The current gold standard technique for SARS-CoV-2 diagnostics is hydrolysis probe-based RT-qPCR. Reliable testing requires reliable control reagents to monitor the efficiency of RNA extraction, reverse transcription and PCR amplification. Here we describe a custom RNA packaging system from the plant virus cowpea mosaic virus to produce virus-like particles that encapsidate specifically designed portions of the genome of SARS-CoV-2, the causative agent of COVID-19. These encapsidated mimics are highly stable particles which can be used either to spike patient swab samples for use as an in-tube extraction and reaction positive control in multiplex RT-qPCR, or alone as a side-by-side mock-positive control reagent. The selection of sequences in the packaged pseudogenomes ensures that these mimics are compatible with the most commonly used primer/probe combinations for SARS-CoV-2 diagnostics . The plant transient expression system used to produce these encapsidated mimics is inherently low-cost, and sufficiently high-yielding that a single laboratory-scale preparation can provide enough positive control reagent for millions of tests. This inSuitable positive controls for RT-qPCR consist of relatively short nucleic acids with known sequences that match the diagnostic primer and hydrolysis probe combinations. Although naked RNA sequences can be used as a reaction control in the RT-qPCR reaction, they are unsuitable as control for the entire diagnostic process as they are highly susceptible to degradation by RNases. Naked DNA is less labile but cannot control for the reverse transcription stage of the diagnostic test. Neither naked RNA nor DNA can be used as a control for the nucleic acid extraction step involved in SARS-CoV-2 infection diagnosis. Based on the available information from “instructions for use” leaflets of commercially-available kits for RT-qPCR detection of SARS-CoV-2, it appears that most manufacturers use a combination of naked DNA or naked RNA as their control reagents . Some  use armored RNA based on a non-specific RNA packaged inside MS2 bacteriophage particles . Meanwhitrans in the presence of the CPMV RNA-1 – encoded viral replication machinery in vivo in Nicotiana benthamiana plants. By using this technique, the specificity of CPMV RNA packaging is harnessed and allows selective packaging of custom RNA sequences in non-infectious particles. Therefore, we designed an RNA molecule that contains regions of the SARS-CoV-2 genome that are relevant for diagnostic purposes, which we term a pseudogenome, and generated CPMV VLPs that carry this RNA. We have further advanced the CPMV VLP production pipeline in plants to obtain the high-yielding packaging system described here (This manuscript describes a method for the production of RNA-based positive control reagents for SARS-CoV-2 diagnostic tests that relies on virus-like particle (VLP) technology. VLPs are nanoparticulate structures composed of viral structural proteins similar in structure to a native virion but which do not contain the infectious viral genome . Particlbed here .22.1th March, 2020  mock-positive reaction control and an in-tube (IT) extraction and reaction positive control . Both ar2020 see .Fig. 1ThThe SBS pseudogenome has the native sequence of the primer binding sites and amplicons, including probe binding sites. The SBS encapsidated mimic is therefore a mimic for wild-type SARS-CoV-2 and can be used as a non-infectious, non-hazardous control for use alongside patient samples in nucleic acid extraction and RT-qPCR diagnostics. By contrast, the IT pseudogenome includes the same primer binding sites and amplicons, but the probe binding sites have been replaced with different, novel synthetic sequences of the same length and melting temperature. The IT encapsidated mimic, therefore, is intended as an in-tube control to be added directly to a patient swab sample before nucleic acid extraction and RT-qPCR. Due to the modified probe binding site sequences, an additional control probe is added to the RT-qPCR reaction (labelled with a different fluorophore than the diagnostic probe) and the IT control can then be detected on a different fluorescence channel than the SARS-CoV-2 in a multiplex reaction. The sequences of the SBS and IT pseudogenomes can be found in Supplementary Fig. S1.2.2in vivo using Agrobacterium tumefaciens - based transient expression in N. benthamiana plants (The SBS and IT pseudogenome sequences are flanked by the native 5’- and 3’-untranslated regions (UTR) of the CPMV RNA-2 genomic component. This allows the pseudogenomes to be packaged inside CPMV VLPs through replication-dependent packaging a plants . Each psa plants , which sa plants . Negativa plants  revealeda plants .Fig. 2SD2.3Previously published work has shown that CPMV-based particles are stable at a broad range of temperatures that are likely to be encountered during production, transport and storage . To veri2.4To assess the extent to which the VLP capsid protects the RNA pseudogenome from enzymatic activity, a throat and nose swab from an individual who had tested negative for SARS-CoV-2 was divided in two, with one aliquot immediately spiked with both IT and SBS VLPs . Both aliquots then underwent nucleic acid extraction, and the eluted nucleic acid from the unspiked aliquot was then spiked with SBS and IT VLPs . This resulted in two eluted nucleic acid samples from the same initial swab, but one sample contained pseudogenomes from VLPs that had undergone nucleic acid extraction (Extracted) while the other contained equivalent amounts of VLPs that had not undergone nucleic acid extraction (Intact). Both samples (Extracted and Intact) were then used as template in RT-qPCR, where the two different pseudogenomes were detected in the same tubes by multiplexing with different fluorophores. RNA from intact, non-extracted VLPs was detected about 10 cycles later than RNA from extracted VLPs . This co2.5Excess Diagnostic Material (EDM) from patient nasopharyngeal swabs collected at St. George’s University Hospital  was used to assess the utility of the IT encapsidated mimic as an in-tube control reagent. Five EDM samples were used, two of which (EDM 1 and 5) had been diagnosed positive for SARS-CoV-2 and three of which  had been diagnosed negative. The EDM samples were then each split into two aliquots, one of which was spiked with 50 ng of IT VLP . RNA was then extracted from all aliquots then used as template for two sets of RT-qPCR reactions using the RdRp and E assays described by These results indicate that in the positive EDM samples, the IT pseudogenome was orders of magnitude more abundant than SARS-CoV-2 RNA. These results suggest there is competition for primers between SARS-CoV-2 genomic RNA and the IT pseudogenome RNA, resulting in the signal for SARS-CoV-2 being outcompeted. To test this hypothesis, SBS encapsidated mimics were used as mock SARS-CoV-2 in mixes of SBS and IT VLPs at various ratios. RNA was extracted from 200 μL VLP mixes  and this RNA was used as template for RT-qPCR using the i.e. the 1:1 ratio was a mix of 100 ng of each VLP and the 1:1000 ratio was a mix of 0.1 ng of SBS and 100 ng of IT VLP). After RNA extraction, 5 % of the eluted RNA was used in each RT-qPCR reaction (2.5 μL of 50 μL eluate), which corresponds to RNA from 5 ng of VLP for the more abundant VLP. Similarly, in the experiments with spiked EDM  of VLP in a 200 μL mix was always used as starting material for the more abundant VLP  until after RNA extraction. This means that both types of encapsidated mimics are suitable for use as controls for the RNA extraction process. Moreover, a serial dilution experiment on four separate preparations of encapsidated mimics revealed that using 1 ng of VLP for RNA extraction, then 5 % of the eluate (corresponding to RNA from 0.05 ng of VLP) in each RT-qPCR reaction consistently allowed detection at 35 Ct (± 1.7 Ct) with the RdRp protocol from It should be noted that the majority of the VLPs produced by transient expression do not contain pseudogenome RNA but instead contain either CPMV RNA-1 or are devoid of any RNA. While the ratios of each of the subtypes of VLPs is consistent from batch to batch, this ratio can be heavily affected by the specific purification procedure used. For example, some techniques such as solvent extraction steps can disproportionately destabilise (and ultimately remove) RNA-free VLPs compared to RNA-containing VLPs. Any large-scale or commercial deployment of these VLPs (which may use a different purification procedure as the one described here) may therefore result in a different pseudogenome-to-VLP protein ratio, and this would need to be assessed. If required, it is possible to separate CPMV VLPs based on their RNA content by density gradient ultracentrifugation , resulting in pure pseudogenome-containing VLPs. This was not done for the current study and is not expected to be necessary for diagnostic applications.i.e. that the nucleic acid extraction and RT-qPCR reactions took place successfully. The American CDC recommends detecting human RNase P in swab samples alongside SARS-CoV-2 as a swab quality control, but the use of the IT control offers complementary information. Indeed, while the RNase P RT-qPCR assay controls for the swab having been taken and transported properly, it does not control for the reverse transcription reaction or RNA stability, because the recommended primer/probe combination binds entirely to exon 1 of the human RNase P gene (GenBank entry NM_006413.5), and so will amplify genomic DNA. The IT VLP cannot control for the quality of the swab, but it can control for its correct processing, RNA extraction, reverse transcription, and quality of the specific diagnostic primers, since these amplify both viral RNA and the IT pseudogenome. However, the data presented here show that this will only be the case if care is taken to use a small enough amount of IT mimic in each patient swab sample to ensure that any SARS-CoV-2 viral RNA will not be outcompeted by excess IT control pseudogenome, which is amplified using the same primers as SARS-CoV-2 targets during reverse transcription and PCR. Nevertheless, our data indicate that there is a tolerance for a 10-fold excess of one target  compared to the other. Our data therefore support the recommendation that if the IT mimic is used, it should be in an amount where signal from the IT pseudogenome is expected at about 35 Ct or higher (which corresponds to using the amount of RNA extracted from about 0.05 ng of IT VLP in the RT-qPCR reaction). This will minimise primer sequestration and enable SARS-CoV-2 detection at a high sensitivity (many diagnostic tests will use a cut-off Ct of 35–37 for determining whether a sample is positive or negative for SARS-CoV-2).The IT mimic is an internal control intended to provide confidence that a negative RT-qPCR result in a patient sample is valid: The SBS mimic is a more standard type of control that is run alongside (and not within) patient samples, and so can also be used for virus quantification rather than purely Ct results. Unlike the IT mimic, this control can be used as a mock patient sample at a wide range of concentrations, but RNA from 0.05 to 1 ng of SBS VLP in an RT-qPCR reaction is expected to yield a clear, consistent signal. In any event, differences in the characteristics of the patient swab samples, RNA extraction protocols, and RT-qPCR protocols will require situational optimisation of the use of SBS and IT encapsidated mimics. The data presented here supports carrying out these optimisations first and foremost with a range of 0.1−10 ng of VLP added to real or mock patient swab samples. The availability of effective, stable, non-infectious mimics of SARS-CoV-2 should also enable effective proficiency testing enabling the comparison of data obtained using different PCR protocols and machines. The SBS encapsidated mimic is already being deployed as a positive control for large-scale SARS-CoV-2 diagnostic testing by the Norwich Testing Initiative based at the Earlham Institute, Norwich, UK, for large-scale diagnostic testing of a university population .None of the amplicons present in the pseudogenome originated from the Spike (S) gene of SARS-CoV-2, and this pseudogenome is, to our knowledge, still valid against the variants of concern (VOC) that have evolved since the beginning of the pandemic. However, it is possible to develop variant-specific encapsidated mimics by modifying the packaged pseudogenome, and this is not expected to have a detectable effect on the yield or physical/chemical characteristics of the VLPs. Similarly, it is important to note that this technology is not inherently specific to SARS-CoV-2, and can readily be applied to create bespoke VLP-based RT-qPCR control reagents for any RNA virus for which genome sequence information is available.44.1th of March, 2020  was used to identify the primer and probe binding sites for the RT-qPCR – based SARS-CoV-2 diagnostic tests available at the time. These probe and primer sequences were used to identify the corresponding amplicons in the reference SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2), and this allowed the design of the SBS and IT pseudogenomes (sequences available in the Supplementary Information), which were ordered for DNA gene synthesis from GeneArt (ThermoFisher Scientific). The pseudogenomes were then cloned between the wild-type sequences of the CPMV RNA-2 5’- and 3’- untranslated regions (UTRs) in the pEAQ-GFP expression vector (HT-VP60 (E. coli TOP10 cells (Invitrogen) and plasmid DNA from transformed colonies was verified by gene sequencing (Eurofins Scientific) before transformation into electrocompetent Agrobacterium tumefaciens LBA4404. These were then used for transient expression in Nicotiana benthamiana plants via agroinfiltration using a needleless syringe as described previously  website accessed on the 18n vector  to yield, and particles were purified from these by polyethylene glycol (PEG) precipitation and pelleting as described previously . Followi4.3Nucleic acid extraction was carried out using the GeneJET Viral DNA/RNA Purification Kit (ThermoFisher Scientific) as per manufacturer’s instructions, except for the EDM spiking experiments, for which nucleic acid extraction was carried out using the QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR was done according to the protocol described by HP designed constructs and HP and GPL conducted expression tests in plants and purification and analysis of VLPs. HP carried out experiments that did not involve EDM. EG, DC, NE and TP carried out the experiments with EDM. JM provided critical assistance coordinating work between JIC and SGUL. All authors contributed to the manuscript.HP, GPL: Conceptualization; HP, EG, DC, NE: Data curation; All authors: Formal analysis; JM, GPL: Funding acquisition; HP, EG, DC, NE: Investigation; All authors: Methodology; HP, JM: Project administration; HP, GPL, JM: Resources; EG, DC, TP, GPL: Supervision; HP: Writing - original draft; All authors: Writing - review & editing.The data are presented in the figures and in the Supplementary material.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher-order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur.Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are thought to provide a favourable environment for replication to occur, concentrating essential viral structural and nonstructural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3D Loundras et al. report on the fibril components of the RNA-dependent RNA polymerase RdRp from foot-and-mouth disease virus. They demonstrate that higher-order fibril-based interactions create multiple complex structures within which RNA replication can occur. However, the molecular details of the architecture of replication complexes remains unknown and thus we do not fully understand how proteins and viral genetic material are associated during the replication process or how dynamic rearrangements of higher-order assemblies may regulate RNA synthesis during viral replication.The replication of genomic material is a key stage of the lifecycle of all viruses. Some of the molecular details of replication are well defined as a result of elegant in vitro analyses. It has also been demonstrated that for many viruses, replication occurs in macromolecular complexes involving both viral and cellular componentspol. This enzyme  produces negative-strand genomic intermediates, from which new positive strands are synthesised10.The genomes of positive-sense RNA viruses act directly as a messenger RNA and hence are translated immediately after infection. For picornaviruses such as poliovirus (PV), rhinovirus and foot-and-mouth disease virus (FMDV), proteolytic processing of the viral polyprotein ultimately releases the nonstructural proteins involved in genome replication. A key protein is the RNA-dependent RNA polymerase, RdRp, termed 3D11. In common with other DNA and RNA polymerases, the structure of the picornavirus 3Dpol resembles a cupped right hand, with three defined subdomains, termed thumb, fingers and palm13. The finger subdomains are extended in picornaviral 3Dpol structures, when compared to many other polymerases, and contact the tip of the thumb subdomain. This contact is important for maintaining polymerase structure whilst the geometry of the fingers against the thumb form part of the NTP entry channel. The single-stranded template RNA enters 3Dpol through a channel at the face distal to the active site and takes a right-angle turn at the palm active site when the product-template duplex is formed. This emerges from the large front opening19. There is, however, no strand separation system identified from polymerase biochemical-structural data alone, but such a mechanism must presumably exist to prevent the formation of a stable RNA duplex.Biochemical and structural studies have yielded a very good understanding of the stages involved in each step of nucleotide incorporationpol are less defined. It forms part of a larger precursor 3CD, and is essential for RNA replication via genomic cyclisation21. Previous studies have also suggested that 3Dpol (or larger precursors thereof) has an essential cis-acting role in replication , which is distinct from a separate trans-acting role 22. Dimers and higher-order oligomers of RdRp molecules have been reported in a number of positive-sense RNA viruses27. Furthermore, the 3Dpol enzyme of both PV and FMDV has been previously shown to form higher-order fibrillar structures31. PV 3Dpol has also been demonstrated to form planar lattices and higher-order structures have been visualised within infected cells25. It has therefore been postulated that the higher-order structures act as a replication scaffold; however, no RNA has been shown within the PV 3Dpol structures and furthermore, these were demonstrated to form in vitro in the absence of RNA31. In contrast, the FMDV fibrillar structures were only detected in vitro in the presence of RNA. Indeed, our previous studies showed that primer, RNA template and nucleotides were required for fibril formation, thus suggesting that they could be part of an active replication complex29.In contrast to its catalytic role in replication, the non-catalytic roles of 3D33, but distinct membrane-bound replication complexes comprised of viral RNA, structural and nonstructural proteins, and host-cell proteins have yet to be identified for FMDV. Although both viruses are members of the Picornaviridae, the Aphthovirus FMDV has several unique features that distinguish it from other members of the family including an extended 5′ untranslated region and triplication of the primer encoding region.Using immunofluorescent confocal and electron microscopy, FMDV has been shown to dysregulate Golgi and ER-derived membranes in a similar way to PV35. Therefore, a more complete understanding of the composition of the replication machinery is important for therapeutic development. Here, we have trapped the products of a replication assay by crosslinking and thus captured higher-order structures in a range of conformations. Using cryo-electron microscopy, we have calculated the structures of 3Dpol fibrils in 9 conformations at 7–9 Å resolution, with some of these occurring within the same fibril. We also probed the importance of 3Dpol-3Dpol interfaces within the fibril by mutagenesis and we use these data to build a model of conformational changes in higher-order assemblies of 3Dpol that may be important in replication.Picornaviruses are responsible for several important human and animal diseases. For example, FMDV, the causative agent of foot-and-mouth disease, is a significant economic problem, such as occurred in the UK in 2001 pol protein was able to bind a synthetic template RNA and was catalytically active. We produced wild-type (WT) 3Dpol in E. coli as previously described29, alongside 3Dpol with mutations in the active site (3Dpol-GNN) as a control. To assess the affinity of RNA binding by 3Dpol, a fluorescence polarisation anisotropy (FPA) assay was performed. Samples of 3Dpol-WT and 3Dpol-GNN were titrated with FITC-labelled 13mer poly-A RNA .We first sought to demonstrate that the recombinant 3DRNA Fig. , resultiRNA Fig. . These d32P] UTP into RNA, was assessed as described in29 utilising a poly-A template and an oligo-d(T) primer, supplemented with a 5:1 ratio of UTP to [α-32P] UTP. After incubation at 30 °C, polymerase activity was assessed by dot-blot and quantified by scintillation counting 29. To investigate the assembly of the fibrils, we employed glutaraldehyde crosslinking both to trap higher-order oligomers and stabilise the fibrils. Samples of recombinant 3Dpol were incubated at 30 °C in the presence of glutaraldehyde and the RNA template-primer used for the activity assay described above. At time intervals, the reaction was stopped by the addition of Laemmli buffer. The samples were resolved by gradient SDS-PAGE and 3Dpol-WT or 3Dpol-GNN multimers detected by anti-3Dpol immunoblot . Reconstructions ranged in resolution between 7.3 Å and 9.5 Å Fig. .Table 1Tpol (PDB ID 2EC0) revealing that all the solved fibril conformations are right-handed 2-start helical assemblies, each comprising a lattice of repeating 3Dpol dimers . It is also important to note that the RNA entry and exit sites are not occluded (except by this putative RNA density).Within the narrow and broad classes, fibrils were found to form either tightly packed or open conformations. N3–N8 were tightly packed fibrils in which flexing of protofilaments resulted in a progressive tightening of packing from N3 to N8, with N8 being the most densely packed of the narrow fibril forms. N9 shows a rather different assembly with a longer axial rise and, although the fibril is narrower, a more open structure with large gaps between protofilaments. Similarly, B1 and B2 showed tight and open forms respectively, with B2 being narrower than B1 and having a longer axial rise per subunit. Three-dimensional classification did not distinguish more subtle variations for broad fibrils (similar to those seen in the tightly packed narrow fibril classes (N3-N8)), possibly a consequence of fewer broad fibrils being present in the dataset. In both N9 and B2 (the open fibril conformations) the spaces between protofilaments contained noisy density that was not accounted for by fitting the atomic model for 3DMovie pol indicated that fibrils each comprise two protofilaments that are ribbons of dimers. Analysis of contacts and clashes between 3Dpol monomers in our models confirms this. These data are also consistent with fibril formation by dimer addition as shown in Fig. n represents a monomer and then n’ is the twofold symmetry-related monomer within a single dimeric subunit . For conformations N3-N9 the transformation from n to n + 1 and all subsequent increments is defined by the left-handed 1-start helical parameters: a rotation of ∼-158° and an axial rise of ∼15–17 Å. For the C2 symmetric broad fibrils, which do not show 1-start helical symmetry, the transformation from n to n + 1 (and subsequent odd-numbered monomers) is simply a rotation of −180°. The transformation from n + 1 to n + 2  is a rotation of ∼-140° and an axial rise of ~27–31 Å. Under this notation, for all fibril conformations, n + 2, 4, 6 etc. are successive monomers on the same protofilament as n, while n + 1, 3, 5 etc. are on the other. The dimer interface is between thumb subdomains, predominantly at the C-terminal helix α16 (residues 453-467) of n and n’  fibrils, helical reconstructions of which have shown a 6-start helix of protofilaments formed of interface 1 dimers31. However, it should be noted that the PV 3Dpol was untagged, whereas the FMDV 3Dpol used here was C-terminally his tagged. 470 residues (of 476 excluding the tag) were modelled. Although density extends beyond this, it is not possible to determine the position of the tag.Comparison of our structures for fibrils formed of FMDV 3Dpol fibrils revealed considerable conformational flexibility. Many attempts were made to achieve sufficient resolution as to allow ab initio atomic modelling, these were not however successful.The cryo-EM data suggested that the fibril protofilaments are assembled via two main intermolecular interactions, a thumb-to-thumb interaction involving residues Y394, G395, T396, F398, P436, Y455, R456, S457, Y459, L460, V463, N464, V466, C467, D469, and A470, and a fingers/thumb-to-palm interaction . It should be noted that these contact residues were identified by the fitting of high-resolution data into an intermediate resolution map, thus no account is taken for potential rearrangement of sidechains upon polymerisation. Helical reconstruction of FMDV 3DThe third interaction site (between protofilaments) is highly variable. Our fitting experiments led to the definition of contact interfaces for tight forms comprising four residues. The considerable flexing of protofilaments argues for a weak interaction at this site. The open forms show a more intriguing potential interaction—formation of an extended antiparallel β-sheet via hydrogen bonding between residues 4–8. Mutagenesis to target this interface would require disruption of secondary structure. For these reasons, we chose to focus our mutagenesis studies on those interactions that support protofilament formation.37. We hypothesised that if the polymerase intermolecular interactions described here are biologically important, disruption of these interactions would impair function, resulting in reduced replicon replication. We therefore selected several residues from both the thumb-to-thumb interface  and the fingers/thumb-to-palm interface  for mutation. Selecting residues at random from both interfaces prevented biasing the outcome by focusing the mutagenesis on specific secondary structures or amino acid physical properties; however, it should be noted that all of the residues are conserved between FMDV isolates. Each of the selected residues was individually mutated to alanine in the context of a FMDV replicon in which the structural proteins were replaced by GFP. Measuring GFP expression therefore allows the replication competency of each mutation to be assessed. From each mutant replicon, RNA was generated by T7 transcription and transfected into BHK-21 cells. Replicons with 3Dpol-WT and 3Dpol -GNN were included as controls, the latter for the level of translation from the input RNA. RNA replication was monitored by GFP fluorescence, with replication shown as the number of GFP positive cells at 8 h post-transfection, analysed according to our established protocols37 42 in Codon plus (+)BL21 using methods described previously15. For lysis, the cells were suspended in 20 mM Tris-HCl [pH 8] lysis buffer with 20 mg/ml lysozyme before sonication as described previously15. Lysates were clarified and the supernatant filtered through 0.22 µm filters and subjected to nickel affinity chromatography using 1 ml HisTrap HP columns . Elution was carried out via a 25–500 mM imidazole gradient in 50 mM Tris [pH 8], 500 mM NaCl buffer. Fractions containing the protein were pooled, dialyzed and stored in aliquots at -20 °C as described previously19. A replication-deficient enzyme  adapted from published protocols43. FPA measures the tumbling rate of a fluorescently labelled molecule. After excitation by polarised light, the excited fluorophore emits light with the degree of polarisation inversely proportional to the tumbling rate of the fluorescent molecule in the solution. Therefore, the smaller the molecule, the higher the tumbling rate resulting in the depolarisation of the emitted light as the fluorophore reorients in solution during the lifetime of the excitation. FPA was undertaken in a black opaque 384-well format (Perkin Elmer). 20 µl of RNA binding buffer (20 mM Tris-HCl (pH 8), 100 mM NaCl, 0.01 % Triton X-100) was added to each well followed by a serial dilution of purified 3Dpol or mutant 3Dpol-GNN. 20 µl of 20 nM 3′FITC-labelled 13mer poly-A (Dharmacon)  was overlaid into each well and samples incubated at room temperature for 1 h. Control samples that contained no fluorescent RNA to establish background polarisation were included. Polarisation was measured using a Tecan plate reader with a 480-nm excitation filter and 530 nm S and P channel emission filters.The ability of FMDV 3Dpol was assessed by the ability of the protein to incorporate [α-32P] UTP into nascent RNA transcripts, as previously described44. The final concentration of 3Dpol that was included in the assays was 18.52 µM. Reactions were stopped by the addition of 1.5 µl 500 mM EDTA at the indicated times. For scintillation, 2 µl of each reaction was blotted onto filter paper (Whatmann) and allowed to dry prior to being washed as described previously44. The dry filter paper was exposed to film for imaging before the individual dots on the filter paper were excised and scintillated with the addition of 3 ml scintillation fluid (Perkin Elmer). Radioactive counts were measured as scintillation counts per minute.Activity of purified FMDV 3Dpol activity assays, substituting [α-32P] UTP with 4 mM UTP were employed. Samples were incubated with 5 mM glutaraldehyde at 30 °C for increasing time periods and reactions stopped by the addition of 30 µL 2x Laemmli buffer. The samples were denatured and analysed by gradient SDS-PAGE . Immunoblotting was performed as described previously45 with primary rabbit anti-3D 397 polyclonal antibody  and detected with anti-rabbit HRP (Sigma-Aldrich).For glutaraldehyde crosslinking, 3D2O for 5 seconds prior to staining with 10 µl 2% uranyl acetate for 1 min, the excess was then removed with filter paper. TEM was analysed on the Jeol 1400 (Jeol USA Inc.) at 30,000 x magnification.Carbon-coated copper grids were prepared by glow discharge at 25 mA for 30 seconds. The activity assay master mix was prepared as described for the crosslinking assay. Samples were incubated for 30 min at 30 °C and then immediately 10 µl was adsorbed onto carbon-coated copper EM grids for 1 min. The excess sample was removed and the grids washed twice with 10 µl dH−/Å2 /second and a defocus range of −1.5 to 2 microns.Activity assay master mix was prepared as described above and incubated at 30 °C for 30 min. 10 µl of the master mix was loaded on a C-flat (protochips) CF1.2/1.3-4 C grid (1.2 µm holes) that had been prepared by addition of a continuous carbon support film and glow discharged at 25 mA for 1 min. The grids were blotted for 6 seconds at blot force 10, using an FEI Vitrobot Mark IV in chamber conditions of 4°C and 95% relative humidity. These were screened and initial analysis performed using a JEOL 2200 TEM equipped with a Direct Electron DE20 camera, optimised grids were then imaged on a Thermo-Fisher Titan Krios in the Astbury Biostructure Laboratory using a Falcon III detector in integrating mode operated at 40 frames per second. Data were collected at nominal 75,000 x magnification, giving a sampling frequency of 1.065 Å/pixel. Two-second exposures were collected at a dose rate of 54 ePreliminary analysis of a small dataset collected on a JEOL 2200 was performed using Relion 2.0.3. A single 2D class average was used to generate a starting model using SPIDER. Initial estimates of approximate helical parameters were derived by taking measurements from 2D class averages (twist = 40° and rise per subunit of 29 Å). Calculation of an initial helical reconstruction was performed giving rise to an interpretable density map with twist = 42.4° and rise of 30 Å. Inspection of this preliminary map revealed a two-start helical assembly Fig. .46. CTF estimation was performed using GCTF47. Helical reconstruction was performed using Relion 3.0 followed by 3.149. Following 2D classification the dataset was reduced to 247,623 fibril section images that had contributed to sharp class averages. These 2D class average images were carefully realigned to ensure that in all images the helix was parallel to the y axis, using SPIDER50, they were then projected to one dimension (1D) using Bsoft51. Radial density for each class average was plotted using Prism 8 (GraphPad), and fibril diameters were estimated as the distance between the minima on each side of the helix axis. Based on this 2D classification analysis, a dataset of fibril images that gave sharp class averages was selected. 3D classification was performed with k = 15, leading to the definition of nine classes of fibrils having diameters that reflected the full distribution of diameters identified by measurements of the radial density profiles. Inspection of the resulting reconstructions revealed that maps corresponding to the narrower class of fibril (Ø = 20–21.5 nm) could be described as left-handed 1-start helices with twist values of ~-158.8° and axial rise values of ~15–17 Å. The broader class of fibrils (Ø = 21.7–22.3 nm) were twofold symmetric about the helix axis and were processed as right-handed 2-start helices  with C2 symmetry imposed. 3D automated refinement led to the calculation of density maps for each class at resolutions ranging between 7.6 and 10.9 Å. As the reconstructions comprised an asymmetric unit that was a dimer, there was a twofold symmetry present that could not be accommodated within the helical reconstruction process. This was subsequently imposed on half-maps using UCSF Chimera52. Briefly, two copies of each half-map were opened, one was rotated 180° about the x axis (perpendicular to the helix axis), the fit in map function was then used to align the two maps and the density was averaged. Symmetrized half-maps were then imported into Relion for postprocessing analysis, resolution estimates ranged between 7.3 and 9.5 Å.Collection of the substantially larger dataset was undertaken at the Astbury BioStructure Laboratory. 5417 micrographs were recorded from which 266,263 images of fibril sections were selected. Motion correction was performed using Motioncor2 with dose-weightinghttps://github.com/attamatti/relion_particle_backtrace).Fibril images contributing to each reconstruction were traced back to their original micrographs using rln-parts-trace.py  was used to setup T7 in vitro transcription reactions, as previously described37. T7 RNA transcripts were purified using a Zymogen RNA Clean and Concentrator-25  according to manufacturer’s instructions and the RNA quality was determined by MOPS-formaldehyde agarose gel electrophoresis.To generate in vitro transcribed RNA for transfection, replicon plasmid DNA linearized with 37. Fluorescent reporter protein expression was monitored using an IncuCyte Zoom Dual Colour FLR (Essen BioSciences), within a 37 °C humidified incubator, scanning hourly up to 24 h post-transfection. Images were captured and analysed using the associated software for fluorescent protein expression. Control transfections (untransfected and the 3Dpol-GNN transfection to assess the level of input translation) were used to determine fluorescence thresholds and identify positive objects from background fluorescence. The number of positive cells per well was determined from the average of up to nine non-overlapping images per well.To assay replicon replication, BHK-21 cells seeded into 24-well tissue cultures plates were allowed to adhere for 16 h before transfection in duplicate with in vitro transcribed replicon transcripts using Lipofectin reagent as previously describedt test and two-way analysis of variance (ANOVA) where appropriate. Statistics were performed using GraphPad Prism 8.The methods for statistical analysis and sizes of the samples (defined as n) are specified in both the results section and individual figure legends for all of the quantitative data. All values are presented as mean ± SEM with the indicated sample size where applicable. Statistical differences were determined using two-tailed unpaired Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesMovie S1Movie S2Movie S3Supplementary Data 1Reporting Summary


The blood–brain barrier (BBB) is a highly selective and restrictive semipermeable network of cells and blood vessel constituents. All components of the neurovascular unit give to the BBB its crucial and protective function, i.e., to regulate homeostasis in the central nervous system (CNS) by removing substances from the endothelial compartment and supplying the brain with nutrients and other endogenous compounds. Many transporters have been identified that play a role in maintaining BBB integrity and homeostasis. As such, the restrictive nature of the BBB provides an obstacle for drug delivery to the CNS. Nevertheless, according to their physicochemical or pharmacological properties, drugs may reach the CNS by passive diffusion or be subjected to putative influx and/or efflux through BBB membrane transporters, allowing or limiting their distribution to the CNS. Drug transporters functionally expressed on various compartments of the BBB involve numerous proteins from either the ATP-binding cassette (ABC) or the solute carrier (SLC) superfamilies. Pathophysiological stressors, age, and age-associated disorders may alter the expression level and functionality of transporter protein elements that modulate drug distribution and accumulation into the brain, namely, drug efficacy and toxicity. This review focuses and sheds light on the influence of inflammatory conditions and diseases such as Alzheimer’s disease, epilepsy, and stroke on the expression and functionality of the BBB drug transporters, the consequential modulation of drug distribution to the brain, and their impact on drug efficacy and toxicity. The blood–brain barrier (BBB) is a complex cellular barrier composed of a tightly sealed monolayer of specialized brain capillary endothelial cells lined on a basement membrane and surrounded by adjacent perivascular astrocytes, pericytes, and microglia. The major function of the BBB is to separate the circulating blood from the brain extracellular fluid and maintain homeostasis in the central nervous system (CNS) .2, a total length of capillaries of ~400 miles, microcapillaries with a diameter of 7–10 µm, and an average inter-capillary distance of ~40 µm ..13].Passive diffusion is the movement across a concentration gradient from high to low concentrations, without expenditure of biological energy or involvement of a carrier protein . PassiveThe second mechanism is endocytosis, which is defined as the vesicular transport across the BBB through receptor-mediated, adsorptive, or bulk-phase endocytosis ,18. ReceThe third mechanism is carrier-mediated transport, through which the solute molecules move from high to low concentrations across the BBB. This membrane-bound protein-mediated transport system is an energy-independent system that could be uniporter , symporter , or antiporter . This type of transporter is of utmost importance for CNS access to essential nutrients such as glucose through the glucose transporter 1, GLUT1 (SLC2A1) . CentralThe fourth mechanism is active transport across the BBB through a carrier protein. This process is energy-dependent and often coupled with ATP hydrolysis; it enables the movement of substances against their concentration gradient . There aWhile the four mechanisms are essential for maintaining brain homeostasis, the transport proteins expressed on the luminal (blood-facing) and abluminal surface of the BBB magnify the protection provided and strictly regulates the transcellular transport. Unfortunately, this process is affected by aging and age-associated disorders . Both thThe gatekeepers of the BBB are the endothelial cells lining its vasculature that express a variety of transporters that strictly regulate the influx and efflux of endogenous nutrients and exogenous toxins including xenobiotics and drugs . Below, The ATP-binding cassette (ABC) superfamily of transporters is perhaps the most studied family of active transporters that exists in nearly all living organisms ,25. The MDR1), ABCG2 , and ABCCs ; they are all efflux transporters, limiting therapeutic drug entry into the brain and preventing successful response to several agents intended to treat neurological disorders. Moreover, decreased expression or function of these transporters has been described in several CNS disorders .,41.40,41ABCG2 is expressed on the luminal side of BBB capillary endothelial cells and acts as an ATP hydrolysis-dependent efflux transporter of a wide range of structurally and chemically distinct compounds, including physiological substrates such as sulfate and glucuronide conjugates of estrone and dehydroepiandrosterone . In addiThe ABCCs are ~190 kDa proteins whose physiological functions include organic anions and nucleotide-based analogs transport, signal transduction, and toxin secretion . Althougβ-glucuronide), and glutathione disulfide (GSSG) -verapamil have initially assessed the function of Abcb1 activity at the BBB [11C]-verapamil concentration and binding to various sections of the brain is associated with decreased expression or function of the BBB. A recent clinical PET study compared the activity of ABCB1 at the BBB in AD patients to healthy subjects. The results of this study revealed a compromise transport activity of ABCB1 and suggested that ABCB1 downregulation might contribute to the progression of Aβ deposition in the brain; they observed a 23% increase in binding potential of [11C]-verapamil in the cortical region and higher binding potentials in other smaller brain regions of AD patients -verapamil efflux and ABCB1 activity was significantly reduced in AD patients Aβ into the brains of Abcb1a/b-/- double knockout (Abcb1-null) mice and wild-type controls -verapamil as a probe substrate for ABCB1 activity, Feldmann et al. studied the plasma-to-brain transport rate constant in a group of patients with drug-resistant mesial temporal lobe epilepsy and patients with adequate seizure control using medications [Epilepsy is a chronic neurological condition associated with excessive neuronal discharges resulting in spontaneous seizures observed clinically or recorded on electroencephalograms. Around 30% of patients with epilepsy experience resistance to available therapeutic options and continue presenting seizures . These oications . Compareications . A signiMounting evidence in epilepsy suggest that a high degree of mechanistic complexity and multiple pathways drives the pathological alterations observed. The impact of epilepsy on transporters other than the ABC family also showed an abnormal expression pattern. Using immunogold electron microscopy and analysis of epileptic patients undergoing cortical resection, Cornford et al. provided evidence that the glucose transporter SLC2A1 is downregulated in regions within and surrounding the seizure focus . EarlierAntiepileptic drugs such as carbamazepine are substrates of drug transporters such as ABCB1 and ABCG2. Although upregulation of these transporters has been demonstrated in clinical studies before, it is not known whether this leads to reduced anti-seizure drug concentrations in interstitial fluid. Rambeck et al. attempted to determine concentrations of antiepileptic drugs in serum, subarachnoid cerebrospinal fluid (CSF), and extracellular space (ECS) of epileptic tissues . SpecifiFollowing the notion that increased expression of transporters may result in decreased distribution of anti-seizure medications to the brain, it is logical to propose that inhibiting drug transporters can help increase brain accumulation of anti-epileptic drugs. Accordingly, Dickens et al. used the ABCB1 inhibitor tariquidar to determine whether it could modulate the transport of phenytoin, levetiracetam, and/or carbamazepine in seven different cell systems. Results obtained in all seven cell lines demonstrated that neither lamotrigine nor carbamazepine are substrates of ABCB1. Phenytoin showed characteristics of a weak substrate in polarized kidney cells (MDCK and LLC-PK1); however, it was not a substrate in any other cell models . SubsequCC genotype at ABCB1 3435, (impaired function) may be associated with drug resistant epilepsy [ABCB1 3435 position with anti-seizure drug response. These authors did not find a significant relation between the CC genotype and drug non-responders (drug resistant epilepsy) [Although these preclinical data were encouraging, similar results have not been reported in patients. An attempt to relate ABCB1 activity to antiepileptic drugs’ efficacy has been made using pharmacogenomic studies. It was initially reported that the epilepsy . Sills epilepsy) . Thus, tpilepsy) ,149.The impact of drug transporter-induced pharmacoresistance in epileptic patients could not be only limited to antiepileptic drugs. For instance, in patients with brain tumors , the presence of epilepsy is considered the most significant risk factor for chronic disability ,151. TheGlobally, stroke is the second leading cause of death and a major cause of serious long-term disability ,155. AboABCB1 (MDR1) gene expression and increased ABCB1 protein expression through activation of hypoxia-inducible factor alpha (HIF-1α) and nuclear factor κB [Further studies described below have tried to correlate upregulation of transporters to drug delivery into the ischemic brain and suggested a potential signaling pathway that induced the upregulation ,163. Foractor κB ,163. Spuactor κB . In theiactor κB . They foactor κB .Another family of transporters that may have utility in delivering neuroprotective drugs across the BBB is the SLCO family. In rodent brain, Slco1a4 is the primary drug transporting Slco isoform expressed at the BBB . In rat Amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, is a progressive neurodegenerative paralytic disease characterized by degradation and gliosis of the upper and lower motor neurons in the brain and the spinal cord, respectively . The etiIn the mid-1980s, clinical studies reported that ALS patients had an altered BBB integrity and permeability ,181,182.Riluzole (RLZ), one of the two FDA-approved drugs for ALS management, is a substrate of ABCB1 and ABCG2 ,193. A sMultiple sclerosis (MS) is a chronic inflammatory autoimmune CNS disease that cause demyelination, axonal loss, and neurodegeneration . MS is eSimilar to chronic neuroinflammation, MS compromises the integrity and function of the BBB. Evidence suggests that dysfunctional transporters at the BBB have been implicated in MS; however, the exact mechanism is uncertain. Several studies identified an important role for ABCB1, ABCG2, ABCC1, and ABCC2 in neuroinflammatory processes underlying MS pathology ,201,202.As the body’s central processing unit, the brain is equipped with a very efficient and complex firewall system named the BBB. In this review, we discussed the critical role of the BBB with a major focus on transporter proteins and their central physiological function in preserving cerebral homeostasis. The interplay between physiology and pathophysiology of transporters in the human brain has gained much interest in recent years. Although the role of the BBB drug transporters in regulating distribution of therapeutics in the CNS has been known for several decades, it is becoming increasing clear that BBB transporters are involved in the onset and progression of several neurological diseases (major findings are summarized in Recent research and neuroimaging studies greatly advance our understanding of BBB drug transporters, making them potential targets for several neurological diseases. Our awareness has also heightened about the impact of BBB dysfunction in the pathology of a wide range of CNS diseases; this provides invaluable insights into targeting BBB transporters as a way to prevent or slow the progression of certain neurological diseases. At the same time, it remains challenging to understand the complex physiological functions of these transporters. From many examples discussed in this review, various neurological diseases can have two completely different effects on the activity and expression of BBB transporters. Therefore, a better understanding of molecular and cellular mechanisms underlying function and expression and of these transporters will significantly improve the way we think about brain diseases and could lead to the discovery of novel BBB targets and approaches for therapeutic solutions.


The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, yet its RNA structural features remain relatively unexplored. Since RNA elements guide function in most RNA viruses, and they represent potential drug targets, it is essential to chart the architectural features of SARS-CoV-2 and pinpoint regions that merit focused study. in silico pipeline to predict regions of high-base-pair content across long genomes and to pinpoint hot spots of well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity for stable RNA folding is exceptional among RNA viruses, superseding even that of hepatitis C virus (HCV), one of the most structured viral RNAs in nature. Furthermore, our analysis suggests various levels of RNA structure across genomic functional regions, with accessory and structural open reading frames (ORFs) containing the highest structural density in the viral genome. Finally, we took a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts, which, given the technical difficulty of experimentally separating cellular mixtures of subgenomic RNA (sgRNA) from genomic RNA (gRNA), is a unique advantage of our in silico pipeline. The resulting findings provide a useful roadmap for planning focused empirical studies of SARS-CoV-2 RNA biology and a preliminary guide for exploring potential SARS-CoV-2 RNA drug targets.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive (∼30-kb) SARS-CoV-2 RNA genome. In this study, we analyzed the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an IMPORTANCE The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, yet its RNA structural features remain relatively unexplored. Since RNA elements guide function in most RNA viruses, and they represent potential drug targets, it is essential to chart the architectural features of SARS-CoV-2 and pinpoint regions that merit focused study. In this study, we found that RNA folding stability of SARS-CoV-2 genome is exceptional among viral genomes and we developed a method to directly compare levels of predicted secondary structure across SARS-CoV-2 domains. Remarkably, we found that coding regions display the highest structural propensity in the genome, forming motifs that differ between the genomic and subgenomic contexts. Our approach provides an attractive strategy to rapidly screen for candidate structured regions based on base pairing potential and provides a readily interpretable roadmap to guide functional studies of RNA viruses and other pharmacologically relevant RNA transcripts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive-strand RNA virus and the etiological agent of COVID-19 24. This vFollowing viral entry and uncoating, the genomic RNA serves as the template for translation of a multicomponent replicase-transcriptase complex that is responsible for synthesizing the viral transcriptome, which includes a series of subgenomic RNAs from which other virion components and accessory protein factors are expressed . Consist8–In line with previous reports on other coronaviral genomes , SARS-Co15–18–in vivo.In light of the pervasive importance of RNA structural elements in the life cycle of RNA viruses, it is essential to understand the relative distribution of RNA secondary structure in the SARS-CoV-2 genome on both global and local scales. A particularly useful way to evaluate the “structuredness” of a viral RNA genome is to compare its global folding stability to that of well-studied RNA sequences using minimum free energy Z-scores . In thisHere, we show that the potential for stable RNA folding of the SARS-CoV-2 genome supersedes even that of HCV and discuss the potential biological consequences of this unprecedented level of global structural complexity. We developed a convenient pipeline to analyze the base pair content of any long RNA and to rapidly identify regions with predicted well-defined structure across kilobase transcripts. We used this pipeline to scan the SARS-CoV-2 genome and pinpoint regions with a high propensity to form stable secondary structures, enabling direct comparisons of structural content among the functional domains of this massive viral genome. We observed a remarkable enrichment of structured regions within open reading frames (ORFs) that encode accessory and structural proteins, and we elaborate on the potential roles these structures might play in the course of viral infection. Finally, we demonstrate that SARS-CoV-2 ORFs can adopt different structures in the genomic and subgenomic contexts.in silico predictions and experimental chemical probing experiments. While we illustrate the utility of this approach by predicting stable architectural features within the SARS-CoV-2 genome, the pipeline can be implemented for characterizing the architectural landscape of any long RNA, and it is particularly valuable during early stages of discovery, when little is known about a virus or transcript. Therefore, our pipeline complements and guides parallel experimental approaches for identifying regulatory and therapeutic targets , which  genomes  and also genomes . In very genomes  and stud genomes , 27, 28.The Z-score distribution for the SARS-CoV-2 genome is shifted far into the negative range , indicatrel) for each section across the SARS-CoV-2 genome .To map and visualize the entire SARS-CoV-2 RNA structural network, we developed a pipeline for quantitating and comparing relative levels of base pair content (BPC) and secondary structural features throughout the genome . Initial2 genome . We defirel distribution across the SARS-CoV-2 genome in conjunction with the corresponding Shannon entropy profile  and correlate them with Shannon entropy values below the global median, resulting in a metric we define as “high BPC/low Shannon” . This definition, which is analogous to the “low SHAPE/low Shannon” designation for flagging probable regions of uniquely determined secondary structure . In this way, we confirmed that the SARS-CoV-2 RNA structural network has a high level of thermodynamic stability.In order to assess the relative thermodynamic stability of specific structured regions defined by this approach, we calculated the relative enrichment in stable base pairs as defined by the ScanFold-Fold analysis in the work of Andrews et al.  and compGiven the abundance of secondary structural units that correlate with low Shannon entropy values in SARS-CoV-2 : indeed, 61% of the 5′ UTR and 41% of the 3′ UTR are characterized by well-defined structures , which iHowever, unlike conventional mRNAs or flaviviral RNAs, RNA structural elements are not predominantly confined to the UTRs, as they are observed throughout all coding regions of SARS-CoV-2 . DiffereThe downstream third of the SARS-CoV-2 genome, which contains ORFs for structural and accessory proteins (the subgenomic RNA [sgRNA]-encoding region), displays a much higher overall secondary structural content than ORF1ab and has 36% of its sequence folding into well-determined structures . RemarkaSimilar to patterns observed for ORF1ab, we also observed RNA structural modules that span multiple ORFs. One example is a module that spans the junction between S ORF and ORF3a, including the transcription regulatory sequence (TRS) at the intersection between them. Similarly, part of ORF6 folds into a substructure that includes elements of ORF7a/b and -8, resulting in an extended structured region that includes three TRS elements. These observations indicate that some TRSs in this region might engage in structures with their surrounding ORFs, a feature that is likely to influence sgRNA synthesis and replication. Taken together, these results suggest the formation of numerous modules of well-determined RNA structure throughout the SARS-CoV-2 genome and reveal important structural trends across its genomic sections.in silico results , which confirms the predicted distributions of well-defined structural hubs throughout the genomic subdivisions of ORF1ab. These results provide an additional experimental validation of the methods we describe here, extending them to applications using experimental data and suggesting broad applicability for characterizing structural trends in long viral RNAs.In order to evaluate the validity of our conclusions using an orthogonal data set, we applied the same computational pipeline to compute the fraction of high-BPC/low-Shannon-entropy nucleotides across ORF1ab (including the 5′ UTR) using experimentally determined chemical probing data obtained in infected cells . We then results . DespiteIntrigued by the abundance of predicted RNA structures within the ORFs of SARS-CoV-2, we asked whether individual ORFs might adopt different structures depending on the context in which they are inserted, i.e., in genomic versus subgenomic RNAs. As a direct application of base pair content analysis, we calculated the predicted folded structure of one specific structural ORF that is present in both genomic RNA (gRNA) and subgenomic RNA. We chose the Nucleocapsid ORF because it forms the most abundant sgRNA (N sgRNA), which is estimated to be at least 1 order of magnitude more abundant than other sgRNAs .When comparing the N ORF base pair content in both the genomic and subgenomic contexts , one obsin silico predictions  reagents and then selectively amplified upstream regions of the N ORF in the sgRNA context and then separately in the gRNA context. In this way, it was possible to obtain experimentally determined secondary structural models for this same region of the N ORF in both distinct contexts and compare them with our dictions . StrikinThese results suggest that the same RNA sequences can adopt completely different structures in the subgenomic and genomic contexts, thereby potentially diversifying functionality of the viral genome, with significant implications for RNA stability, processing, and molecular mechanism. A compact structure with high BPC in the upstream segment of the N sgRNA might contribute to its stability as an independent transcript, potentially explaining the unusually high abundance of this sgRNA. It will be important to conduct genetic studies to assess this model and to perform analogous studies on the other sgRNA/gRNA combinations to evaluate how they might influence viral function. These limited studies exemplify the presence of context-dependent differences in the structures of specific viral RNA sequences, and they provide a framework for sifting through vast coronavirus genomes (and other genomes) to identify discrete elements of dynamic RNA secondary structure.The majority of biophysical and structural studies being conducted on coronaviruses focus on the viral proteins, such as the virion constituents and components of the replication-transcription machinery –36. Howe–38–It is interesting to consider why the largest RNA genome might also be the most highly structured. One hypothesis is that the idiosyncratic SARS-CoV-2 genomic architecture serves a protective function. Biophysical studies have shown that extensively structured viral RNAs like HCV adopt highly condensed states in solution and that these are inaccessible to external probe hybridization , which hAnother consequence of the highly structured, compact SARS-CoV-2 RNA genome is that its reduced dimensionality would facilitate interactions between RNA structural elements that are otherwise far apart from one another in primary sequence. Bringing genomic elements into close spatial proximity of one another will support the formation of long-range interactions between distant segments of the genome. Much like the topologically associating domains (TADs) in the chromatin of eukaryotes , “RNA TAin silico predictions and confirm the structural trends reported for ORF1ab  Z-scores for SARS-CoV-2, HCV, West Nile virus (WNV), and human mRNAs  were calculated with the ScanFold program . ScanFolP > 99%) are used as hard constraints in this step. Finally, a consensus secondary structure is obtained by outputting base pairs consistently predicted during windowed folding, i.e., occurring in more than one-half of windows. The Shannon entropy for each nucleotide is computed with base pairing probabilities derived from the partition function calculation.Secondary structure predictions for the full-length SARS-CoV-2 RNA genome, SARS-CoV-2 nucleocapsid (N) subgenomic RNA, and full-length HCV RNA genome were obtained using SuperFold with default settings as described by Smola et al. , which arel, i.e., the relative base pair content for a given nucleotide, was calculated with an in-house script by computing the percentile of the nucleotide’s absolute BPC among the global set of BPC values comprising the entire RNA. Briefly, 100,000 values were resampled with replacement (bootstrapping) from the set of BPC values. The bootstrapping is used as a convention to approximate the percentile of BPC values across a common denominator, regardless of the RNA tested. For each nucleotide, we then computed the fraction of bootstrapped values that lie below that nucleotide’s BPC score. By doing this, the median BPC value for a given RNA is “normalized” to 0.5, making for an intuitive measure of the relative significance of the magnitude of a given nucleotide’s BPC value. In case the median value itself is present multiple times in the BPC data set, the standardized median threshold shifts beyond 0.5, which can be corrected by resetting all median BPC values with a BPCrel of 0.5. In this way, the rescaled BPC values (BPCrel) can be used for direct quantitative comparison of the structural content across any RNA.The base pair content (BPC) was calculated on Excel from the predicted secondary structure in sliding windows of 51 nt tiling the RNA in steps of 1 nt. For each nucleotide, we define BPC as the fraction of base-paired nucleotides within the window centered about that nucleotide. For the terminal 25 nucleotides at both ends of the RNA (window sizes < 51 nt), sliding windows were truncated accordingly. The BPCrel values, nucleotides with BPCrel greater than 0.5, i.e., above the global median of the distribution, and Shannon entropy values below the global median were flagged as high BPC/low Shannon. The fraction of well-defined structure in a given section of the RNA was then defined as the fraction of high-BPC/low-Shannon nucleotides in that section. For the comparison between in silico and experimental results for ORF1ab  from the ScanFold-Fold analysis of SARS-CoV-2 reported by Andrews et al. (avg scores for SARS-CoV-2 were downloaded from the Moss lab RNAStructuromeDB (https://structurome.bb.iastate.edu/). In order to match the criteria we used to flag well-defined structures , low Zavg values are defined here as those occurring below the distribution median of Zavg values, i.e., regions with folding stability above the average. The statistical significance of the overlap between both methods was evaluated on MATLAB by running simulations of randomly distributed elements in both groups. The P value was estimated by computing the number of times an overlap equal or greater than the observed value was obtained and then dividing it by the number of simulations.It was important to assess the overlap between nucleotides in well-defined regions as defined by our approach (high BPC/low Shannon) and nucleotides with low average Z-scores ; cDNA was then amplified with a forward primer targeting the 5′ end of SARS-CoV-2 5′-leader sequence (5′-ATTAAAGGTTTATACCTTCCCAG-3′) and a reverse primer targeting the 3′ end of the SARS-CoV-2 3′ UTR (5′-TTTTTTGTCATTCTCCTAAGAAG-3′). In conjunction with a short PCR extension time (2 min), this RT-PCR design allows for selective amplification of the N sgRNA sequence and cannot efficiently amplify intervening regions in the gRNA or other sgRNAs. To verify selective amplification of the N sgRNA, the correct amplicon size  was confirmed by gel electrophoresis. Libraries were sequenced on the Illumina NextSeq 500/550 platform using a 2 × 75-bp paired-end sequencing. Sequencing data were analyzed using the ShapeMapper 2 analysis pipeline . Human beta-actin mRNA (NM_001101.5), human GAPDH mRNA (NM_002046.7), human HPRT mRNA (NM_000194.3), human α-tubulin 1 mRNA (NM_006009.4), West Nile virus genome  and HCV genome  sequences were used for Z-score analysis.The SARS-CoV-2 reference genome from Wu et al.  was usedhttps://github.com/pylelab/SARS-CoV-2_global_local_structure.Data sheets, sequence files, and the script used to calculate relative BPC values are available at the GitHub repository:


In this review, we describe our current knowledge about viral and host factors associated with tobamovirus RNA replication and discuss the replication mechanisms. We also mention the usefulness of tobamovirus genomes as vectors.Tobamovirus is a positive-strand RNA virus of plants. Its single-stranded RNA genome replicates  Tobamovirus includes many virus species, such as Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Cucumber green mottle mosaic virus (CGMMV), Sunnhemp mosaic virus (formerly TMV-Cc), Odontoglossum ringspot virus (ORSV), Youcai mosaic virus (recently proposed to be the same species as the crucifer-infecting tobamovirus TMV-Cg), Tobacco mild green mosaic virus (TMGMV), and the rakkyo-infecting tobamovirus (TMV-R).via full-length negative-strand RNA, which is complementary to the genomic RNA. The 30 K protein and CP are synthesized by translation of the respective subgenomic RNAs, which are 3’-co-terminal with the genomic RNA Initially, both the 130 K and 180 K proteins were thought to be involved in the replication of tobamovirus RNA, based on the observation that they were the only proteins that were translated directly from the genomic RNA prior to replication. The most convincing experimental evidence that both these proteins are involved in tobamovirus replication was obtained using artificially constructed mutants. Ishikawa Osman and Buck purified a crude membrane-bound RNA-dependent RNA polymerase (RdRp) from ToMV-infected tomato leaves by sucrose density gradient centrifugation. This polymerase, which contained the viral 130 K, 180 K proteins, as well as several host proteins and double-stranded ToMV RNA, incorporated labeled ribonucleotides into both double-stranded and single-stranded ToMV RNA. The membrane-bound polymerase became dependent on exogenously added ToMV RNA when treated with micrococcal nuclease. This template-specific ToMV RdRp was inhibited by the addition of antibodies against the 130 K or 180 K proteins. These results indicate that the 130 K and 180 K proteins are components of the tobamovirus RdRp.12)7GTP using S-adenosylmethionine as the methyl group donor. These reactions are considered to be important for the capping of TMV RNA.E. coli are capable of unwinding duplexed RNA,24)The amino acid sequence of the read-through region of the 180 K protein contains a motif that is found in RdRp.in vitro. A region immediately downstream of the core methyltransferase domain of the 130 K protein is responsible for this binding (25)In addition to the enzymatic activities discussed above, the 130 K and 180 K proteins also have the ability to bind specifically to the 3’-noncoding region of the ToMV RNA  binding . A ToMV ΔClaI’ region in cis action of replication proteins may represent a general strategy for template selection in monopartite positivestrand RNA viruses.et al. found that replication-defective TMV mutants that expressed the intact 130 K protein, in which the read-through region of the 180 K protein gene was deleted, could be transcomplemented by a replication-competent mutant that expressed the functional 180 K protein but not the 130 K protein; these transcomplemented TMV mutants accumulated at moderate levels both in protoplasts and in plants. However, these defective viruses could not be transcomplemented by the wild-type TMV. Knapp et al. also found that replication-defective TMV mutants that expressed only the ΔClaI region of the 130 K protein, with the remainder of the replication protein-coding region being deleted, could be transcomplemented by the wild-type TMV. These mutants accumulated at high levels in protoplasts but not in plants, which suggests that these defective viruses cannot move in plants. These results may explain why defective tobamoviruses are rarely found in nature.32)Some of the replication-defective 130 K- and 180 K-defective mutants can be trans-complemented by self-replicatable helper tobamoviruses.et al. prepared template-specific TMV RdRp by solubilization of the P30 fraction of TMV-infected tobacco leaf extracts with the anionic detergent taurodeoxycholate, followed by immunoaffinity purification using antibodies against the TMV replication proteins. This RdRp preparation contained a heterodimer of the 130 K and 180 K proteins.Watanabe N gene-mediated hypersensitive response in tobacco plants.36)The 130 K and/or 180 K proteins are also known to be involved in the elicitation of the A priori, these terminal regions have been considered to be important for the replication, translation, and stability of tobamovirus RNA,The tobamovirus genomic RNA harbors 5’- and 3’-noncoding regions of approximately 70 and 200 nucleotides, respectively. in vitro.et al. have demonstrated that the tRNA-like structure is important for in vitro minus-strand RNA synthesis by the template-dependent ToMV RdRp.in vivo infectivity of tobamovirus RNA,Turnip yellow mosaic virus, which belongs to the Tymovirus genus in the alpha-like virus superfamily, suggests that aminoacylation of the genomic RNA itself is not an absolute requirement for RNA replication.39)The 3’-proximal 100-nucleotide region of tobamovirus RNA is folded into a tRNA-like structure, and is a substrate for cellular tRNA-modifying enzymes, which include aminoacyl-tRNA synthetase and :tRNA terminal nucleotidyltransferase. Indeed, tobamovirus RNA is aminoacylated with histidine or valine et al. introduced several deletion mutations in this region. The mutant in which five out of the six double-helical segments were deleted, such that the pseudoknot structures were no longer formed, was still able to replicate. This indicates that the complete set of pseudoknot structures is not essential for replication. However, it has been shown that either deletion of the double-helical segment immediately upstream of the tRNA-like structure or a base substitution that destabilizes the helix results in loss of infectivity.et al. have demonstrated that this pseudoknot region enhances the translation of the genomic RNA,In the 3’-noncoding region of the tobamovirus RNA, three consecutive pseudoknot structures, each of which is composed of two double-helical segments, are present immediately upstream  of the tRNA-like structure.et al. constructed ToMV-derived chimeras, in which the 3’-noncoding region was replaced by the corresponding regions of either TMV RNA, CGMMV RNA, or TMV-Cc RNA. ToMV, TMV, and CGMMV RNA have tRNA-like structures that are histidylated, and TMV-Cc RNA has a tRNA-like structure that is valylated. The three chimeric viruses were able to multiply in both tobacco protoplasts and plants, although the multiplication levels of the latter two constructs were lower than that of ToMV.Brome mosaic virus (BMV), which belongs to the Bromovirus genus in the alpha-like virus superfamily, and that has a tRNA-like structure that is tyrosylated, was used to construct ToMV chimeras. Although the level of virus accumulation was very low, the chimeric virus was able to replicate in protoplasts.cis by the 130 K protein, as discussed above.Ishikawa et al. analyzed the infectivity of ToMV mutants that had nucleotide deletions in the 5’-noncoding region. Mutants with deletions of about ten nucleotides, whereby these ten nucleotides were located in the region between nucleotides 10 and 71 (referred to as “short deletion mutants”), maintained infectivity in tobacco protoplasts, while the deletion of nucleotides 2 to 8 caused a loss of infectivity.et al. inoculated one of the short deletion mutants into Samsun tobacco plants, which represent a systemic host for ToMV. Although some of the inoculated plants showed severe mosaic symptoms, the remaining plants showed no symptoms. The severity of symptoms paralleled the levels of virus accumulation. Analysis of the progeny virus from the plants with severe mosaic symptoms revealed that they were revertants that had re-acquired CAA repeats. Thus, this region probably controls the ability of ToMV to systemically infect whole plants.et al. have shown that the 5’-noncoding sequence of TMV contains a translational enhancer, and that the specific binding of HSP101 protein to this region is necessary for this enhancement.The 5’-noncoding region of ToMV RNA lacks internal guanosine residues and contains CAA repeats. It is believed that the complementary sequence of the 5’-noncoding region carries information that is important for the initiation of genomic RNA synthesis. Takamatsu cis-acting sequences, genomic position is known to have a significant impact on the transcription of subgenomic RNAs.51)Subgenomic RNA transcription is initiated internally from the full-length negative-strand RNA. Sequences upstream and downstream of the transcription initiation sites  are necessary for transcription.The multiplication of eukaryotic positive-strand RNA viruses involves a complex interplay between the virus genomes, virus-coded proteins, host proteins, and intracellular membranes.et al. contained host proteins of 220-kDa and 34-kDa,Osman and Buck purified taurodeoxycholate-solubilized ToMV RdRp from infected tomato tissues, and found that the 56-kDa, 54-kDa, and 50-kDa host proteins were associated with the RdRp. The 56-kDa host protein cross-reacts with the anti-yeast GCD10 protein antibody, and this antibody inhibits RdRp activity.et al.et al.Arabidopsis thaliana, in which TMV-Cg multiplication was reduced to low levels. These mutants carry the independent recessive mutations, tom1 (tobamovirus multiplication 1) and tom2. Since these mutations are recessive and active at the protoplast level, their wild-type gene products (TOM1 and TOM2) probably function to support the multiplication of TMV-Cg within host cells.Turnip yellow mosaic virus or Turnip crinkle virus (genus Carmovirus),Ishikawa TOM1 gene encodes a protein with seven predicted transmembrane regions.tom2 mutant identified two distinct genes, TOM2A and TOM2B, which are involved in intracellular tobamovirus multiplication. TOM2A is predicted to be a four-pass transmembrane protein, and TOM2B appears to be a small basic protein.via the helicase domain,35)Positional cloning studies have demonstrated that the tom1 mutants that have been independently isolated to date carries a mutation that is predicted to result in a complete loss of function, TMV-Cg and ToMV can still multiply at low levels in these mutant plants. Yamanaka et al. further mutagenized one of the tom1 mutants, to obtain a mutant line in which the multiplication of TMV-Cg and ToMV was completely inhibited. Genetic analysis revealed that this mutant carried another recessive mutation (named tom3) in addition to tom1. The loss of the TOM3 gene alone did not noticeably affect TMV-Cg or ToMV multiplication. Positional cloning of the TOM3 gene has revealed that it encodes a protein that is closely related to TOM1. Therefore, TOM1 and TOM3 probably have parallel and essential roles in tobamovirus multiplication.61)Although each of the three et al. proposed that TOM1 tethers the 130 K and 180 K replication proteins to membranes, and TOM2A facilitates the binding of the replication proteins to TOM1 and/or the formation and maintenance of the replication complex.Subcellular localization analysis using GFP fusions of TOM1 and TOM2A has shown that these proteins are localized mainly on tonoplasts (vacuolar membranes). Iodixanol density gradient centrifugation analysis demonstrated that TOM2A was almost exclusively present on membranes that fractionated to the low buoyant density fractions that contained the tonoplasts. TOM1 fractionated to the low buoyant density fractions, but also to higher buoyant density fractions that contained multiple types of membranes, including the endoplasmic reticulum (ER) and Golgi body. The fractionation of lysates of ToMV-infected tobacco protoplasts showed that the fractionation patterns of the 130 K and 180 K proteins and viral RdRp activity were similar to that of TOM1. Based on these results, Hagiwara Tm-1 is a semi-dominant tomato gene that inhibits the intracellular multiplication of ToMV. Tm-1 inhibits ToMV multiplication in protoplasts, even in the presence of actinomycin D, which suggests that the molecules required for resistance are not induced but are pre-existing in uninfected cells.Tm-1 tomato plants, carries mutations that result in amino acid changes (Gln 979 to Glu and His 984 to Tyr) in the helicase domain of the replication proteins proteins .64),65) et al. reported that a single amino acid substitution  in the 130 K and 180 K proteins of ToMV altered host specificity. This ToMV mutant (TLIle) was unable to replicate in tomato protoplasts, whereas it replicated normally in tobacco protoplasts.Hamamoto After invasion of the host cell and uncoating, the genome of the eukaryotic positive-strand RNA virus is translated, to produce the virus-coded replication proteins. The set of replication proteins recruits the genomic RNAs as replication templates to the cytoplasmic surfaces of intracellular membranes, isolates them from the translation machinery and other cytoplasmic macromolecular machinery, and forms replication complexes. in vitro. The latter fraction is composed of the majority of the replication protein pool during the late stages of infection, when most of the immunocytological analyses have been performed.62)The membranes upon which the replication complexes are formed vary from virus to virus. However, for each virus, the replication complex is formed on specific organelle membranes, which suggests a specific molecular interaction between the replication proteins and the membranes. For tobamoviruses, the membranes on which the replication complexes reside remain controversial. Immunostaining tobamovirus-infected cells with anti-replication protein antibodies has demonstrated that the replication proteins are localized to cytoplasmic granular inclusion bodies,Consistent with the importance of membranes for the formation and maintenance of replication complexes, the solubilization of replication complex-containing membranes results, in most cases, in the loss of RdRp activity, or in RdRp that can synthesize only the complementary strand RNA to exogenously added positive-or negative-strand RNA templates of the homologous virus. In exceptional cases, detergent-solubilized RdRp catalizes the complete replication cycle of exogenously added homologous viral genomic RNA.et al. established a cell-free translation-replication system for tobamovirus RNA. This system utilizes lysates of non-infected plant protoplasts, from which the vacuoles have been removed by stepwise density gradient centrifugation. Importantly, this lysate contains membranes, since the ultracentrifugation and gel filtration steps that remove membranes are omitted in the preparation of the lysate. In this lysate, ToMV RNA is translated to produce the 130 K and 180 K proteins, followed by the addition of 32P-labelled ribonucleoside triphosphates. This reaction produces internally 32P-labeled ss ToMV genomic and subgenomic RNAs and the ds replicative-form RNA, in a similar pattern to that observed for ToMV-infected protoplasts.in vivo processes of: (1) selection of genomic RNA as the replication template by the 130 K and 180 K proteins; and (2) formation of the replication complexes on membranes. This system will facilitate studies on the mechanisms of tobamovirus replication.Recently, Komoda Drosophila DICER, to generate 21–25-nucleotide RNA molecules, which are called ‘small interfering (si) RNAs’. This siRNA is incorporated into an enzyme complex, which is named the ‘RNA-induced silencing complex’ (RISC), and the RISC degrades target RNA in a sequence-specific manner. siRNA is also thought to act as a primer of target dsRNA synthesis by the host RdRp, which results in amplification of the RISC.75)Plant cells have the ability to degrade RNA in response to specific ds or highly-structured RNAs that have nucleotide sequence homology to the target RNA. This phenomenon, called RNA silencing, has also been found in various eukaryotic organisms, including animals and fungi, and is thought to function to protect the cells against invasion by viruses or transposable elements. During RNA silencing, dsRNA is first degraded by enzymes that are related to et al. established tobacco plants that were transformed with a cauliflower mosaic virus 35S RNA promoter-driven green fluorescent protein (GFP) gene cassette, in which GFP expression was suppressed by RNA silencing. The transgenic tobacco plants were susceptible to ToMV, and showed mosaic symptoms, as did non-transformed tobacco plants. In the leaves of the ToMVinfected transgenic tobacco plants that showed mosaic symptoms, RNA silencing was suppressed  within the light-green area where ToMV accumulated, while RNA silencing was still active in the dark-green area, in which there was no ToMV accumulation. In contrast, the attenuated ToMV strain L11, which has a causal mutation in the coding region of the 130 K protein (76)In order to multiply successfully, viruses have evolved a variety of strategies to escape RNA silencing. protein , showed via the plasmodesmata, in a process that requires the 30 K protein. Hirashima et al. have shown that a chimeric TMV carrying the helicase domain region of TMV-R can replicate and express the 30 K protein in protoplasts, although this virus cannot move from cell to cell.After replication of the genomic RNA within an initially infected cell, the genomic RNA moves to the neighboring cells et al., and consisted of a simple gene replacement vector, in which the coat protein gene was replaced by the bacterial chloramphenicol acetyltransferase (CAT) gene. This chimeric virus replicated, spread from cell to cell, and produced active CAT within the inoculated tobacco leaves. However, due to the lack of the CP, the virus could not spread systemically. 82)As a consequence of the extremely high efficiencies of multiplication and gene expression of tobamoviruses, the tobamovirus genome has been utilized as a vector for foreign gene expression in plants.et al. constructed a chimeric TMV, in which the coat protein-coding region was replaced with a foreign gene, and a second CP mRNA promoter, followed by a CP gene, was inserted behind the foreign gene sequence. However, upon infection of plants, homologous recombination deleted the region between the twice-repeated subgenomic RNA promoter sequences, which included the foreign gene.et al. employed a DNA shuffling method, to specifically mutagenize the 30 K protein gene, and obtained mutant vectors with improved movement properties.Solanaceous plant species.89)In order to create a vector that was capable of systemic infection, Dawson et al.et al.et al. have designed a vector in which a polypeptide-coding sequence is fused to the C-terminus of the CP via a leaky termination codon. The resulting virus multiplies systemically in host plants, and the wild-type CP and fusion protein co-assemble into virus particles.One attractive approach to the production of vaccines is to display immunogenic epitopes on the surfaces of plant virus particles. Tobamoviruses are suitable for this purpose, since large quantities of pure virus particles can easily be purified from infected plants. Turpen via epigenetic expression of foreign genes.A. thaliana were constructed in both the sense and antisense orientations using a tobamovirus vector. These libraries will be useful in identifying the functions of a large number of A. thaliana genes.98)In addition to the production of foreign proteins or peptides, pioneering work has been performed with tobamovirus vectors with respect to demonstrating virus-induced gene silencing


Nuclear small RNAs have emerged as an important subset of non-coding RNA species that are capable of regulating gene expression. A type of small RNA, microRNA (miRNA) have been shown to regulate development of the ovarian follicle via canonical targeting and translational repression. Little has been done to study these molecules at a subcellular level. Using cell fractionation and high throughput sequencing, we surveyed cytoplasmic and nuclear small RNA found in the granulosa cells of the pig ovarian antral preovulatory follicle. Bioinformatics analysis revealed a diverse network of small RNA that differ in their subcellular distribution and implied function. We identified predicted genomic DNA binding sites for nucleus-enriched miRNAs that may potentially be involved in transcriptional regulation. The small nucleolar RNA (snoRNA) SNORA73, known to be involved in steroid synthesis, was also found to be highly enriched in the cytoplasm, suggesting a role for snoRNA species in ovarian function. Taken together, these data provide an important resource to study the small RNAome in ovarian follicles and how they may impact fertility.The online version contains supplementary material available at 10.1186/s13048-021-00802-3. Increasing ovarian follicle size has long been an important indicator of oocyte quality, with oocytes obtained from large follicles showing consistently higher in vitro and in vivo developmental potential than their counterparts from small follicles , 51. In Following cell fractionation, intact nuclei were visualized by phase-contrast and fluorescence microscopy, after chromosome staining with Hoescht 33342 Fig. a. BeforeFour nuclear and four cytoplasmic RNA pools were sequenced from both LGCs and SGCs. After trimming adapter sequences, reads with a sequence length less than 18 nt were discarded. Remaining sequencing reads had lengths between 18 and 50 nt Fig. a. The maS. scrofa small RNA databases and looked broadly at RNA species breakdown in nuclear and cytoplasmic fractions  between the two technologies  whereby these small RNA can be recruited to complex nucleic acid scaffolds to recruit and stabilize transcriptional co-factors . Gene onollicles , consistollicles . In mousollicles . Roles follicles , 68. Givollicles . Althougollicles , althougollicles , 27, 37,We also profiled the snoRNA and piRNA that were captured by our sequencing data. The most studied function of mammalian piRNAs is the maintenance of genomic integrity in gametes . In spitA similarly-limited focus has been placed on the role of snoRNA in the ovary. As both intact structures, and as a source of short regulatory RNAs, the role of snoRNA in cells continues to expand . A studygithub.com/derektoms/s3RNA, and we anticipate that it will be a valuable resource for others studying the role of small RNA in the pig ovary. Increasing granularity in small RNA profiling, including from single cells   for 1 h, following by incubation with secondary antibody . Membranes were again washed with TBST three times for 10 min each, incubated with Clarity chemiluminescence substrate (Bio-Rad), and imaged on the ChemiDoc MP, and then the bands were detected and analyzed with ImageLab 4.1.6 granulosa nucleus or 100 μl cytoplasm lysate were mixed with 350 μL of Buffer RL, then vortexed for up to 60 s to ensure the mixture becomes transparent. 200 μL of 96–100% ethanol was added into to the lysate following by vortexing for 10–30 s. Lysate with ethanol was added onto the column and centrifuge for 1 min at 3,500 x g to allow the entire lysate to pass through the column. RNase-Free DNase I Kit was used to minimize genomic DNA contamination in according with the manufacture’s On-Column DNA Removal Protocol . After genomic DNA removal, the column was washed and centrifuge twice. 50 μL of elution solution was used to recover the total RNA. Quality and quantity of total RNA were determined using a NanoDrop ND-1000 Spectrophotometer at the Genomics Facility in the University of Guelph’s Advanced Analysis Centre. Using Agilent 2100 Bioanalyzer DNA and RNA chips, we verified that DNA concentration in the nuclear fraction was >100X that of the cytoplasmic fraction and that 28S ribosomal RNA peak fluorescence in the cytoplasmic fraction was >10X that of the nuclear fraction. These two ratios for the same samples imply good fractionation.Total RNA Purification Kit  was used to isolate total RNA from both cytoplasm and nuclear samples. Briefly, around 3 × 10Small RNA library preparation and sequencing was performed by Personalized Genomics and Innovative Medicine in Toronto, Canada.Briefly, small RNAs ranging from 18 to 52-nt were gel-purified and ligated to the 39 adaptor  and 59 adaptors (59-GUUCAGAGUUCUACAGUCCGACGAUC-39). Ligation products were gel-purified, reverse transcribed, and amplified using Illumina’s sRNA primer set . Samples were sequenced on an Illumina 1G Genome Analyzer.http://igenomes.illumina.com.s3-website-us-east-1.amazonaws.com/Sus_scrofa/UCSC/susScr3) using the mapper module of miRDeep2 package with the following parameters (−e -h -i -j -k TGGAATTCTCGGGTGCCAAGG -l 18 -m -p –s –t –v). Followed by alignment of the collapsed reads against the pig reference genome (susScr3) using bowtie to generate mapped output in ARF format and collapsed reads in fasta format. The core miRDeep2 module is used to detect known and novel small RNAs, that involves the following input files, the mapped output (ARF), collapsed reads (FASTA), pig reference genome (FASTA) and the miRBase v21 mature and hairpin reference sequences (FASTA). In-house perl scripts were written to detect and quantify all types of isomiRs, which were treated as reads for a single mature miRNA.Samples were sequenced on the rapid run mode of Illumina HiSeq 2500 sequencing platform. Base calling (BCL) files from sequencer were converted to the FASTQ files using bcl2fastq2-v2.17.1.14 tool. The raw reads generated were single-end and of length 51-nt. Raw reads quality in FASTQ format was assessed using FastQC tool v0.11.0  and idenDESeq2  was uspheatmap package based on the Euclidean distances between transformed expression values using a complete linkage method . For om method .Sus scrofa by identifying the sequence within the genome of Sus scrofa using the “BLAST” feature. The selected genes were then searched in the Ensembl Sus scrofa database to determine the target sequences’ relative location to the start codon, ATG in order to confirm the target sequence is indeed in the promoter region, which is ~ 1000 bp upstream of ATG and ~ 200 bp downstream of ATG.Target genes were predicted using computational prediction software MicroPIR2 (biotech4/2). This software contains a database with over 80 million miRNA predicted targets in the promoter sequences of the human genome. MicroRNA targets were searched by miRNA name, with an average p-value ≤0.05, average conservation score ≥ 0.85, and a maximum number of four unpaired nucleotides. The binding pattern and sequence was observed with consideration towards near perfect matching in the seed region. Ensembl, a genetic database, was then used to determine if the predicted human promoter target sequence is conserved in Additional file 1.Additional file 2.