Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin β1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression. The SARS-CoV-2 ORF6, an antagonist of interferon-mediated antiviral signaling, directly binds to STAT1 and prevents its nuclear localization. Coronaviridae family3. The genome of SARS-CoV-2 is ~29.7 kb long with short untranslated regions (UTR) at the 5′ and 3′ termini, and encodes nonstructural (nsp1–16), structural , and accessory proteins 5.The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a single-strand RNA virus belonging to the 6. Several studies have recently shown that both the SARS-CoV and SARS-CoV-2 ORF6 proteins antagonize the host innate immune system via the Janus activated kinase 1 (JAK1)- and JAK2-signal transducers, and activators of transcription (STAT)12. STAT1 is a key mediator of cytokine-induced gene expression as it is activated by cytokines including type I and type II interferons (IFNs)14. Activation of JAKs associated with type I IFN receptor results in the tyrosine phosphorylation of STAT1 (PY-STAT1), leading to the formation of a STAT1-STAT2 heterodimer, while Type I interferon (IFN-α or -β) and type II interferon (IFN-γ) induce the formation of the PY-STAT1 homodimers. Both the hetero- and homo-dimer STAT1 complexes translocate to the nucleus to bind the IFN-stimulated response elements (ISRE) or IFN-γ-activated site (GAS)14. Previous studies have reported that ORF6 inhibits the nuclear transport of PY-STAT1 to suppress primary interferon signaling11.Among them, ORF6 is a small protein of approximately 7 kDa, which consists of 61 amino acids and exhibits a 69% homology with the SARS-CoV ORF6, from which it differs due to a two amino acid deletion at the C-terminus18. The process of protein import into the nucleus commonly involves the recognition of a classical nuclear localization signal (cNLS) by the importin α/importin β1 heterodimer20. Cargos containing the cNLS are recognized by the adaptor molecule importin α . Following the entrance of the cNLS-containing cargo/importin α/β1 trimeric complex into the nucleus through the NPCs, the cargo is released from importin α by binding of GTP-bound small GTPase Ran (RanGTP) to importin β121. After the complex dissociation, importin α is exported to the cytoplasm by cellular apoptosis susceptibility gene product  with RanGTP, and the importin β1/RanGTP complex also returns to the cytoplasm, where it is reused in the subsequent rounds of transport21.Constitutive and signal-dependent protein transport through nuclear pore complexes (NPCs) embedded in the nuclear envelope are mediated by members of the importin  superfamily22. Based on sequence similarity, each importin α protein is assigned to one of three conserved subfamilies. In humans, clade 1 consists of importin α5 (encoded by the KPNA1 gene), importin α6 (KPNA5), and importin α7 (KPNA6). Clade 2 consists of importin α1 (KPNA2) and importin α8 (KPNA7), and clade 3 consists of importin α3 (KPNA4) and importin α4 (KPNA3)21. The members of each subfamily display cargo specificity and are differentially expressed in different tissues and cell types24. Differences in the usage of “importin α” or “KPNA” and the number of proteins in humans have often been a source of confusion. Therefore, in the present study, we uniformly use the human nomenclature for “importin α” to refer to the protein and use the italic term “KPNA” to refer to the gene, while the normal term “KPNA” is described according to the way in which it is used in the citation.Seven importin α proteins have been identified in humans, whereas six have been discovered in mice28. According to the several studies on the viral proteins that inhibit nuclear transport of PY-STAT1, SARS-CoV ORF6 has been reported to tether KPNA2, but not KPNA1, to ER to sequester importin β1 into endoplasmic reticulum (ER)/Golgi apparatus, resulting in the suppression of nuclear import of PY-STAT17. Recently, SARS-CoV-2 ORF6 has also been shown to interact with KPNA2 and KPNA111, further supporting its interference with the nuclear transport of PY-STAT1. In addition, SARS-CoV-2 ORF6 has been reported to interact with the NPC components Nup98 and RAE1 to inhibit the nuclear transport of a broad range of proteins, including PY-STAT130. However, the exact molecular mechanisms of the effects of SARS-CoV-2 ORF6 on the nucleocytoplasmic trafficking remains largely unclear. In particular, our understanding of that ORF6 induces the remarkable nuclear exclusion of STAT1 with high specificity remains poor.It has already been demonstrated that nuclear transport of the PY-STAT1 as a homodimer or a heterodimer with STAT2 is mediated by specific clade 1 subtypes of importin α such as importin α5 (referred to as KPNA1 in some papers)In the present study, we characterized the effect of SARS-CoV-2 ORF6 on nucleocytoplasmic protein transport. We observed that ORF6 directly binds to STAT1 to suppress IFN-induced nuclear localization and nuclear shuttling in the absence of IFN-stimulation. In addition, the direct binding of ORF6 to importin α1 significantly reduces cNLS-cargo transport, whereas the inhibitory effect of ORF6 is more prominent for STAT1. The results reveal that ORF6 has distinct strategies of disrupting nucleocytoplasmic trafficking of STAT1 and importin α1-mediated cargos. Finally, the results from a newly produced replicon system and a hamster model demonstrate that ORF6 acts as a virulence factor via the regulation of nucleocytoplasmic trafficking to accelerate viral replication.11. Here, we attempted to verify the inhibitory effects of SARS-CoV-2 ORF6 on a type-II IFN (IFN-γ)-activated STAT1. An AcGFP-fused ORF6 was transfected into HeLa cells, and the subcellular localization of PY-STAT1 was observed when the cells were stimulated with either IFN-β or IFN-γ. Whereas the PY-STAT1 was localized in the nucleus of AcGFP-transfected cells in response to each treatment, the distribution shifted markedly to the cytoplasm in AcGFP-ORF6-transfected cells  stimulationlls Fig. . The flulls Fig. .Fig. 1InIP-10)31 mRNA 6 h post-transfection or later, upon stimulation . In comparison to the AcGFP-transfected control cells, the AcGFP-ORF6-transfected cells showed significant down-regulation of IFN-γ-inducible protein 10  49 to 52 (referred to as ORF6-M1) retained the inhibitory effect over PY-STAT1, while the other mutants with alanine replacing a.a. 53 to 55 (ORF6-M2) and 56 to 61 (ORF6-M3) did not retain their inhibitory functions  Fig. . The rep32, was also examined. Under non-stimulated conditions (untreated), Flag-STAT1 was mainly localized in the cytoplasm in either AcGFP-, AcGFP-ORF6-, or AcGFP-ORF6Δ9-transfected cells , which has been reported in previous studieslls Fig. . Upon IFlls Fig. . In contlls Fig. , suggestlls Fig. .Fig. 3ORSubsequently, to determine whether ORF6 directly binds to STAT1, bacterially purified recombinant STAT1 protein was incubated with either recombinant GST-GFP or GST-GFP-fused ORF6 full-length protein (GST-GFP-ORF6). Figure 11. However, as described above, ORF6 binds directly to STAT1 in the absence of IFN stimulation in the cytoplasm, which raises a possibility that the binding of ORF6 to importin α proteins might not be required for the inhibition of nuclear accumulation of STAT1. Therefore, we explored the interplay between ORF6 and importin α proteins. Since ORF6 has been shown to alter the distribution of importin α1 (KPNA2) and/or importin α5 (KPNA1)10, we first validated the findings concerning all human importin α subtypes. Consistent with the previous reports, in AcGFP-transfected cells (control), the overexpressed Flag-importin α proteins were mainly localized in the nucleus eus Fig. . In conteus Fig. . The anaeus Fig. . The dat25. Overall, there are importin α subtype specificities, with ORF6 mainly binding to importin α1, in contrast to the low affinity for importin α5, corresponding to the subcellular distribution change for each importin α.Next, we examined whether ORF6 directly binds to importin α proteins. Purified recombinant GST-GFP-ORF6 was incubated with Flag-importin α1 or Flag-importin α5, respectively, and then the GST-proteins were pulled down using glutathione Sepharose beads (GST-beads) to determine whether the Flag-importin α proteins were co-precipitated or not. The pull-down assay results indicated that ORF6 is certainly bound to Flag-importin α1, while only a faint band was detected for Flag-importin α5 Fig. . We also7. Indeed, the cytoplasmic localization of endogenous importin α1 was accelerated in ORF6-transfected cells  for 1 h. Under oxidative stress conditions, the cytoplasmic localization of Flag-importin α1 was markedly shifted to the nucleus in the ORF6-transfected cells  together with AcGFP or AcGFP-ORF6, and then the nuclear intensities of the fluorescent substrate were measured. Although the majority of mCherry-NLS was observed in the nucleus of AcGFP-ORF6-transfected cells, its cytoplasmic proportion was significantly increased compared to that observed in the AcGFP-transfected control cells Fig. .Fig. 6ORNext, we compared the binding ability of importin α1 to the cNLS-containing cargo (GST-NLS-GFP) and ORF6 (GST-GFP-ORF6). The GST pull-down assay showed that importin α1 more efficiently interacted with cNLS than ORF6 Fig. . To asse37. First, HeLa cells were transfected with AcGFP or AcGFP-ORF6, and then treated with 200 μM cobalt chloride (CoCl2) for 5 h to induce the nuclear accumulation of HIF-1α. In addition, the nuclear migration of NF-κB p65 was investigated using AcGFP- or AcGFP-ORF6-transfected cells treated with 20 ng/mL tumor necrosis factor-α (TNF-α) for 30 min. As a result, the nuclear accumulations of both HIF-1α  and Nuclear factor-kappa B (NF-κB) component p65/RelA, since they have been shown to be transported into the nucleus by several importin α proteins including importin α1-1α Fig.  and NF-κ-1α Fig.  were sli-1α Fig. . The resTo further examine the roles of ORF6 in the viral life cycle of SARS-CoV-2, the viral genome encoding ORF6 was replaced by that of NanoLuc, and a SARS-CoV-2 variant that does not express ORF6 (SARS-CoV-2/ΔORF6) was generated using the circular polymerase extension reaction (CPER). As a parental virus, a recombinant virus expressing ORF6 fused with NanoLuc (NLuc) and Porcine teschovirus 2 A peptide (SARS-CoV-2/NLuc2AORF6) was generated  or SARS-CoV-2/ΔORF6 was intranasally inoculated in 4-week-old hamsters. Hamsters infected with SARS-CoV-2/ΔORF6 showed significant weight gain 5 days post-infection, whereas those infected with the WT virus showed no change in the body weight Fig. . In addiIn the present study, we report that ORF6 acts on the nucleocytoplasmic signaling via two distinct ways. That is, first, ORF6 inhibits the nuclear translocation of one of the key signaling molecules for COVID-19, STAT1, through its direct binding to antagonize the IFN signaling. Second, ORF6 has potential to impair the nuclear transportation of cNLS-containing cargos including signaling molecules such as HIF-1α and NF-κB p65, through affecting the function of importin α.7. Since the nuclear transport of PY-STAT1 is known to be mediated by importin α526, and we demonstrate here that importin α1 can enter the nucleus even in the presence of ORF6 under oxidative stress conditions, it is unlikely that SARS-CoV-2 ORF6 tethers importin α1 to the ER to cause the nuclear exclusion of STAT1.A previous study reported that SARS-CoV ORF6 tethered KPNA2 (importin α1 in this study), but not KPNA1 (importin α5), to the ER, and, as a result, sequestered importin β1 into the ER/Golgi segment through the interaction with KPNA2, resulting in the inhibition of PY-STAT1 nuclear import40. Moreover, in this study, we found that the bacterially purified STAT1 proteins, which are not phosphorylated, bind to ORF6. Since the phosphorylation of STAT1 has been shown to be unaffected by ORF6 upon the IFN stimulation11, the interaction between ORF6 and STAT1 might occur in a phosphorylation-independent manner. Collectively, we propose a scenario that the nuclear exclusion of STAT1 is caused by the direct binding with ORF6, independent of importin α proteins.In contrast, we found that the Flag-STAT1 was more abundantly localized in the cytoplasm in the absence of IFN stimulation in the ORF6-transfected cells than in the control cells. It has previously been demonstrated that unphosphorylated STAT1 shuttles between the nucleus and the cytoplasm, and this shuttling might play an important role in regulating the expression of IFN stimulated genes37.Conversely, we observed that the subcellular localization of all importin α subtypes were altered in ORF6-transfected cells, suggesting that ORF6 directly or indirectly influences the importin α/β1-mediated nuclear transport pathways. Consistently, we further found that the nuclear accumulation of the mCherry-NLS substrate was significantly reduced in the ORF6-transfected cells. In addition, using the semi-intact nuclear transport assay, the addition of excess amounts of ORF6 inhibits the nuclear transportation of GST-NLS-mRFP. Furthermore, ORF6 negatively regulates the nuclear import of HIF-1α and NF-κB p65, which have already been shown to be mediated by importin α proteins41. Indeed, our data revealed that the nuclear distribution of CAS, a nuclear exporter of importin α, was enhanced in the ORF6-transfected cells, suggesting that the nuclear export system may be affected considerably in the presence of ORF6. Conversely, we could not identify the dramatic suppression of p65 in SARS-CoV-2-infected cells. Since several recent papers have reported that viral components, such as Nsp5 or ORF7a, enhance cytokine expression through activating the NF-κB signaling pathway43, the inhibitory effect of ORF6 may be counteracted by the other components. Overall, at least in the classical nuclear import pathway, the inhibitory effects of ORF6 could be limited, in contrast to the specificity for the STAT1-signaling pathway. Since the nucleocytoplasmic trafficking is vital for cell survival, SARS-CoV-2, therefore, should evade antiviral immune signaling without affecting cell survival to develop COVID-19.Recently, it has been shown that the specific interaction of ORF6 with NPC components, Nup98 and RAE1, might disrupt the nuclear trafficking of a broad range of proteins, in particular for the host mRNA export systems10, further studies are required to understand how ORF6 coordinates binding with either importin α, Nup98, or STAT1 itself through its C-terminal region to achieve the nuclear exclusion of STAT1. Understanding the effects of SARS-CoV-2 proteins on the nucleocytoplasmic trafficking system might facilitate the development of novel COVID-19 therapeutics.Our microinjection analysis showed that only the C-terminal sequence of ORF6 could inhibit the nuclear localization of STAT1, whereas the nuclear exclusion of STAT1 was not likely completely achieved in the full-length ORF6-expressing cells. Interestingly, the GST-GFP-fused M0, M1, and M2 proteins all migrated into the nucleus. In contrast, the GST-M3-GFP protein localized only to the cytoplasm, like that with the control GST-GFP protein. This indicates that the C-terminal region of ORF6, in particular residues 56–61, has a potential as an NLS, but it is different from a classical NLS sequence based on a basic amino acid cluster. Notably, the M2 mutant lost its inhibitory effect on STAT1 nuclear localization. Specifically, ORF6 separately influences the STAT1-signal pathway and the importin α-mediated pathway. This is supported by our finding that although ORF6 suppressed the nuclear trafficking of the classical NLS-containing cargos through binding with importin α, the activity was more prominent with respect to STAT1 signaling. Together with the finding that the arginine substitution at methionine residue 58 of ORF6, which is deficient in Nup98 binding, abolishes its IFN antagonistic functionAll animal experiments using the SARS-CoV-2 virus were performed in biosafety level 3 (ABSL3) facilities at the Research Institute for Microbial Diseases, Osaka University. The animal experiments and the study protocol were approved by the Institutional Committee of Laboratory Animal Experimentation of the Research Institute for Microbial Diseases, Osaka University (R02-08-0). Throughout the study, we focused on minimizing animal suffering and reducing the number of animals used in the experiments. Four week-old male Syrian hamsters were purchased from SLC .44. An 8-week-old female WKY rat was injected with 100 μL of emulsions containing ORF6 peptide (CEEQPMEID)-conjugated KLH and Freund’s complete adjuvant into the rear footpads. Seventeen days after the first immunization, an additional immunization of SARS-CoV-2 ORF6 peptide-KLH was administered without an adjuvant into the tail base of the rat. Four days after the second immunization, cells from the iliac lymph nodes of the immunized rat were fused with mouse myeloma Sp2/0-Ag14 cells at a ratio of 5:1 in 50% polyethylene glycol. The resulting hybridoma cells were plated onto 96-well plates and cultured in HAT selection medium . The SARS-CoV-2 ORF6-specific antibody was screened using ELISA, western blotting, and immunostaining of hybridoma supernatants. Finally, hybridoma clone producing the monoclonal antibody, later named 8B10, was selected. Using a rat isotyping kit the MAb 8B10 was found to be an IgG 1 (k) antibody subtype. The monoclonal antibody against SARS-CoV-2 NP (3A9 clone) was generated by Cell Engineering Corporation . Western blotting for the protein in cells infected with different viral strains, which were obtained from the National Institute of Infectious Diseases (NIID) in Japan, Hong Kong (HK)/VM20001061, USA-CA2, Germany/BavPat1, New York (NY)-PV09197, NY-PV08410, and NY-PV08449  and performed according to CEC Animal Experimentation Regulations. A rat monoclonal antibody that specifically recognized the SARS-CoV-2 ORF6 protein was generated using the rat medial iliac lymph node method2 in DMEM  containing 10% fetal bovine serum , and penicillin/streptomycin . The viral stock was generated by infecting VeroE6/TMPRSS2 cells at a MOI of 0.1. The viral supernatant was harvested two days post infection and the viral titer was determined using plaque assay.The SARS-CoV-2 (2019-nCoV/Japan/TY/WK-521/2020) strain was isolated at the National Institute of Infectious Diseases (NIID). The Germany/BavPat1/2020, USA-CA2/20200 (USA-CA2), NY-PV08410/2020, HK/VM20001061, NY-PV08449/2020, and NY-PV09197/2020 strains were obtained from BEI Resources . The different strains of SARS-CoV-2 were used to infect VeroE6/TMPRSS2 cells cultured at 37 °C with 5% COThe AcGFP and HA were amplified and cloned into pCAGGS vector designed as pCAG AcGFP-HA. The cDNA of ORF6 was obtained from Vero-TMPRSS2 cells infected with SARS-CoV-2. The wild type ORF6, ORF6-M1, ORF6-M2, and ORF6-M3 were amplified and cloned into pCAG AcGFP-HA designed as pCAG AcGFP-ORF6-HA, pCAG AcGFP-ORF6M1-HA, pCAG AcGFP-ORF6M2-HA, and pCAG AcGFP-ORF6M3-HA, respectively. The ORF6Δ9 was constructed using a splicing method achieved by overlap extension (ORF6Δ9-N and -C). The primers used throughout the study are described in Supplemental Table 46 using the primers listed in Supplemental Table 47. Human importin αs including importin α1 (KPNA2) and importin α5 (KPNA1) were cloned into a pcDNA5/FRT/FLAG expression vector, as previously described47. For constructs encoding the SV40T antigen NLS , the relevant oligonucleotides  was cut off by EcoRI and Spe1, and then subcloned into the pGL4.10 plasmid .The pISRE-TA-Luc plasmid was constructed as previously described49. Construct integrity was confirmed by DNA sequencing. For constructs encoding the C-terminus of ORF6 (M0) and its alanine replacing mutants , the relevant oligonucleotides  gene at the multicloning site; thus, producing the pGEX2T-NLS-mRFP vector.The cDNAs of full-length ORF6 and the C-terminal mutant in which the amino acids 49–61 were deleted (ORF6ΔC) were amplified using the specific primers described in Supplemental Table 50. Cleavage of the GST tag to induce cleaved fusion proteins was performed using PreScission protease  or thrombin protease . Importin β1, p10/NTF2, and GDP-bound Ran were purified as previously described50.Purification of bacterially expressed recombinant proteins was performed as previously described50. Bacterially produced FLAG-h-importins α1 and α5 or Flag-h-STAT1 recombinant proteins (100 pmol each) were incubated with GST-GFP, GST-GFP-ORF6, GST-M0-GFP, or GST-NLS-GFP immobilized on glutathione-Sepharose 4B beads , in 200 μL of transport buffer  with 0.1% Triton X-100 at 4 °C for 1 h. After the beads were washed five times with TB containing 0.1% Triton X-100, they were suspended in SDS-PAGE loading buffer %-BPB, glycerin, anion surface acting agent, and reducing agent, Nacalai Tesque, Kyoto, Japan).Solution binding assay using bacterially produced recombinant proteins was performed as previously describedThe following primary antibodies were used in the present study: Phospho-STAT1 (Tyr701)  Inc., Danvers, MA, USA), STAT1 , importin α1/KPNA2 , BD Transduction Lab., San Jose, CA, USA), HIF-1α , NF-κB p65 , importin β1 , CAS , Lamin A/C , Flag , GFP , GFP , NP , Actin , and HA .Horseradish peroxidase (HRP)-conjugated anti-rabbit (#111-035-003), anti-mouse (#115-035-003), or anti-rat (#112-035-003) secondary antibodies  were used for western blotting. The secondary antibodies used for indirect immunofluorescence were as follows: Alexa Fluor Plus 488 conjugated anti-rabbit (A32731) or anti-mouse (A32723), and Alexa Fluor 594 conjugated anti-rabbit (A21207) or anti-mouse (A21203) (Invitrogen).51 were cultured in Dulbecco’s modified Eagle’s medium , containing 10% FBS  at 37 °C in 5% CO2. The cells were plated onto 18 × 18 mm coverslips  in 35-mm dishes for immunofluorescence or 60-mm dishes  for qRT-PCR 2 days prior to transfection. The transfections were performed using Lipofectamine 2000 DNA Transfection Reagent (Thermo Fisher Scientific) or the TransIT-LT1 Transfection Reagent  following manufacturer’s instructions.HeLa cells (ATCC), HEK293 cells (NIBIOHN), Huh7 cells , Huh7-ACE2 which were generated by infection with lentivirus expressing human ACE2, VeroE6/TMPRSS2 cells , and Vero E6 replicon stable cells2. The reagents used for indirect immunofluorescence were as follows; IFN-β and IFN-γ , TNF-α , hydrogen peroxide , and Cobalt(II) chloride hexahydrate . Following fixation with 3.7% formaldehyde in PBS for 15 min, cells were treated with 0.1% Triton X-100 in PBS for 5 min and then blocked in PBS containing 3% skim milk for 30 min. For the anti-Phospho-STAT1 antibody , cells were permeabilized with 100% methanol at -20 °C for 20 min and then blocked in 3% skim milk in PBS. Cells were incubated with primary antibodies (1:200) with 3% skim milk in PBS overnight at 4 °C. The following day, the cells were incubated with the Alexa-Fluor-488 plus- or Alexa-Fluor-594-conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with DAPI  for 20 min at 25 °C. The coverslips with fixed cells were mounted on glass slides using ProLong Gold Antifade . Cells were examined under a confocal microscope . Using Leica Application Suite X, cells only expressing AcGFP were extracted and then the fluorescence intensities were identified at a region of interest in the nucleus, as well as in the whole cells. The relative fluorescence intensity values in the nuclei against the whole cells were calculated.HeLa cells were cultured on 18 × 18 mm coverslips  in 35-mm dishes (IWAKI) and incubated for 48 h at 37 °C in 5% CO52. The membranes were incubated with primary antibodies (dilutions ranging from 1:1000 to 1:2000) diluted in Can Get Signal Immunoreaction Enhancer Solution 1  overnight at 4 °C. The used HRP-conjugated secondary antibodies  were diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) at 25 °C for 1 h.Western blotting was performed as previously described7) were suspended with 1 mL RIPA buffer  and lysed by successive passage through 26-gauge needles (3 times each). The samples were kept on ice for 20 min and then insoluble material was removed by centrifugation at 13,000 × g at 4 °C for 15 min. The supernatant was precleared with 20 μL of Dynabeads  at 4 °C for 1 h, followed by incubation of the pre-cleared cell lysates with 20 μL Dynabeads and 2 μg of a primary antibody specific for flag  at 4 °C for 2 h. The beads were washed five times with 1 mL Lysis buffer and bound proteins eluted with the addition of SDS-PAGE loading buffer .HEK293 cells  according to the manufacturer’s instructions. One microgram of total RNA and the PrimeScript RT reagent kit (Takara Bio.) were used to perform the first-strand cDNA synthesis. The PCR reaction was performed as previously described−ΔΔCT method. β-actin was used as the housekeeping gene. The primers used are described in Supplemental Table For detection of N2 in SARS-CoV-2, total RNA of Huh7-ACE2 or lung homogenates were isolated using ISOGENE II . Real-time RT-PCR was performed with the Power SYBR Green RNA-to-CT 1-Step Kit  using an AriaMx Real-Time PCR system . The relative quantification of the target mRNA levels was performed using the 253. Briefly, 5 μL of culture supernatants were mixed with 5 μL of 2× RNA lysis buffer  and incubated at 25 °C for 10 min. Next, 90 μL of RNase free water were added to the mix. A volume of 2.5 μL of the diluted sample was added to 17.5 μL of reaction mix. Real-time RT-PCR was performed using the Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) and an AriaMx Real-Time PCR system (Agilent).The amount of RNA copies in the culture medium was determined using a qRT-PCR assay as previously described with slight modifications2 for overnight. The supernatants were serially diluted using inoculated medium and incubated for 2 h. Next, the culture medium was removed, fresh medium containing 1% methylcellulose (1.5 mL) was added, and the cells were cultured for 3 more days. Lastly, the cells were fixed with 4% paraformaldehyde in PBS  and the plaques were visualized by using a Giemsa’s azur-eosin-methylene blue solution .Vero-TMPRSS2 were seeded into 24-well plates  at 37 °C in 5% CO6 PFU (in 60 μL) SARS-CoV-2 via intranasal routes. The body weight was monitored daily for 5 d. Five days post-infection, all animals were euthanized, and the lungs were collected for histopathological examinations and qRT-PCR.Syrian hamsters were anaesthetized with isoflurane and challenged with 1.0 × 102O2 in PBS. After treatment with 5% skim milk in PBS for 30 min at 25 °C, the sections were incubated with mouse anti-NP antibody . EnVision+ system-HRP-labeled polymer anti-mouse secondary antibody  was used. Lastly, the sections were counterstained with hematoxylin and the positive signals were visualized using the peroxidase–diaminobenzidine reaction.Lung tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. For immunohistochemical staining, 2 μm thick sections were immersed in citrate buffer (pH 6.0) and heated for 20 min with a pressure cooker. Endogenous peroxidase was inactivated by immersion in 3% H54, with some modifications51. Briefly, seven DNA fragments covering the SARS-CoV-2 genome (excluding the region spanning the S gene to ORF8 gene) were amplified using PCR, and subcloned into a pCR-Blunt vector (Invitrogen). The DNA fragments containing cytomegalovirus (CMV) promoter, a 25-nucleotide synthetic poly(A), a hepatitis delta ribozyme, as well as a bovine growth hormone (BGH) termination, and a polyadenylation sequences . For the RNA interference experiment, small interfering RNAs (siRNA) targeting STAT1  and control siRNA ) were purchased from Sigma-Aldrich. Huh7 cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen), and the downregulation of the target protein expression was analyzed by western blotting. For the transient replicon assay, the cells were seeded onto 24 well plates and transfected with siRNA overnight. Subsequently, the cells were transfected with the replicon vector, together with AcGFP or AcGFP-ORF6 vector, and harvested 24 h post-DNA transfection.55 with some modifications. Briefly, 14 SARS-CoV-2 (2019-nCoV/Japan/TY/WK-521/2020) cDNA fragments (#1-#13) were amplified using PCR and subcloned into a pBlueScript KS(+) vector. The primers used are described in Supplemental Table SARS-CoV-2 recombinants were generated by CPER reaction as previously describednts #1-#1 were ampHuh7 cells were seeded into a 24-well plate and incubated at 37 °C for 24 h. The cells were transfected with either pISRE-TA-Luc or pGAS-TA-Luc, and pRL-TK (Promega), as well as pCAG AcGFP-HA or pCAG AcGFP-ORF6-HA using TransIT-LT1 reagents (Mirus), according to the manufacturer’s instructions. The cells were incubated for 24 h after transfection and treated with IFN-γ (50 ng/mL) for 12 h.VeroE6/TMPRSS2 cells were seeded into a 24-well plate and incubated at 37 °C for 24 h. The cells were transfected with either pISRE-TA-Luc or pGAS-TA-Luc and pRL-TK (Promega). After 24 h, the cells were infected with SARS-CoV-2 and treated with IFN-γ (50 ng/mL) for 12 h.Luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Relative luciferase values were calculated based on the firefly/Renilla luciferase values of AcGFP.47. The NLS substrate GST-NLS-mRFP (4 pmol) was used in 10 μL of reaction mixture, and the competitive substrate AcGFP-ORF6 was added to the assay with 20 pmol, 40 pmol, and 80 pmol, which represented 5×, 10×, or 20× the NLS-substrate dosages, respectively.A digitonin-permeabilized in vitro nuclear transport assay was performed as previously describedHuh7 cells were cultured in a 35-mm glass bottom dish (Matsunami), and either GST-GFP, GST-M0-GFP, GST-M1-GFP, GST-M2-GFP, and GST-M3-GFP recombinant proteins  were microinjected into the cytoplasm with AlexaFluor555-conjugated antibody as an injection marker . The cells were incubated with or without IFN-γ  for 30 min.P < 0.05, **P < 0.01, ***P < 0.001.For most analyses, data are shown as the mean ± standard deviation (SD) from 2 to 4 independent experiments. Data were analyzed with Prism 7.0 software . Statistical significance was evaluated by one-way Analysis of Variance (ANOVA) or two-way ANOVA for comparison of multiple groups, and the Student t test for two groups, *Further information on research design is available in the Peer Review FileSupplimental informationDescription of Additional Supplementary FilesSupplementary Data 1Reporting summary


High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples.There is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes . In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination.i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or −80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples.The quantity and quality of RNA differed by sample type (Our findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies. Gut microbial communities play important multifactorial roles in animal physiology. They are important in controlling pathogen colonization and in immune system development. They also help in digestion by metabolizing the compounds in diet, which could not be broken down through enzyme production by the animal host, and in the production of vitamins, such as vitamins B12, B5, and K .Characterizing the livestock gut microbiota in terms of taxonomy and phylogeny has been carried out in a large number of studies by sequencing of the 16S ribosomal RNA subunit gene, of which either the full length or its hypervariable regions are targeted for sequencing and used for taxonomic classification . Shotgunetc.  fecal samples. These sample matrices were chosen because the vast majority of livestock microbiome efforts are performed on fecal samples. We selected two currently used storage media RNALater stabilization reagent (referred to as RL) and DNA/RNA Shield™ (referred to as ZM) used in two storage conditions (overnight at 4 °C or 2 weeks at −80 °C), and in combination with four commercially available microbial RNA extraction kits, the QIAGEN RNeasy Power Microbiome total RNA Kit (referred to as PM) , the Norgen Biotek stool RNA Kit (referred to as NO) , the ZymoBIOMICS RNA miniprep Kit (referred to as ZY) , and the Macherey-Nagel NucleoSpin RNA stool Kit (referred to as MN) . We evaluated the performance of these approaches using metrics that have been previously shown to be affected by the storage media and extraction method including RNA yield, purity and more importantly RNA integrity .n = 1) and 55 days old chicken (n = 8) were obtained from conventional slaughterhouses. Subsequent to the euthanasia of the animals, the gastrointestinal tract was removed, stored on normal ice (4 °C) and immediately transported to the laboratory (~15 min transportation time). Samples from the digesta in the distal part of the large intestine, herein referred to as feces, were gathered. The pig fecal sample, as well as the pooled chicken fecal samples, were homogenized and then transferred into the tubes containing the defined storage media including RNALater  stabilization reagent (RL) and DNA/RNA Shield™  reagent (ZM). After an overnight storage at 4 °C, to mimic the real field and transportation condition, half of the tubes in each storage media were immediately subjected to the RNA extraction procedure, while the other half was stored at −80 °C for 2 weeks until extraction. The duration of the freezing period was considered of minor importance in this study and was chosen to reflect realistic conditions based on in-house work routine  per each animal type. The combinations were the following: RL stabilization + short storage ; RL stabilization + long storage ; ZM stabilization + short storage; ZM stabilization + long storage. The tubes containing samples were thawed on ice and then vortexed for homogenization. The content of each tube was transferred into individual 2 ml Eppendorf tubes and centrifuged with 15,000 × g (Eppendorf Centrifuge 5424 R) for 5 min at room temperature (25 °C) to pellet out the feces and to remove the supernatant, which could contain traces of stabilizers. The RNA extraction from all samples was carried out following kit protocols with minor adjustments: Firstly, 150 mg of sample were used as initial starting material, and secondly, 80 μl of RNAse-free water was used instead of 100 µl for the elution of all the extracted RNA.Extraction of total RNA was done using four different commercially available kits, namely the RNeasy PowerMicrobiome kit, , the Stool Total RNA purification kit , ZymoBIOMICS™ RNA Miniprep kit , and the NucleoSpin RNA stool kit . The RNA extraction was conducted in triplicate, yielding twelve samples . The 260/230 ratio was used to estimate the presence of contaminants . Samples with a 260/230 ratio in the range of 2.0–2.2 were considered to contain a sufficiently pure RNA. Secondly, the concentration and the quality of the total RNA was determined with the Qubit 4 fluorometer . The total RNA was quantified according to the Qubit RNA XR Assay Kit protocol. The RNA integrity and quality were quantified according to the Qubit RNA IQ Assay Kit protocol. Thirdly, the RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system  with capillary electrophoresis, using the RNA 6000 Nano kit . Bioanalyzer RNA 6000 Nano assay provides a measurement of RNA integrity  quantified by the RNA Integrity Number (RIN). The RIN and concentration were checked following the manufacturer’s protocol.Three methods were used to check the RNA quantity (concentration) and quality (purity and integrity) in each of the 96 samples. Firstly, the Nano-Drop spectrophotometer  was used to check the absorbance at different wavelengths. The 260/280 ratio was used to estimate the purity of RNA regarding compounds absorbing UV light , or pig  ribosomal protein L32 (RPL32) gene was performed. Details on the qPCR assays  are summarized in To determine if the extracted RNA was functional, Reverse Transcription (RT-qPCR) was applied, using a previously established assay targeting the bacterial 16 S rRNA gene . To furtRPL32 genes in two duplex reactions .After assay optimization and evaluation in the corresponding matrices, an equivalent volume from all samples, three biological replicates per extraction method and host organism, were then assayed by qPCR in technical triplicates both with the BactQuant assay and the assays against the chicken and pig P-value of <0.05, we performed subsequent linear regression stratified by sample type, followed by a pairwise analysis. The normality of the data was checked with Shapiro-Wilk-Test , a linear regression was performed where the predictors were sample type, extraction method, storage media, storage temperature and an interaction term between the sample type, extraction method, storage media, storage temperature to determine if the final yield or quality of the extracted RNA was impacted by interaction of the variables. In case the interaction term had a ilk-Test  and wherilk-Test .P = 0.02), storage media (P = 1.6e−07), storage temperature (P = 1.1e−05) and the extraction kits (P = 1.4e−11) on the final RNA yield. The sum of the square value indicated that among the different experimental variable, the extraction kit accounted for the majority of the variability in the RNA yield (SSR = 12.50), followed by storage media (SSR = 5.22), storage temperature (SSR = 3.41) and the sample type (SSR = 0.78). In general, a significantly higher amount of RNA was recovered from the chicken feces samples compared to the pig fecal material (P = 0.19), suggesting that one specific combination of the storage media and storage temperature followed by a specific RNA extraction methodology resulted in a higher yield of RNA, regardless of the sample type. In fact, the highest yield of the RNA was obtained from the samples stored in RL for 2 weeks at −80 °C and extracted with the MN kit  after adjusting for the storage media and the storage time .Linear regression analysis showed a significant effect of the sample type . Regardless of the sample type, the combination of the storage in ZM buffer for 2 weeks at −80 °C and extraction using the ZY kit resulted in a higher integrity of the extracted RNA . The second main predictor for the chicken fecal samples was the storage temperature , while the second main driver of the RIN  for the pig feces samples was the storage media. The chicken samples stored at 4 °C, extracted by the PM kit showed significantly (P ≤ 0.01) higher integrity of the RNA, regardless of the storage media, although sample storage in RL resulted in a higher RIN number. However, for the samples stored at −80 °C, a storage media dependent effect was observed, mainly for the ZY kit (P = 0.014). Regardless of the storage media the MN and ZY extraction kits demonstrated a good performance in terms of the integrity of extracted RNA , while the NO kit revealed a low bacteria to host RNA ratio.Next to quantity, purity and integrity, RNA extracts were further evaluated for functionality by detecting the bacterial 16S rRNA gene. To further quantify the extent of coextraction of eukaryotic RNA, thereby adding an additional quality metric allowing to select conditions with high amounts of bacterial RNA, the chicken, respectively swine RPL32 mRNA by RT-qPCR for this study, constitutes a severe technical issue in metatranscriptomic analysis. Obtained results uncovered coextraction of host RNA in all samples, regardless of the extraction kit and storage condition, with the PM kit showing overall the highest bacterial to host RNA ratio.In this study, we compared the impact of the storage media, temperature and length as well as RNA extraction approaches on the recovery and integrity of microbial RNA extracted from chicken and pig fecal samples. Our findings showed that the quantity and quality of RNA varied significantly by the extraction kit. Pig feces samples showed the lowest variability and chicken feces samples exhibited the greatest variability regarding differences in the extraction method. Overall, the PM extraction kit in combination with storing the fecal samples in RL at either 4 °C (for 24 h) or −80 °C (up to 2 weeks) performed best for both chicken and pig samples considering the RNA yield, the RNA integrity and finally the level of host contamination, as an additional quality metric. The presence of host RNA, as estimated by targeting the chicken and pig Our results further underscored the importance of a standardized protocol or reporting guidelines for sample storage, duration and RNA extraction approach. This is very important as the possible effect of these technological measures can potential mask the actual biological heterogeneity to be described in the microbiome studies. Human microbiome researchers have already advocated for reporting guidelines and standardization procedures in their field . DevelopTo the best of our knowledge, our study is the first one investigating the impact of sample storage and RNA extraction strategies on the recovery of high-quality microbial RNA for livestock gut microbiome analysis. However, there are a few studies that have been performed on human stool or biofilm samples . In 2015via RNA sequencing is often impaired by the low abundance of prokaryotic mRNA. As the mRNA accounts only for a small subset of the total RNA, the removal of rRNA before starting library preparation has already been implemented in various studies on the gut microbiome to avoid wasting reads in the sequencing process and allow cost-effective MTX analysis  constitutes a technical issue: Commercially available rRNA depletion kits might not provide a sufficient high level of enrichment in bacterial mRNA, which can affect the sequencing depth for the microbiota, increase overall processing costs and complicate downstream analysis  for metatranscriptomics on human, pig, chicken and elephant feces samples as well as on infant gut and cattle rumen samples . Based oIn many studies fecal samples are used as a proxy to study the gut microbiota. However, these may not be fully representative of the whole gastrointestinal tract. In this study we used colon fecal samples, so our results may not apply to protocols where the recovery of high-quality RNA is performed from other chicken or pig gastrointestinal regions such as duodenum, jejunum, ileum or cecum and they may have suffered from the small size of our sampling material (one pig and eight chicken). Sample size in terms of the number of animals was small, we cannot exclude the likelihood that the results observed here represent those from a larger number of individuals. Although there are gut microbiota studies that reveal good correlation between 16S rRNA and mRNA stability , rRNA isIn this study, we highlighted how key stages of conducting a metatranscriptomics study on livestock microbiome, including the sample storage and RNA extraction, can potentially affect the results and therefore their possible biological interpretation. Our results suggest that the shipping of chicken and pig fecal samples on normal ice (4 °C) from farm to laboratory within 24 h, while stabilized in RNALater buffer, followed by extraction with Rneasy Power Microbiome kit could be established as a standard practice for large cohort livestock microbiome studies.10.7717/peerj.13547/supp-1Supplemental Information 1Total RNA yield, integrity (RIN) and quality measured using a Bioanalyzer 2100 system obtained from chicken, pig feces using four different RNA extraction kits , two different stabilization kits (RL and ZM), two different storage temperatures (−80 °C and 4 °C).Click here for additional data file.10.7717/peerj.13547/supp-2Supplemental Information 2Detailed results from BacQuant (16S rRNA) and RPL32 gene transcripts; Primer and probe sequences of BactQuant and RPL32; RT-qPCR protocol includes reagents and PCR cycling conditions.Click here for additional data file.


The lung is one of the most frequent distant metastasis sites in colorectal cancer (CRC) patients; however, lung metastasis risk and prognostic factors have not been comprehensively elucidated. This study aimed to identify the homogeneous and heterogeneous lung metastasis risk and prognostic factors in CRC patients using the Surveillance, Epidemiology, and End Results (SEER) database.CRC patients registered in the SEER database between 2010 and 2016 were included to analyse risk factors for developing lung metastasis by using univariable and multivariable logistic regression. Patients diagnosed between 2010 and 2015 were selected to investigate prognostic factors for lung metastasis by conducting Cox regression. Kaplan–Meier analysis was used to estimate overall survival outcomes.A total of 10,598 (5.2%) patients with synchronous lung metastasis were diagnosed among 203,138 patients with CRC. The median survival time of patients with lung metastasis was 10.0 months (95% CI 9.6–10.5 months). Older age, unmarried status, uninsured status, poor histological differentiation, more lymphatic metastasis, CEA positivity, liver metastasis, bone metastasis and brain metastasis were lung metastasis risk and prognostic factors. Black patients and those with left colon, rectum, and stage T4 disease were more likely to develop lung metastasis, while patients with right colon cancer and no surgical treatment of the primary tumour had poor survival outcomes.The incidence of lung metastasis in CRC patients was 5.2%. CRC patients with lung metastasis exhibited homogeneous and heterogeneous risk and prognostic factors. These results are helpful for clinical evaluation and individual treatment decision making.The online version contains supplementary material available at 10.1186/s12876-022-02270-5. Colorectal cancer (CRC) has become the third most commonly diagnosed cancer worldwide and is the second leading cause of cancer-related death . PatientCRC patients with lung metastasis usually have poor survival outcomes. A previous study revealed that the 1-year cause-specific survival rate of CRC patients with and without lung metastasis was 55.5% and 90.2%, respectively, which was worse than that of patients with liver metastasis . CRC patThe purpose of this study was to analyse the risk factors for lung metastasis and estimate the lung metastasis-associated prognosis in patients newly diagnosed with CRC based on data from the Surveillance, Epidemiology, and End Results (SEER) database. We further identified the heterogeneous and homogeneous risk and prognostic factors.https://seer.cancer.gov/seerstat/)  was used to generate the patient list. CRC patients diagnosed with lung metastasis between 2010 and 2016 were included in this study. Patients who were diagnosed at autopsy or via a death certificate were excluded. Patients with unspecified follow-up, a primary tumour outside of the colorectal region, and unavailable lung metastasis information were excluded. A flowchart showing the patient inclusion and exclusion process is presented in Fig. In this population-based study, CRC patient data were acquired from a US National Cancer Institute (NCI) open public database, the SEER database. SEER*Stat version 8.3.5 ; sex ; race ; marital status (unmarried and married); insurance status (uninsured and insured); site of primary tumour ; histological types ; N stage ; T stage ; carcinoembryonic antigen (CEA) ; without or with liver metastasis; without or with bone metastasis; without or with brain metastasis; and surgical treatment for the primary cancer (yes or no).P < 0.05. Statistical analyses were conducted with the IBM Statistical Package for the Social Sciences (SPSS) version 23.0 software package for Windows . MedCalc 18.0 was used to generate survival curves.Quantitative data are presented as the mean ± standard deviation (SD), and categorical data are described as numbers and percentages . Univariate and multivariate logistic regression were used to identify the factors associated with synchronous lung metastasis. The Kaplan–Meier method was used to estimate overall survival outcomes. Univariate and multivariate Cox regression analyses were conducted to identify potentially associated prognostic factors. Statistically significant levels were two-tailed and set at A total of 203,138 CRC patients were initially identified between 2010 and 2016. Of these patients, 10,598 (5.2%) patients were initially diagnosed with lung metastasis, and 192,540 (94.8%) patients were without lung metastasis. The mean age of all patients was 64.88 ± 14.32 years. A total of 105,727 (52.0%) patients were male. A total of 51.3% were married . Over half of the patients were white . Most patients were insured . Regarding the site of the primary tumour, 40.3%  of cancers were located in the right colon, 33.3%  were in the left colon, and 23.5%  were in the rectum. Most CRC patients were diagnosed at grade III , N0  and stage T3 . The detailed demographic and clinical characteristics are displayed in Table The univariate logistic regression analysis showed that age, sex, race, marital status, insurance status, primary site, histological grade, lymphatic metastasis, T stage, CEA, liver metastasis, bone metastasis and brain metastasis were all correlated with the occurrence of lung metastasis. The multivariate logistic regression confirmed that older age, black race, unmarried status, uninsured status, site, poor histological differentiation, more lymphatic metastasis, T4/T1 stage, CEA positivity and liver metastasis, bone metastasis and brain metastasis were associated with lung metastasis (see Table P < 0.001). Kaplan–Meier analysis was performed for CRC patients with lung metastasis . The 1-year, 3-year, and 5-year survival rates for lung metastasis patients were 44.3%, 13.5%, and 5.2%, respectively. When the cancers were located in the right colon or had poorly differentiated grade, were CEA positive, or involved different metastatic organs, the median survival of lung metastasis patients was reduced   but highAccurately identifying the population at high risk for lung metastasis is helpful for subsequent individualized treatment. Our results showed that patients with older age, black race, left colon, rectum, poorly differentiated grade, more lymphatic metastasis, T4 stage, CEA positivity and liver metastasis, bone metastasis and brain metastasis were more likely to develop lung metastasis. Given that stage T1 and stage T2 CRC patients have much lower risk of metastasis, another univariate and multivariate logistic regression analyses were performed which only included patients with stage T3 and stage T4 CRC .


Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N Pneumoviridae family [Respiratory syncytial virus (RSV) is a significant viral pathogen most commonly responsible for inducing lower respiratory tract diseases in infants, older adults, and immunocompromised individuals globally ,2,3,4. Ie family . The gene family . From 3’e family . Each gee family .0) with its N-terminal domain (PNTD) and with the NC with its C-terminal domain (PCTD) [Paramyxoviridae  [Orthomyxoviridae  [Filoviridae  [Bunyaviridae  [The RSV N protein consists of 391 residues and has a molecular weight (M.W.) of about 43 kDa ,13. The n (PCTD) ,18. Prion (PCTD) . The aton (PCTD) . Certainn (PCTD) . Positivn (PCTD) . Residuen (PCTD) . These in (PCTD) . The RNAn (PCTD) . A priors virus) , Orthomya virus) ,21, Filoa virus) , and Buntavirus)  have beeNTD can be exploited as a chaperone to block N association with non-specific RNAs via the co-expression of PNTD and N, thus successfully obtaining RNA-free N0 in RSV on a large scale and in a trackable manner [Despite the successful reconstitution of the RSV polymerase transcription assay in vitro, the templates used were naked RNA sequences . Additioe manner . We havee manner . Howevere manner ,26. In c0) with PNTD (P1-126) for well-defined, homogeneous N-RNA construction in vitro with short, specific RNA sequences. NC morphologies in vitro were qualitatively distinguished between using negative-stain EM imaging. We demonstrated that the optimal construction of homogeneous NCLPs depends upon multiple variables, including incubation time, length, the sequence of the encapsidated RNA, buffer salt concentration, and buffer pH. We demonstrated that the efficient assembly of homogeneous RSV N-RNA ring particles in vitro is favored by co-incubating adenine-rich RNA sequences greater than 6 nt in length with RNA-free N0PNTD for greater than 3 days, a buffer NaCl concentration between 200 and 400 mM, and a pH value of between 7 and 8. We believe that the formation of stable, homogeneous NCLPs in vitro can be further used to construct a high-resolution structure through cryo-EM and serve as a platform for exploring both of the mechanisms involved in RSV RNA synthesis and elucidating methods for antagonizing the NC complex for therapeutic benefit.This study aimed to successfully reconstitute RNA-free nucleoprotein , which allowed for the expression of the N0-TEV-P1-126-10xHis fusion protein. The E. coli BL21 (DE3) chemically competent strain was utilized for producing N0-P1-126. Cells were cultured at 37 °C in Miller’s Luria Broth medium until the A600 absorbance value reached 0.8. Expression was induced after lowering the temperature to 16 °C and incubating with 0.5 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight. Cell lysis was conducted through sonication in lysis buffer A  and subsequent centrifugation. The supernatant was then loaded onto a cobalt column equilibrated with lysis buffer. The cobalt column  was washed with buffer B  and buffer C . The protein was eluted from the cobalt column with buffer D . Further protein purification was performed via Q column , followed by TEV (tobacco etch virus) cleavage with dialyzation against buffer E . The TEV enzyme was removed by re-running the Q column . Finally, purification was completed using a Superdex 200 gel-filtration column equilibrated with buffer F (20 mM of HEPES at pH 7.4 and 200 mM of NaCl). The purified protein N0-P1-126 complex was concentrated using a centrifugal concentrator  with a molecular weight cut-off (MWCO) of 10 kDa.The N0-P1-126 complex and RNA were co-incubated with a molar ratio of 1:1.5. The sample was used directly to make the negative stain grids for screening.RNA oligos were purchased from Integrated DNA Technologies and saved as stock at a concentration of 100 µM. In order to assemble the NCLPs in vitro, the protein N0-P1-126 complex was preincubated in the presence of RNA at 4 °C overnight, then incubated for one hour at room temperature in the presence of RNase A .For RNase A  treatment, the Nw/v) uranyl formate. Negative stain EM imaging was performed using an FEI Talos L120C electron microscope  operating at 120 keV and equipped with an FEI Ceta 4k × 4k charge-coupled device camera. Micrographs were collected at nominal magnifications of 73,000 (1.97 Å/pixel). The images were acquired at a defocus value of −1.2~−2.0 µm and electron doses of −25 e−/Å2.First, 3 µL of each of the samples was applied to the clean side of freshly glow-discharged grids and subsequently stained with 1%  with a fixed molar ratio of RNA. For these complexes, a fusion protein comprised of full-length N (N1-391) and the N-terminal domain of P (P1-126) was utilized, separated by a TEV cleavage domain with a C-terminal 10-His tag. A fixed molar ratio of protein-to-RNA was utilized in this study to achieve saturation of N with RNA when assembling the NCLPs, and protein concentrations were held constant between samples to allow for qualitative comparison. The peak corresponding to the NP1-126 complex in the gel filtration profile showed an A260nm/A280nm ratio of less than 1, implying no RNA contamination in the sample (0P1-126 complex has been outlined (0) with a fixed molar ratio of RNA. Negative stain EM imaging was used to analyze the NCLPs ring assembly. To qualitatively describe the particle quality, we use the term “efficiency”, referring to the proportion of particles within a sample that resemble a complete ring-like N-RNA oligomeric assembly. To qualitatively define the energetic favorability of oligomerization, we use the term “propensity”, referring to the raw proportion of the specific type of oligomeric structure observed within the sample, including complete N-RNA ring assemblies, incomplete N-RNA ring assemblies, and particle aggregates.NCLPs were assembled by incubating RNA-free N  tested A,F. NotaIn order to test the stability of the RSV NCLPs assembled in vitro, RNase A was used to digest the RNA in the NCLPs with RNA sequence lengths greater than 7 nt, reducing the sample proportion of all observed oligomer types. For the 7 nt, 8 nt, and 11 nt sequences tested, RNase A treatment of the N-RNA complexes resulted in a reduction in the sample proportion of incomplete ring oligomers and particle aggregates . A signiIn this study, we show that it is possible to construct high-quality, homogeneous NCLPs assemblies within certain ranges of select buffer parameters and RNA characteristics, which can further serve as a manipulatable base unit for performing NC functional studies, such as in vitro RNA polymerase assays. An RNA-free NP fusion protein consisting of full-length N and the N-terminal domain of P A was use0P with the 7 nt A/C/G/U randomized sequence resulted in a seemingly lowered propensity for particle oligomerization relative to the A and C randomized RNA. This suggests that when using 7 nt RNA sequences, A has a greater binding affinity to RSV N, making N more amenable to RNA encapsidation through certain mechanisms, such as favoring P displacement from N0.Our results agree with our previously published results  in that 0P with short polyA RNA sequences of varying lengths: the first well-defined N-RNA rings were observed when using poly7A RNA. This suggests that shorter RNA sequences may not stabilize the equilibrium from the RNA-free N conformation to the RNA-bound N conformation required for oligomerization. Interestingly, ring formation propensity increases, whereas formation efficiency reduces when incubating N0P with poly8A; ring efficiency is restored despite an even greater oligomerization propensity when incubating N0P with poly11A. This is potentially suggestive of different N-RNA binding modalities that may depend on encapsidated RNA length. In RSV, when using an RNA length equivalent to the length of RNA bound by each N monomer, which is 7 nt, the encapsidation conformation may follow that of other viruses in the Mononegavirales order. For instance, in the Measles virus, each N monomer binds 6 ribonucleotides such that its 15,894 nt genome is completely encapsidated and is nuclease-resistant [0 conformation within the ring and do not bind RNA. Results from the RNase A assay showed that N-RNA ring assembly propensity with poly7A RNA was impacted with RNase A treatment to a far lesser extent than for either of the longer polyA sequences tested: poly8A and poly11A. There was a reduction in the proportion of all observed morphologies regardless of polyA length, which may indicate that even with one specific RNA length, multiple binding configurations can be adopted depending on energetic favorability. This indicates that longer RNA may form overhangs that can potentially serve as nucleation sites for oligomerization, which are otherwise eliminated with RNase A treatment. However, further work needs to be performed concerning structural analyses of N-RNA interactions at the RNA-binding groove to ascertain any such possibility.We also compared N-RNA ring formation propensity and efficiency when incubating Nesistant ,29. AlteWe then modified the incubation time and two buffer parameters: NaCl concentration and pH. While complete N-RNA rings were observable at 1 day, 3 days, and 15 days, the propensity for ring formation was much greater, as was the efficiency of well-defined N-RNA rings. At 15 days, there were minimal particle aggregates, suggesting that thermodynamic stability may have been attained for many N-RNA conformers. Thus, it may be the case that particle aggregates may be a result of kinetically favorable interactions, or perhaps particle aggregates are the predominant particle morphology corresponding to kinetically trapped intermediates. Our data suggest that complete N-RNA rings are the thermodynamically stable conformation for RSV NCLPs.0 and non-specific binding to random cellular RNAs [0 form [Mononegavirales order of viruses was described as involving a dual interaction between the N C-arm and the RNA binding domain as well as the binding of PNTD to N, such that interactions with other N monomers and non-viral specific RNAs are impaired [As elaborated on in an earlier section, RSV P functions in preventing both the self-oligomerization of Nlar RNAs ,31, and  [0 form ,25. Inte0P complex. However, within infected cells, the N0P complex assembly occurs concomitantly with RNA synthesis, which may impact NCLPs formation dynamics. Despite this, in vitro studies are still highly informative as they allow for greater NCLPs manipulation, enabling N to encapsidate specific RNA structures/sequences of choice. The ability to construct high-quality NCLPs in vitro and manipulate the RSV RNA synthesis machinery within this environment is, thus, a powerful method that can better inform strategies for constructing other oligomeric structures assumed by the RSV NCLPs within infected cells, such as helical filaments. Optimizing the construction of different NCLPs morphologies will ultimately assist in recreating the native RSV NCLPs structure bound to specific genomic RNA sequences in vitro, which can be used as a model for developing therapeutic interventions against RSV infection.While our current study provides a framework of conditions for efficient in vitro N-RNA ring construction , further


Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach. Pattern recognition receptors (PRRs) specifically recognize molecular patterns associated with pathogen invasion or cell damage. Based on their protein domain homology in vertebrates, PRRs are classified into toll-like receptors [TLRs], nucleotide oligomerization domain (NOD)-like receptors [NLRs], retinoic acid-inducible gene-I (RIG-I)-like receptors [RLRs], C-type lectin receptors [CLRs], and absent in melanoma-2 (AIM2)-like receptors [ALRs] , althougFollowing traditional cytotoxic therapies such as chemotherapy and radiotherapy, cancer cells can exhibit remarkable resistance to treatment due to multiple factors, including an immune-suppressive tumour microenvironment. Therefore, immunotherapy has emerged as standard combination cancer therapy. The innate immunity induced by RNA sensing may potentiate adaptive anticancer immunity  so RNA sSeveral molecular delivery systems have been developed to improve the therapeutic index and cellular uptake to lower the dosage of the molecule . Electro2 humidified atmosphere at 37°C. Cells were regularly tested for the presence of Mycoplasma spp. with the Myco-Sniff PCR Detection Kit  and found negative.4T1 mouse mammary cancer cells , Manassas, VA, USA) were cultured in ATCC formulated Roswell Park Memorial Institute Medium (RPMI-1640) supplemented with 5% foetal bovine serum . WEHI 164 murine fibrosarcoma cells (ATCC CRL-1751) were cultured in RPMI-1640 (Gibco) supplemented with 5% FBS. All cells were cultured in a 5% CO7/ml in culture medium with a final concentration of 0.4 mg/ml poly(I:C). Six 100 μs pulses with a voltage to distance ratio of 1300 V/cm and a frequency of 4 Hz [Poly (I:C)  was suspended to 2 mg/ml in molecular grade water. Cells were suspended to a concentration of 2.5^10 of 4 Hz  were appTwenty-four hours after transfection, the medium was replaced with 100 µl medium containing PrestoBlue , incubated for 1 hour, and viability quantified as determined by fluorescence intensity with a CLARIOstar microplate reader . The survival of cells in the experimental groups was normalized to the control group.Total RNA was extracted from cells 4 hours after transfection using Trizol Reagent  then purified on RNeasy columns . After quantification, 250 ng of total RNA was reverse transcribed into complementary DNA  according to the manufacturer’s instructions and diluted 10-fold. Messenger RNA was quantified on an Applied Biosystems QuantStudio 3 real-time thermal cycler  using custom primers  in SYBR Green Master Mix . Primer sequences are listed in Supplementary Table S1. Spleen RNA acted as a positive control. Relative quantification was performed by comparison to the reference genes β-actin and glyceraldehyde 3-phosphate dehydrogenase using the ΔΔCt method .The secretion of the pro-inflammatory proteins interferon beta  and interleukin-6 , into the medium was measured 4 hours after poly(I:C) transfection according to the manufacturer’s instructions.The total protein concentration in the medium was normalized and 25 µg of each sample was analysed using a premixed multiplex for CCL2, CCL4, CCL3 and CXCL10  on a MAGPIX System  per manufacturer’s instructions.Four hours after poly(I:C) electroporation, cells were lysed in RIPA buffer per the manufacturer’s instructions . Protein concentrations of the lysates were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) then adjusted with RIPA buffer. Equal amounts of protein were loaded into the wells of a 10% acrylamide SDS-PAGE gel and the separated proteins transferred to nitrocellulose membranes . The membranes were blocked with 3% (w/v) bovine serum albumin in tris buffered saline and then incubated overnight at 4°C with primary antibodies diluted 1:500 (Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb #12390, Cell Signaling Technologies, Danvers, MA, USA), p-IRF3 . The membranes were washed with phosphate buffered saline Tween  and incubated with a horseradish peroxidase-conjugated rabbit anti-mouse IgG  for 30 min at room temperature. The membrane was then exposed to HRP chemiluminescent substrate  and the protein bands on the gels and membranes were visualized after separation and transfer using the signal accumulation mode in a ChemiDoc MP System . Image Lab  was used to measure the adjusted intensity of the total protein bands after SDS-PAGE and the chemiluminescent signals from the western blots and intensity quantified by Image J software .GraphPad Prism 9.1.0  was used for statistical analysis and graph preparation. Since the data were normally distributed, significance was determined by a two-tailed t-test or one-way ANOVA test followed by a Tukey-Kramer post-test. A p < 0.05 was considered statistically significant.We initially examined the viability of both mammary carcinoma and fibrosarcoma cells 24 hours after the delivery of poly(I:C) into the cells using a metabolic assay . No statWe then compared the cell line responses to transfection of this dsRNA analogue. After 4 hours, we quantified the relative mRNA expression of pro-inflammatory cytokines and chemokines by RT-qPCR . NeitherWe next sought to evaluate the profiles of the IFNβ and IL-6 protein secretion by ELISA. After poly(I:C) transfection, the levels of protein expression of IFNβ and IL-6 on mammary carcinoma cells were significantly upregulated . We obseThe expression of many pro-inflammatory proteins is induced by RNA sensing ,10. TherThe fact that Type I IFN and pro-inflammatory cytokine and chemokine mRNAs were upregulated and pro-inflammatory proteins were secreted implicated the activation of RNA sensing by poly(I:C) transfection ,10. We tWe next evaluated the mRNA expression levels of several molecules important to RNA sensing and the pro-inflammatory response . IRF1 coMessenger RNA upregulation does not necessarily translate to increased protein activity. We quantified phosphorylated and therefore transcriptionally active IRF3 and IRF7 in mammary carcinoma cells using western blots  delivery via electroporation. We found that the effect of poly(I:C) on cell death was amplified by transfection. The levels of mRNAs or proteins of several pro-inflammatory molecules were also amplified after poly(I:C) electroporation. The mRNAs of several RNA sensors were present and several of them, and several of them were significantly upregulated. Components of RNA sensing signal transduction pathways were present or regulated. These observations and the fact that intratumour DNA delivery has been developed preclinically as a cancer therapy and is now in clinical trials for solid tumours ,21 suppoCell death is a potential outcome of RNA sensing ,10. We dPoly(I:C) mimics viral infection and elicits the secretion of type I IFN and pro-inflammatory cytokines, which play an essential role in inducing immune response activation (Yu and Levine 2011). We found that the mRNAs of the pro-inflammatory molecules Cxcl10 and IL-6 were upregulated in both cell types, while TNFα mRNA was upregulated only in mammary carcinoma cells when cells were transfected with poly(I:C) . We alsoPro-inflammatory signalling implicates the activation of RNA sensing by the dsRNA synthetic analogue poly(I:C). Although many RNA sensors are unassigned, several currently known RNA sensors are classified as TLRs, RLRs, and ALRs . RNA helThe dsRNA sensor TLR3 is expressed in the endosomes of the cell . Like maThe RLRs Ifih1/MDA5 and Ddx58/RIG-I function as cytoplasmic sensors of viral RNA  and haveFinally, RNA sensing signal transduction pathways extensively overlap with potential cross-talk between sensors, making it difficult to predict specific outcomes. We chose to assay several representative signalling pathway proteins. IRF1 protein induces transcription of interferon stimulated genes ,26. SevePRR activation induces the innate immune system, which may potentiate adaptive immunity. The coordinated release of cytokines could intensify an inflammatory response to increase tumouricidal activity. Consequently, ligands for PRRs such as poly(I:C) and CpG motif DNA have been used as analogues of pathogen RNA and DNA, respectively, to create immunomodulators as sole or combination cancer therapies.www.clinicaltrials.gov).The function of poly(I:C) in tumour immunotherapy has been explored for several decades. In the 1970s, studies in mammary carcinoma preclinical models demonstrated some success ,57, but We report that poly(I:C) transfection decreased cell viability in both mammary carcinoma and fibrosarcoma cell lines with a concomitant pro-inflammatory response. Electroporated Poly (I:C) stimulated the mRNAs of several sensors and signalling pathway proteins. In the future, determining the contribution of the emerging RNA sensors to their direct RNA-sensing roles in the cell metabolism as well as the mechanism by which different RNA sensors cross-talk will enhance our understanding of how RNA sensing and metabolism and their activities cooperate in the induction of nucleic acid-induced immunity.Click here for additional data file.


It is concluded that lipid-based nanoplatforms will play a key role in the development of RNA neuro-therapies.RNA-delivery is a promising tool to develop therapies for difficult to treat diseases such as neurological disorders, by silencing pathological genes or expressing therapeutic proteins. However, in many cases RNA delivery requires a vesicle that could effectively protect the molecule from bio-degradation, bypass barriers i.e., the blood brain barrier, transfer it to a targeted tissue and efficiently release the RNA inside the cells. Many vesicles such as viral vectors, and polymeric nanoparticles have been mentioned in literature. In this review, we focus in the discussion of lipid-based advanced RNA-delivery platforms. Liposomes and lipoplexes, solid lipid nanoparticles and lipid nanoparticles are the main categories of lipidic platforms for RNA-delivery to the central nervous systems (CNS). A variety of surface particles’ modifications and routes of administration have been studied to target CNS providing encouraging results  Indeed, the first gene therapy clinical trials resulted in serious adverse effects or even deaths that lead the US National Institute of Health (NIH) to suspend the trials in pre-clinical stages  such as tumors or neurodegenerative pathologies, have high mortality rates as there are no medicinal products to cure them . For exaAlthough many approaches to deliver RNA molecules that do not contain viral vectors exist such as chemically modified antisense oligonucleotides (ASO), N-acetylgalactosamine  ligand-modified short interfering RNA (siRNA) conjugates  or polymLipid-based nanoparticles are the most well-studied non-viral platforms for the delivery of RNA molecules . This brLiposomes are self-assembled pseudospherical vesicles in which the lipid bilayers surround the aqueous core. Liposomes have been widely and for a long time used as drug delivery systems for the treatment of a plethora of diseases. Indeed, more than 15 liposomal drugs are currently on the market . HoweverLiposomes can be categorized by their size, charge, key surface molecules or surface modifications to modulate circulation time and avoid detection and elimination by the immune system. The development of liposomes for gene therapy often involves the use of cationic lipids which interact electrostatically with the negatively charged genetic material . The comc) and rabies virus glycoprotein fragment (RVG-9r). As it is presented in the literature, RVG selectively interacts with the nicotinic achetilocholine receptor on the surface of the neuronal cells while the addition of a positively charged arginine residue at the carboxyl terminus of RVG (9r modification) allows the electrostatic interaction of the modified peptide and the siRNA chains  that decrease the amount of neuronal cellular prion protein ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate (DOSPA) and 1,2-Dioleoyl-sn-glycerophosphoethanolamine (DOPE)]. The platform offers favourable loading capacity, displays efficient cellular uptake and low in vitro cytotoxicity in human cortical cell lines  due to opsonization and activation of the complement system and toxicity issues connected with cell apoptotic mechanisms, enhanced reactive oxygen species (ROS) levels, have prompted the development of the ionizable cationic lipids and the lipid nanoparticles (LNPs) . Cationimolecule . Finallyll lines .Solid lipid nanoparticles (SLNs) are another category of lipidic nanoparticles. They from liposomes in that SLNs do not have an aqueous central cavity but a solid lipid:surfactant one . Dependiin vitro mucoadhesiveness and permeability properties than the non-coated SNPs  adsorptive-mediated transcytosis (AMT), 2) receptor-mediated transcytosis (RMT), and 3) carrier-mediated transcytosis (CMT) . Concernon liposomal nanovesicles. However, other platforms, such as LNPs may be better choices for the transport of genetic material in the brain due to the better encapsulation of a sensitive RNA cargo. For instance, although many of the studies mentioned above indicate that LNPs containing RNA molecules could play an important role in the treatment of CNS diseases, all of them used a patient unfriendly route of administration and more work is necessary for the development of platforms that are not only safe and efficacious but also not demanding for the patient. Moreover, in vivo toxicity studies after repeated administration is necessary to verify the safety of the platform as in many cases the therapeutic regimen would entail multiple, rather than single, dose protocol. Nevertheless, lipidic nanoparticles that carry RNAs hold great promise as advanced therapeutic medicinal products that could provide effective results in the treatment of neurodegenerative disorders, such as Alzheimer’s disease, or brain tumors like glioblastoma.The majority of recent and current studies related to the delivery of small molecules or bio-molecules in the CNS with the aid of nanotechnology have focused


NONO and SFPQ are involved in multiple nuclear processes . These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein–protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer’s RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment. Non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline- and glutamine-rich (SFPQ) are RNA-binding proteins (RBPs) belonging to the Drosophila behavior human splicing (DBHS) family. Together with paraspeckle protein 1 (PSPC-1), they are core protein components of the paraspeckles, with subnuclear bodies observed in the interchromatin space of mammalian cells placed between larger nuclear speckles and chromatin . AlthougIn vitro studies demonstrated that DBHS protein interaction mediates specific functions, depending on the cellular context or developmental stage, by modulating protein expression . Indeed,Until today, few data are available regarding the predisposition of DBHS proteins to homo- and hetero-dimerization. In vivo studies suggest that the NONO::SFPQ complex would be preferred among all the DBHS complexes . The preTo equilibrate the crystallographic structures and analyze the dynamical behaviors of the NONO::NONO and SFPQ::SFPQ homodimers and NONO:SFPQ heterodimer, we carried out three 3 μs MD simulations. The evaluation of the dimer stability during the simulations was based on both geometric and energetic parameters, as described below.The general stability of the three dimers is confirmed by the tendency of the MD simulations to reach convergence within a short simulated time frame. In detail, the RMSD values reach a plateau around 2.5 Å for all dimers within 1 µs a. HoweveTo better describe the dynamical behaviors of the NONO and SFPQ proteins within the homo- and hetero-dimers, we calculated the root mean square fluctuations (RMSF) of each residue within the simulations b. ProteiWhen taken together, these data show that, for all dimers, the lowest RMSF values are associated with α-helices and β-sheets, while the highest values are associated both with random coils and N- and C-termini.From these analyses, we detected a highly mobile stretch of residues between RRM2 and coiled-coil regions (NOPS). These residues  form an α-helix facing the RRM2 of the other monomer d,e. NoteBy analyzing the molecular interactions throughout the simulations, we observed that all the dimers conserve over 50 H-bond interactions during the MD simulations . In partHowever, the number of polar interactions is not sufficient to fully characterize the interaction profiles between the homo- and hetero-dimers, as solvation effect and hydrophobic and short-range interactions may significantly affect dimer formation. For this reason, we calculated the dimer formation energy, in order to evaluate whether the homo-/hetero-dimerization of NONO and SFPQ is a thermodynamically favored process, as well as whether there could be any inconsistency, in terms of energy, between complexes. Dimer formation energy was calculated by comparing the energetic features between MD simulations of DBHS monomers and dimers, resulting in the energy difference between the reactants and products of the dimerization process. Being performed on explicit solvent MD simulations, this procedure should implicitly capture the role of solvation in dimers stability. More details on dimer formation energy calculation are described in To investigate the residues involved in the dimerization process, we integrated molecular dynamics simulation interaction analyses with single point potential energy calculations and an in silico alanine scan. We recorded how long each residue of each monomer was involved in interactions with its binding partner, which was averaged over the simulated time, considering the hydrogen bonds, salt bridges, and Pi–Pi and Pi–cation interactions. Cluster analysis of the trajectories allowed us to identify the most stable conformations by extracting the medoid of the largest clusters. On these configurations, we performed a single point energy calculation and decomposed the potential interaction energies at the residue level. On the same structures, we then performed an in silico alanine scan experiment, in order to individuate the hot spot residues necessary for the dimer’s stability. First, we identified persistent backbone-to-backbone hydrogen bond interactions within the N-terminal loops for only the NONO::NONO homodimer a, in accIn all the investigated dimers, we were able to observe RRM1 domains interacting with each other at the level of ɑ-helix 2 b, both tOverall, multiple (conserved) interactions could be found between the RRM2s of one monomer and the coiled-coil and NOPS domains of the other c,d. SpecAs expected, coiled-coil helices primarily interact with themselves e; howeveThe analysis of hydrophobic interactions  also revWe investigated the recognition mechanism between RRM1s and RNA by simulating the dynamics of homo- and hetero-dimers in complex with a short guanosine pentanucleotide mimicking the NEAT1 guanine-rich regions ,24. We sBy recording per-residue protein::RNA interactions along trajectories, we were able to identify the conserved residues that are likely involved in RNA recognition . Both NOwww.rcsb.org) were subjected to a pre-processing procedure consisting of the following steps: (i) addition of ACE and NMA capping groups to N- and C- termini, (ii) addition of hydrogens and optimization of the protonation state using PROPKA [The three-dimensional crystallographic structures of the NONO::NONO (PDB ID: 5IFM-1) and SFPQ::SFPQ (PDB ID: 6OWJ) homodimers and NONO::SFPQ (PDB ID: 7LRQ) heterodimer  pMolecular dynamics (MD) simulations were carried out with Desmond  . Systemsvrun module and Schrodinger python API. An in-house Python implementation of the GROMOS clustering algorithm [Trajectory energetic and geometric data were extracted using the Desmond lgorithm  was usedProtein::protein and protein::RNA interactions were calculated using the Schrödinger Python API to analyze MD trajectories. Measured interactions include hydrogen-bonds, salt bridges, and pi–pi and pi–cation interactions. Hydrophobic interactions were measured on MD medoids using a protein::protein interaction analysis web server . Per-resFor each dimer, three trajectories were considered: the dimer itself and two constituting monomers . For each chain of these systems, we computed the protein–solvent (including ions) and protein–protein interaction energies (in case of dimeric systems). We also computed the internal energy of each protein chain to keep account of the energy stored or released during its rearrangement. We then defined dimer formation energy This represents the energy balance of the transition from two separate monomers to the dimer configuration and, when negative, indicates a favorable process. For this calculation, we considered only the second (equilibrated) half of the trajectories.wt complex, reporting it as an ΔAffinity value.In silico mutagenesis was performed to produce an alanine scan of each protomer in the NONO and SFPQ homo- and hetero-dimers. The methodology was carried out with Prime . One at a time, each residue was mutated into alanine, and all residues within 6 Å from the mutation site were minimized; finally, the interaction energy between dimer protomers was computed and compared to the −1 Å−1) of NONO and SFPQ RRM1s in both homo- and heterodimers to the structure of CUG-binding protein 1 (CUGBP1) in complex with RNA (PDB: 3NNA). Then, we built the polyG pentaribonucleotide from scratch using the Maestro 3D Builder graphical interface and aligned it to the co-crystalized RNA molecule in 3NNA  using Maestro flexible ligand alignment tool. Finally, we merged the structures of the homo- and heterodimers with the aligned pentanucleotide. This procedure yielded four models of NONO and SFPQ homo/heterodimers, with an RNA molecule proximal to each RRM1: NONO::NONO(::polyG), SFPQ::SFPQ(::polyG), NONO(::polyG)::SFPQ, and NONO::SFPQ(::polyG). These models were then refined by energy-minimizing residues, within 6 Å from the RNA ligand, using Prime implementation of MM/GBSA, and submitted to 500 ns molecular dynamics.To provide an initial guess for the RNA-bound structure, we aligned the equilibrated structures  probe that mimics the guanine-rich regions of NEAT1.By performing MD simulations and energy calculations on NONO::NONO, SFPQ::SFPQ, and NONO::SFPQ dimers, we were able to confirm dimer stability and the different interactions between homo and heterodimers; in particular, we show that the formation of the NONO::SFPQ heterodimer is a more favorable process, compared to homodimers formation. Although our models are truncated at the N-termini, due to the lack of full-length crystallographic structures, and other phenomena may regulate hetero/homodimerization processes in vivo, our findings are in accordance with the experimental results showing a higher abundancy of DBS proteins heterodimers, compared to homodimers.We also hypothesize that NONO and SFPQ RRM2s are not required for primary RNA interaction. In fact, RRM2s lack the typical RNP signature; instead, we show that the RRM2 residues that are in the place of the RNP signature are mostly involved in protein–protein interactions with the other DBHS protomer; meaning that RNA binding is primarily due to RRM1s.The simulation of RNA binding to NONO and SFPQ validated the importance of the RRM1 RNP signature but also highlighted other key residues in β2 and β4 strands that could be required for specific recognition of RNA sequences. Moreover, we show that the NOPS region and other protomer’s RRM2 β2/β3 loop may also be required to strengthen RNA interactions.Our results shed light on the interactions between RNA and DBHS dimers, which will be required for the rational design of small molecules that are able to modulate the activity of these proteins. In fact, the follow-up of this work will be aimed at the pharmacological disruption of NEAT1 binding using RNA-mimetics, which are able to selectively bind to NONO and/or SFPQ RNA-recognition site, impairing paraspeckle formation, thus being a first step towards the rational design of new drugs for the treatment of multiple myeloma.