had previously been demonstrated to be culturable, suggesting that members of this genus can remain viable in indoor air [Psychrobacter and Veillonella, both of which have been detected previously in low levels in bioaerosols of different BEs [Pseudomonas), which may suggest species or strain-level variations in activity that may or may not have ecological, physiological, or clinical importance [Sphingomonadales  had the lowest RNA:DNA ratios, further suggesting that these abundant members have lower metabolic potentials in the environment sampled.The RNA:DNA abundance ratios for OTUs present in both DNA and RNA populations were calculated to estimate their metabolic potentials. The ratios for these OTUs ranged from 0.002 to over 400  also presented OTUs that were significantly associated with other taxa in the viable network. Significant and positive correlations  within members of the same genus were only observed in the DNA population, whereas OTUs of different genera could be involved in both co-occurrence and negative  associations in the DNA and RNA-based networks , which may be possible as a result of resuspension of surface microbiomes (some of these microbes may be active) from the resident into the adjacent air [Source-tracking analysis was performed to estimate how viable populations of nearby surface sources potentially contributed to the air sink viable communities and vice versa. In general, the surface closest to the air samplers  showed approximately 50% community overlap with the air communities Table . Interescent air , 70, descent air , 72. Noncent air , 63, 73,Sphingomonas, OTUs of which were overrepresented in DNA population and classified as having low RNA:DNA ratios in our residence bioaerosols, are commonly detected in BE samples, both in the air [In summary, our residential analysis revealed strong variations in DNA and RNA components of the residential microbiomes in terms of community structures, taxonomies, and associative networks. As our in vitro tests suggested, methodological variations may have contributed to the observed DNA and RNA community differences. However, the increased abundance of host-associated taxa in the RNA population in our household analysis is not biologically improbable. Skin shedding, and talking and coughing from indoor occupants introduce host-associated organisms that may persist and remain viable and/or active for extended periods of time onto BE surfaces and into the air , 73, 74. the air , 37, 61  the air –77, whicEstimating metabolic potential by calculating the genetic pool to viable population ratio has been reported across ecosystems , 78, 79.2 was the preferred method for long-term sample storage, although strikingly, significant nucleic acid degradation was noticed in all storage methods by the first week. Despite the potential logistical difficulties, our strongest recommendation for minimizing technical biases is to perform the nucleic acid extraction within the first week, and then store the nucleic acids in preservation solutions until further analysis.This study confirms the high level of technical variability, similar for both DNA and RNA sample processing, and emphasizes the relevance of replicates in molecular-based microbial community studies. Although the FastPrep method of DNA/RNA co-extraction had the highest efficiency of RNA recovery, the overall differences among the methods did not rise to the level of a strong recommendation of one method exclusively. Similarly, no recommendations can be made for sample swab methods due to the similarity of their performance. Flash freezing with liquid NIn spite of the inherent technical biases, the biological variation observed in activity/viability of residential BE samples in this study highlighted the potential roles of key microbial taxa. Specifically, we identified taxa that by DNA-based sequence analysis appeared to be in high abundance, but by rRNA-based sequence analysis suggestive of dormancy or non-viability. Conversely, we identified low-abundance taxa that by rRNA-based sequencing may have important ecosystem functions. Further studies are required to fully demonstrate and understand the effectiveness of rRNA as a proxy for viability, but certainly rRNA-based microbial community studies offer a new dimension of information not accessible by the DNA-based analysis. It is our opinion that future nucleic acid-based BE studies incorporate rRNA preparation and analysis to provide insight into microbial population dynamics of the active/viable taxa , and to Additional file 1:Table S1. Pure strains and primers used in this study. List of the pure strains and primers used in this study and their references. (DOCX 123 kb)Additional file 2:Table S2. Nucleic acids processing protocols. qPCR and retrotranscription reactions and conditions. (DOCX 65 kb)Additional file 3:Table S3. Sampling, extraction, and preservation products and surfaces tested. List of in vitro tests and the products tested, including the manufacturer details. (DOCX 77 kb)Additional file 4:Text S1. Detailed description of optimized DNA/RNA co-extraction protocols. Step by step detailed co-extraction protocols. Steps modified from the manufacturers’ instructions specified. (DOCX 18 kb)Additional file 5:Text S2. Detailed protocols of sample storage test. (DOCX 64 kb)Additional file 6:Table S4. Differentially abundant OTUs and their taxonomic classification between DNA and RNA populations in residential air microbiome. (XLSX 45 kb)Additional file 7:Table S5. RNA:DNA ratio of shared OTUs in residential air microbiome. (XLSX 37 kb)Additional file 8:Figure S1. Network analysis of DNA and RNA components of the microbiome. Each node represents a particular OTU that is involved in significant correlation with other OTU(s) as calculated in SPIEC-EASI. Networks generated using Cytoscape. OTUs are colored by their genera and are connected to other OTUs to represent positive (blue edges) or negative (red edges) correlations. The strength of the correlation is represented by the thickness of the edge. Hub OTUs, and OTUs with high metabolic potential, are indicated with their OTU number and genus-level taxonomy. Taxa with significant associations are not necessarily classified as the abundant. Similar structure properties were detected for both network analyses Additional file 10:Figure S2. Correlation network structure between DNA and RNA components of the microbiome. Density plot of intra-genus (red) and inter-genus (blue) correlations within the (A) DNA and (B) RNA components of the microbiome. Plots are faceted for each microbiome component based on whether the correlation is positive or negative. C–F. Network structure comparison between DNA (blue) and RNA (red) components of the microbiome in terms of (C) degree distribution, and natural connectivity upon removal of network node either (D) randomly or via decreasing order of (E) degree or (F) betweenness centrality. Network structure analyzed using SPIEC-EASI. (PDF 1413 kb)


However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a DOI:http://dx.doi.org/10.7554/eLife.27055.001 Immature virions of HIV-1 and other retroviruses are assembled from the viral Gag polyprotein. This precursor protein consists of matrix (MA), capsid (CA), nucleocapsid (NC) and p6 domains, as well as two spacer peptides (SP1 and SP2) flanking the NC domain. The MA domain binds Gag to the inner leaflet of the plasma membrane , althougAlmost any single-stranded nucleic acid can support virus-like particle (VLP) assembly in vitro . Nonethecis-acting RNA element called the ‘packaging signal’ (‘Ψ') that is required for its selective packaging during virion assembly  for the binding. In fact, most biophysical techniques  require high Gag concentrations (micromolar regime); at these concentrations Gag/RNA complexes will associate into virus-like-particles. We have circumvented this problem by using FCS, in which the concentration of the fluorophore-tagged RNA is low enough that association of Gag/RNA complexes into VLPs or other large structures is not thermodynamically favorable.The goal of the experiments described here was to measure and characterize the binding of recombinant Δp6 HIV-1 Gag protein to short single-stranded RNAs. As noted in the Introduction, addition of RNA to Gag can lead to assembly of VLPs, complicating the measurement of a dissociation constant  of the RNAs in each mixture. In addition, we found that binding of Gag induced quenching of the Cy5 fluorophore; thus the fraction of RNA molecules bound to Gag could be estimated from the normalized fluorescence intensity in each reaction mixture. It has been shown that quenching of this fluorophore results from the steric stabilization of a non-fluorescent photon-induced Cy5 isomer  were added. After overnight incubation at 4° C, the mixtures were analyzed by FCS at room temperature. The autocorrelation curves from Cy5-labeled RNAs were analyzed to obtain the weighted average diffusion constant  and ≈ 200–250 nM Gag with MoMLV Ψ and GRPE RNAs. (D) of the monomeric and dimeric HIV-1 Ψ RNAs were different enough to be resolved from each other by FCS and that the dimerization efficiency was ≈ 90%). Thus, the binding of Gag results in a decrease in the hydrodynamic radius of the RNA. This was somewhat unexpected, as the Gag/RNA complexes must have greater masses than the free RNAs, and implies that binding of Gag to the RNA causes a collapse in the RNA. The increase in the diffusion coefficient is not due to RNA degradation; upon digestion of the Gag/RNA complexes with Proteinase K, the D of the labeled RNA reverts to that of naked RNA  in the Hill Equation for cooperative binding) was greater than one for all RNAs (between 1.7 and 3.4). Therefore, Gag-induced RNA collapse is evidently a cooperative process. It should be pointed out that the fit for the dimeric HIV-1 Ψ is relatively poor, probably because the number of data points in the sigmoidal portion of the curve is very small.Molecular dynamics simulations indicate that RNA collapse by capsid proteins involves protein-protein interactions . If RNA  to zero . These pAs mentioned before, the fluorescence of the RNAs is reduced by addition of Gag see . By usinDK and Hn for each RNA. DsK obtained ranged from 21 nM for dimeric HIV-1 Ψ to 44 nM for GRPE. Furthermore, the Hsn derived from these curves varied between 1.6 and 2.5 , non-cooperative multiple binding and one-to-one binding (see supplementary information).  2.5 see , implyinviz. yeast tRNA. Unless otherwise noted, in the competition experiments the tRNAs and Gag were pre-incubated together for about 10–15 min before addition of the labeled RNAs. The concentration of the tRNAs was 50-fold higher by mass than that of the labeled RNAs (≈120-fold molar excess).To test the in vitro binding specificity of Gag for the HIV-1 Ψ we repeated the titrations in the presence of an excess of an irrelevant competitor RNA, DsK derived from these curves , while the inverse order of addition showed only a slight decrease in the apparent affinity (DK of 44 nM) (red squares). On the other hand, the apparent affinity for the GRPE (green diamonds and squares) is greatly affected by order of addition of the RNAs. Unlike the results with HIV-1 Ψ, addition of tRNAs reduced the apparent affinity of Gag for GRPE by 2.8-fold when added after the GRPE, but 5.5-fold if added prior to GRPE.  . These results confirm that Gag-Gag interaction plays a major role in RNA collapse; taken together with the cooperativity of the binding of WM Gag K) and estimated the net number of ions displaced during binding. The D(1M)K represents the dissociation constant in the absence of electrostatic interactions, which can be understood as the contribution of specific interactions to the overall DK.Binding of a protein to nucleic acids could be driven by non-specific interactions, such as the electrostatic attraction between positively charged side chains in the protein and the phosphate groups on the RNA. It could also be a highly specific interaction; in most cases, these interactions are non-electrostatic. Finally, it might be a combination of both specific and non-specific interactions . The rolomplexes . By usinTo understand the contribution of electrostatic and non-electrostatic interactions to binding we performed Gag titrations at different NaCl concentrations with the RNAs that exhibited the highest and lowest affinity for Gag: the dimeric HIV Ψ and GRPE, respectively. DsK calculated from y-intercept represents D(1M)K. Surprisingly, with our data, these plots are not linear . On theAs mentioned before, when Gag-Gag interactions were weakened (by means of ablating the dimer interface with the WM mutation), the presence of tRNAs revealed binding specificity for the dimeric HIV-1 Ψ . In a siWe also asked whether the specific interactions between Gag and RNA are strong enough to permit binding when MA-RNA and Gag-Gag interactions are both weakened. To do so we combined the 8N and WM mutations in one protein (8N/WM Gag). The green diamonds in DKs of SSHC Gag for the dimeric HIV-1 Ψ and GRPE RNA, respectively, are not significantly different from those of WT Gag. However, increasing the salt concentration drastically interfered with the binding to both RNAs. Binding of SSHC Gag to the dimeric Ψ RNA was undetectable at NaCl >300 mM . The blue triangles in To further analyze the contribution of the NC domain, we used a mutant protein (310 Gag) in which four positively charged amino acids between the two zinc fingers were mutated to alanines  binding of Gag to an RNA results in the physical collapse of the RNA and in quenching of the Cy5 fluorophore covalently linked to the 3’ end of the RNA; (2) in all cases examined, Gag binds cooperatively to RNAs; (3) the affinities of Gag for all the RNAs tested are all very similar to each other at physiological ionic strengths; however, (4) specificity for Ψ is revealed when affinities are measured in the presence of an excess of a competing irrelevant RNA; when Gag-Gag and/or Gag-RNA interactions are impaired by mutations in Gag; or  when thThe results obtained with mutant Gag proteins and with competing tRNA are particularly informative. We found that addition of excess tRNA interferes significantly more with binding to the control RNAs (lacking Ψ) than to either monomeric and dimeric HIV-1 Ψ RNAs. This is in agreement with a previous study showing that addition of an excess of a competitor RNA helps to reveal binding specificity . HoweverDK at 0.25 M NaCl and by the salt-resistance of the binding  retained significant affinity for Ψ, but its ability to bind the GRPE control RNA was drastically curtailed, as indicated by the salt-sensitivity of this interaction . This saDK ~10 µM to DK ~1 mM  non-electrostatic interactions; (II) electrostatic interactions; and (III) the interactions between two or more Gag molecules bound to a single RNA molecule. Interactions of the first type, mediated largely by the NC zinc fingers, impart specificity for Ψ. To a first approximation, the electrostatic interactions are presumably similar for different RNAs; our data show that the basic residues in the MA domain contribute significantly to these interactions. It seems likely that the basic character of the NC domain also plays a role in these interactions. The specificity for Ψ is revealed in the presence of mutations that reduce the strength of either the second or third types of interaction, or by addition of RNAs that compete for these interactions, or in an ionic environment that screens the second type of interaction.Comparison of the effects of added tRNAs or increased ionic strength upon the apparent affinities of WT and 8N Gag for the labeled RNAs  shows theffZ)  ; in thesstraight  and 9. Anditions , 2011b. DKs, and thus we cannot completely exclude the possibility that the actual DK is even lower than our results indicate. However, it is important to note that the curve for WT Gag and GRPE RNA is also bent  is near the measured lso bent , althouglso bent . FurtherDK is a weighted sum of the electrostatic interactions (black line and circles) and strong non-electrostatic interactions (solid green line). In the first binding mode (I) the conformation of Gag is such that binding is mostly non-electrostatic. In the second binding mode (II), the salt-induced conformational change in Gag gradually decreases the contribution of the non-electrostatic interactions (dotted green line). (The details of this model are explained in the supplementary information section.) Again, with 8N Gag/dimeric Ψ, 8N Gag/GRPE, and 310 Gag/dimeric Ψ, only a single slope is visible: this is because we could not detect binding at higher ionic strengths . The DKs at higher salt concentrations in these cases must be >1 µM. In other words, these curves do have a second slope, but this slope is so steep that we could not make measurements at these high salt concentrations.It is particularly surprising that the curves exhibit steeper slopes at higher ionic strengths than at low ionic strength. One might naively expect that as the salt concentration is increased the electrostatic component decreases, ultimately reaching a point at which only the non-electrostatic component remains significant. This scenario would produce a curve with a steep slope  at low salt and a shallow one at high salt; however, our plots  and 9 shet al.  with binding to GRPE RNA, we also characterized binding to monomeric Ψ and to the MoMLV Ψ (RNAs containing MoMLV Ψ are not selectively packaged by HIV-1 Gag protein). In general, our data shows that binding of these two RNAs to Gag and its mutants is intermediate between the dimeric HIV-1 Ψ and the GRPE RNA. Under most experimental conditions the binding affinity of WT, as well as of most mutant Gag proteins, for the dimeric and monomeric HIV-1 Ψ were very similar to each other. Nonetheless, the dimeric RNA always exhibited a higher binding affinity than its monomeric counterpart.  In fact, because the difference in the overall binding character between the monomeric and dimeric HIV-1 Ψ were small, we decided not to test the effects of RNA dimerization on the MoMLV Ψ. It was recently reported  that theet al. , by Topo cloning. To increase the labeling density of the RNAs (see below) a 15-base polyA tail was added between the end of each viral sequence and the NcoI restriction site.The HIV-1 Ψ (nt. 193–368) and the Gag-Pol Frame Shift (GRPE) (nt. 2004–2179) sequences were amplified by PCR from a pNL4-3 plasmid. The Moloney Murine Leukemia Virus Ψ (MoMLV Ψ) sequence (nt. 202–377) was amplified from a proviral plasmid (GenBank J02255.1). A T7 promoter and an NcoI and BglII and the fragment containing the viral sequence was purified from a 1% agarose/TAE gel by using a Nucleo spin kit  and stored at −20°C. In vitro transcription of the fragments was done with a MEGAshortscript kit (ThermoFisher Scientific) following the vendor’s instructions. The RNA was purified by gel electrophoresis (6% polyacrylamide/TBE-UREA gel) and eluted overnight in a Thermomixer  at 30°C and 1000 rpm . The RNAs were further purified by phenol:chloroform extraction and ethanol precipitation  , (v) to remove any nucleic acids a 5% polyethylamine (PEI) solution was added to a final concentration of 0.05% after the ammonium sulfate step (see protocol in  plots were fitted by doing a least squares linear fit using the following formula:The log In most instances the data was not linear over the entire salt concentration regime, thus we used two independent linear equations to fit the data. These two equations were used to obtain the fitting parameters for the shallow and steep slopes.It should be noted that, because of the nature of the interaction of protein with the fluorophore, the quenching is all-or-none. That is, as mentioned in the main text, excitation of Cy5 induces a reversible conversion to a completely non-fluorescent state, evidently stabilized by steric interactions in the Gag-RNA complexes. This is distinct from the quenching experienced by many fluorophores, in which there is a change in emission spectra or the relaxation time of the dye . Since t2 (black circles) was used to approximate the pure electrostatic component of binding of WT Gag to the dimeric Ψ (black line). The salt titration data for WT Gag/Ψ2 (red circles) was used to represent the sum of both electrostatic and non-electrostatic interactions (red line).To explain the concave or ‘upward bent’ shape of these salt titrations  and 9 weThis model assumes that there are two binding modes. In the first  binding mode (I) the interaction between WT Gag and the dimeric Ψ is mostly non-electrostatic (solid green line). This assumption is based on the facts that between 0.2 and 0.4 M NaCl the salt-sensitivity of binding is minimal and that disruption of the zinc fingers (in SSHC Gag) greatly increases the salt sensitivity of binding. In the second binding mode (II) we assumed that the salt-dependent conformational change in Gag gradually reduces the strength of the non-electrostatic interaction (broken green line). This assumption is based on the high salt-sensitivity of this second binding mode.To model the experimental data, measured affinity at each salt concentration was taken to be the sum of the electrostatic component  and a hypothetical non-electrostatic interaction (green line). In the first binding mode (I), the weighted sum of these two interactions was adjusted so that it produced the same slope and intercept as the experimental data; the weight of the non-electrostatic interactions was greater than the electrostatic one and both contributions were kept constant. In the second binding mode (II), the weight of the non-electrostatic interactions was reduced. The ratio of each interaction with respect of each other was chosen to obtain a slope and intercept similar to the experimental data.It is important to point out that the goal of this model is not to fit the experimental data but to show that the concave or ‘upward bent’ shape of the salt-titrations indicates that binding of Gag to RNAs has a significant non-electrostatic component and that the strength of this interaction varies as the salt concentration is increased. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Gisela Storz (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Jeremy Luban (Reviewer #3).Thank you for submitting your article "Dissection of specific binding of HIV-1 Gag to the "packaging signal" in viral RNA" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In addition to the essential revisions noted below, it will be important for the Board and reviewers to understand the importance of the new conclusions you wish to draw.Summary:This paper reports a variety of experiments that score Gag-RNA interactions, as measured by a fluorescence quenching assay, that probe the selectivity of Gag for the viral gRNA, as occurs in assembling virions. This area has a long history, but there remain unanswered questions about this selectivity. The work is carefully done and clearly explained. There are few dramatic new findings: most of the results are confirming earlier work, albeit with better readouts. There are new results concerning Gag-Gag interactions and effects of Gag mutations. Studies of the RNA are not included, except to test monomer and dimer RNAs.The major gorilla-in-the-room problem is the high background of nonspecific binding manifest in Gag: in many simple in vitro binding experiments, no high selectivity is observed. Here we see that binding at physiological salts again shows no selectivity. Adding competitor tRNA reveals selectivity for short RNAs containing the packaging sequences, (as per earlier work). Mutations that reduce Gag-RNA interactions (MA mutations) help in revealing some selectivity. We now also learn that mutations in Gag that reduce Gag-Gag interactions (CA mutations) reveal some small selectivity, which is enhanced by addition of competitor. The nonspecific binding, and not the specific binding, seems to involve Gag-Gag multimers: Gag mutants with reduced Gag-Gag binding revealed a modest reduction in nonspecific binding (though only 2.5x), and no change in Gag-gRNA binding. High salt also reveals selectivity, phenocopying the CA mutations. The relevant salt concentrations tread a narrow window ledge of enhancing selective binding before all binding is lost.The assays (by detecting changes in diffusion coefficient) show a new phenomenon going on in Gag binding: the condensation or "collapse" of the RNA. The Gag binding to nonspecific RNAs seems to induce this more effectively. It is unclear exactly what this means, and in particular how many Gags per RNA are involved.All told these are nice data on a long-standing problem. The bottom line: revealing selective binding requires careful control of the conditions and settings. There are some new conclusions, if no groundbreaking upheavals of dogma.Essential revisions:A number of clarifications, listed below, are in order:1) Reagent validation: Although the lab has much experience in the purification of RNAs and Gag proteins, supplementary figures introduced at the beginning of the Results section to validate the reagents would be desirable. For RNAs, native gels would be useful. For proteins, gels of WT and mutant Gag proteins also would be useful. I'm less concerned about protein purity than about how much protein has aggregated or oligomerized irreversibly at the storage concentration of 20-40 uM.2) MLV Psi analysis: As a dimeric RNA control, did the authors test a MLV Psi2 dimer ligand in their studies? If so, what were the results?3) Fraction RNA bound calculation: As far as I can tell from Equation 8, the authors are assuming a linear proportionality of Gag binding to fluorophore quenching, i.e., that binding multiple Gag proteins to an RNA does not have a disproportionate quenching effect relative to binding a single Gag protein. If I am incorrect, the authors might add an explanatory sentence in the data analysis section. If the authors are assuming a linear proportionality, it would be helpful to know why this assumption is correct.4) 5) 6) tRNA effect on D: The fact that tRNA addition marginally affected WT Gag binding to HIV Psi2, but also reduced "RNA collapse" is consistent with the binding of tRNAs to Gag-HIV Psi2 complexes (subsection “tRNAs reduce the degree of Gag-induced RNA collapse”), but a model for what is happening ought to be included in the Discussion.7) 8) Defining the 310, SSHC, and 8N/WM mutants: This should be done in the first paragraph of the subsection “Decreasing NC-RNA or Gag-Gag interactions reveals specific binding at slightly higher than physiological salt concentrations”.9) WT Gag-HIV Psi2 binding complexes: Have the authors performed sedimentation or other experiments to determine the Gag:RNA stoichiometries? Essential revisions:A number of clarifications, listed below, are in order:1) Reagent validation: Although the lab has much experience in the purification of RNAs and Gag proteins, supplementary figures introduced at the beginning of the Results section to validate the reagents would be desirable. For RNAs, native gels would be useful. For proteins, gels of WT and mutant Gag proteins also would be useful. I'm less concerned about protein purity than about how much protein has aggregated or oligomerized irreversibly at the storage concentration of 20-40 uM.Three new supplementary figures, linked to 2/s, thus by using the Stokes-Einstein equation  we obtained a hydrodynamic radius (hR) ≈ 2.5 nm. This shows that at under these experimental conditions Gag is monomeric and that there is no significant aggregation in solution. Further, Finally, this information has been added to the Materials and methods section  and data analysis”).2) MLV Psi analysis: As a dimeric RNA control, did the authors test a MLV Psi2 dimer ligand in their studies? If so, what were the results?We did not test dimeric MoMLV Ψ in our studies. The main reason for this was that even for HIV-1 Ψ the difference in the binding character between monomeric and dimeric RNAs was small. In fact, the only condition where the monomeric HIV-1 Ψ showed a significantly impaired binding character was in the tRNA competition experiments using WM Gag. This has been addressed in the fifteenth paragraph of the Discussion.3) Fraction RNA bound calculation: As far as I can tell from Equation 8, the authors are assuming a linear proportionality of Gag binding to fluorophore quenching, i.e., that binding multiple Gag proteins to an RNA does not have a disproportionate quenching effect relative to binding a single Gag protein. If I am incorrect, the authors might add an explanatory sentence in the data analysis section. If the authors are assuming a linear proportionality, it would be helpful to know why this assumption is correct.This is a crucial question. Our answer has two parts. First, because of the nature of the interaction of protein with the fluorophore, the quenching is all-or-none. That is, as mentioned in the manuscript , excitation of Cy5 induces a reversible conversion to a completely non-fluorescent state, evidently stabilized by steric interactions in the Gag-RNA complexes. This is distinct from the quenching experienced by many fluorophores, in which there is a change in emission spectra or the relaxation time of the dye . Since the conversion is all-or-none, there is a direct, linear relationship between the number of molecules quenched and the decrease in total fluorescence.The second part of the answer is to point out that the quenching eventually reaches a plateau, where further addition of Gag protein has no further effect on fluorescence; we interpret this to mean that at this plateau, all the RNA molecules are bound by one or more Gag molecules. This stoichiometry is taken as 100% bound RNA in the Gag titrations. Although these are the only assumptions we used in converting the quenching data into binding plots, it should be noted that a model positing cooperative binding gave a far better fit to the binding data than other models .We have discussed these aspects of the data analysis in the Supplementary Information subsection “Fluorescence spectroscopy and data analysis”.4) This is probably because the number of data points in the sigmoidal portion of the curve is very small. This increases the error of the fit. This issue has been addressed in the last paragraph of the subsection “Gag binding causes RNA collapse”.5) H values we obtained. We acknowledge the imprecision in the measurements and note this in the last paragraph of the subsection “Gag binds to RNAs with high affinity but low specificity”.The reviewers are correct that we have not distinguished between the different n6) tRNA effect on D: The fact that tRNA addition marginally affected WT Gag binding to HIV Psi2, but also reduced "RNA collapse" is consistent with the binding of tRNAs to Gag-HIV Psi2 complexes (subsection “tRNAs reduce the degree of Gag-induced RNA collapse”), but a model for what is happening ought to be included in the Discussion.We have added the following model to the Discussion: “It was interesting to note that the presence of excess yeast tRNA greatly reduced the magnitude of RNA collapse . This is7) We have added the following explanation to the Discussion section: “It is somewhat surprising that the apparent affinity of 8N Gag for the dimeric HIV-1 Ψ between 500 and 550 mM NaCl is greater than that of WT Gag . However8) Defining the 310, SSHC, and 8N/WM mutants: This should be done in the first paragraph of the subsection “Decreasing NC-RNA or Gag-Gag interactions reveals specific binding at slightly higher than physiological salt concentrations”.We have now introduced these mutants in the first paragraph of the subsection “Decreasing NC-RNA or Gag-Gag interactions reveals specific binding at slightly higher than physiological salt concentrations”.9) WT Gag-HIV Psi2 binding complexes: Have the authors performed sedimentation or other experiments to determine the Gag:RNA stoichiometries?We have not performed such experiments. The problem is that sedimentation experiments, as well as most biophysical techniques, require higher Gag concentrations; at these concentrations the Gag/RNA complexes will associate into virus-like-particles. We have addressed this at the beginning of the Results section.