Journal: Molecular Therapy. Nucleic Acids

Article Title: A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition

doi: 10.1016/j.omtn.2023.04.011

Figure Lengend Snippet: Screening effective gRNA target sites for cyclin T1 editing: HeLa cells were transfected with pLentiCRISPR-gRNA-1, pLentiCRISPR-gRNA-2, or pLentiCRISPR-gRNA-3 using lipofectamine 2000 as described in methods Our previously reported lentiviral vector pHIV-7-GFP was transfected as control. , 8 days post-transfection, total protein was isolated and analyzed for cyclin T1 protein levels using western blot analyses. pLentiCRISPR-gRNA-2 demonstrates maximal cyclin T1 suppression (A). HIV-infected HeLa-CD4 cells were transfected with pLentiCRISPR-gRNA-1, pLentiCRISPR-gRNA-2, or pLentiCRISPR-gRNA-3. At designated time points, culture supernatants were collected and analyzed for HIV p24. All three gRNAs demonstrate HIV suppression, with pLentiCRISPR-gRNA-2 demonstrating maximal suppression at both time points (B). Cells were trypsinized, followed by trypsin neutralization buffer. Cells were washed to remove trypsin and resuspended in DMEM 10% FBS. The otal number of live cells and percentage of viability were determined by trypan blue staining as described in . Cyclin T1 knockdown does not affect the percentage of cell viability. However, we observed a statistically significant decline in the number of live cells with all pLentiCRISPR-gRNAs, including our most efficient pLentiCRISPR-gRNA-2 (C). n = mean ± SEM from 3 independent experiments. ∗, significant from control; S, significant from each other (p < 0.05).

Article Snippet: Three gRNA sequences targeting cyclin T1 were obtained from Genscript ( https://www.genscript.com/gRNA-detail/904/CCNT1-CRISPR-guide-RNA.html ).

Techniques: Transfection, Plasmid Preparation, Control, Isolation, Western Blot, Infection, Neutralization, Staining, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition

doi: 10.1016/j.omtn.2023.04.011

Figure Lengend Snippet: HIV LTR-hsp70 fusion promoter demonstrates HIV-inducible co-expression of cyclin T1-targeting gRNA and Cas9 (A) Schematic of LTRhsp-CRISPR cassette (not drawn to scale): in presence of HIV Tat, P-TEFb kinase is recruited to the HIV LTR-minimal Drosophila hsp70 fusion promoter, inducing transcription from both promoters. Most of the transcription from the minimal hsp70 promoter will terminate at the minimal polyA to express the cyclin T1-targeting gRNA-2, while transcriptional readthrough will encode the Cas9. The strong eukaryotic translation initiation signal CCACC ensures that the first ATG after this sequence is used for translation initiation. Given the critical importance of cyclin T1 for P-TEFb recruitment by HIV Tat, inactivation of cyclin T1 will irreversibly block all transcription from HIV locking it in a transcriptionally inactive state. Since the fusion promoter also requires Tat-cyclin T1 interaction for co-expression of cyclin T1 gRNA and Cas9, once cyclin T1 is knocked down, transcription from both the HIV proviral DNA as well as the fusion promoter will be completely inhibited, possibly affecting a functional cure and limiting any further expression from the fusion promoter. (B) Uninfected HeLa-CD4 cells or HIV-infected HeLa-CD4 cells were transfected with LTRhsp-CRISPR or the NF-κB or SP1 deletion mutants, (LTRhsp(ΔNF-κB)-CRISPR or LTRhsp(ΔSP1)-CRISPR respectively). 72 h post-transfection, total protein was isolated and analyzed for Cas9 expression by western blot analyses. HIV LTR-hsp70 fusion promoter demonstrates Cas9 expression only in HIV-infected HeLa-CD4 cells and not in uninfected HeLa-CD4 cells, demonstrating HIV-inducible expression. No expression of Cas9 is observed in LTRhsp(ΔNF-κB)-CRISPR-transfected uninfected or HIV-infected HeLa-CD4 cells, suggesting that NF-κB sites are important for transcription of Cas9 from the fusion promoter even in the presence of HIV Tat. Some expression is observed in uninfected HeLa-CD4 cells transfected with LTRhsp(ΔSP1)-CRISPR, suggesting that deleting the SP1 sites promotes leaky transcription from the fusion promoter. (C and D) Uninfected or HIV-infected HeLa-CD4 cells were transfected with LTRhsp-CRISPR or the constitutive pLentiCRISPR-gRNA-2. 6 days post-transfection, total protein was isolated and analyzed for cyclin T1 suppression by western blot analyses. Both LTRhsp-CRISPR and pLentiCRISPR-gRNA-2 demonstrate cyclin T1 suppression in HIV-infected HeLa-CD4 cells (C). However, only the constitutive pLentiCRISPR-gRNA-2 demonstrates cyclin T1 suppression in uninfected HeLa-CD4 cells. LTRhsp-CRISPR does not suppress cyclin T1 in uninfected HeLa-CD4 cells, demonstrating HIV-inducible cyclin T1 knockdown from our fusion promoter (D). (E) HIV-infected HeLa-CD4 cells were transfected with LTRhsp-CRISPR as described in the . Cells were washed four times to remove any residual HIV p24 and resuspended in fresh DMEM with 10% FBS. On day 6 post-infection, culture supernatants were collected and analyzed for HIV p24 by ELISA. LTRhsp-CRISPR suppresses HIV p24 in our stringent model of HIV infection. n = mean ± SEM from 3 independent experiments. ∗, significant from control (p < 0.05).

Article Snippet: Three gRNA sequences targeting cyclin T1 were obtained from Genscript ( https://www.genscript.com/gRNA-detail/904/CCNT1-CRISPR-guide-RNA.html ).

Techniques: Expressing, CRISPR, Sequencing, Blocking Assay, Functional Assay, Infection, Transfection, Isolation, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition

doi: 10.1016/j.omtn.2023.04.011

Figure Lengend Snippet: Cis -cleaving minizymes improve RNA Pol II CRISPR-based HIV suppression (A) Schematic of LTRhsp-CRISPR minizyme-embedded variants. In the original clone ( Figure 2 A), co-expression of cyclin T1 gRNA and Cas9 results in cyclin T1 gRNA with a 5-methyl guanosine cap, which can export the gRNA from the nucleus to the cytoplasm. This can lead to suboptimal CRISPR-mediated knockdown of cyclin T1. Embedding a cis -acting minizyme just downstream of the transcriptional start such that it would cleave the 5′ cap will result in cap removal and retention of the gRNA in the nucleus. The weak ribozyme will ensure that part of the RNA is cleaved to remove the cap while some of the RNA retains the cap and is exported to the cytoplasm to express the Cas9 protein. (B) A schematic representation of the cis -cleaving minizyme-embedded variants and cleavage site upstream of the gRNA to remove the 5′ cap and improve RNA Pol II CRISPR efficacy. Hammerhead ribozymes can cleave any RNA as long as the ribozyme arms can hybridize with the target RNA, and the target contains an NUX triplet where N = A, G, C, or U and X = A, U, or C for optimal cleavage. LTRhsp-MzCRISPR recognizes a canonical GUC cleavage site, while LTRhsp-Mz(Wk)CRISPR recognizes a weaker non-canonical GUG cleavage site. (C and D) HIV-infected HeLa-CD4 cells were transfected with LTRhsp-MzCRISPR or LTRhsp-Mz(Wk)CRISPR. Transfection with the lentiviral backbone pHIV-7-GFP was used as control and to monitor transfection. Transfection with LTRhsp-CRISPR was used for comparison. To mimic a more physiological setting, only the transfection medium was replaced with fresh DMEM with 10% FBS. At designated time points, culture supernatants were collected and analyzed for HIV p24. Both ribozyme cis -cleaving minizyme-embedded variants demonstrate slightly improved HIV suppression compared with LTRhsp-CRISPR (C). Following 12 days of transfection, experiments were terminated, and cell viability was determined. LTRhsp-CRISPR or the minizyme-embedded LTRhsp-MzCRISPR or LTRhsp-Mz(Wk)CRISPR did not affect cell viability and demonstrated similar live cell counts. n = mean ± SEM from 3 independent experiments. ∗, significant from control; S, significant from each other (p < 0.05).

Article Snippet: Three gRNA sequences targeting cyclin T1 were obtained from Genscript ( https://www.genscript.com/gRNA-detail/904/CCNT1-CRISPR-guide-RNA.html ).

Techniques: CRISPR, Expressing, Knockdown, Infection, Transfection, Control, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition

doi: 10.1016/j.omtn.2023.04.011

Figure Lengend Snippet: HIV-inducible CRISPR systems were packaged, as lentiviral vectors demonstrate prolonged and sustained HIV suppression in T cell lines when used in combination with initial antiretroviral treatment CEM T cells were infected with HIV IIIB strain as described in methods. All CRISPR constructs including pHIV-7-GFP and the constitutive pLentiCRISPR-gRNA-2 were packaged as lentiviral vectors. (A) Cells were transduced with HIV-7-GFP or the CRISPR constructs at 100 MOI. After 24 h, medium was changed to fresh medium containing tenofovir (5 μM). 3 days post-transduction, the medium was replaced with fresh medium without tenofovir, and CEM cells were propagated in the absence of tenofovir for the remainder of the experiment. Culture supernatants were collected at the designated time points. Lentiviral vector-based delivery significantly enhances the suppressive effects of our HIV-inducible CRISPR system compared with electroporation- or transfection-based delivery. LTRhsp-CRISPR and LTRhsp-Mz(Wk)CRISPR demonstrated comparable suppression (∼90% HIV suppression) up to day 24. LTRhsp-MzCRISPR-transduced cells demonstrated the best suppression, with viral titers becoming undetectable by day 12 and remaining undetectable until day 21. The constitutively expressed LentiCRISPR-gRNA-2 demonstrated undetectable HIV on days 21 and 24. However, this was most likely due to extensive cell death in this group (B). LTRhsp-CRISPR and LTRhsp-MzCRISPR improved cell viability over controls. However, they demonstrated some decrease in the number of live cells. n = mean ± SEM from 3 independent experiments. ∗, significant from control; ∗∗, significant from pHIV-7-GFP + tenofovir; S, significant from all other CRISPR constructs; ND, not detectable (p < 0.05).

Article Snippet: Three gRNA sequences targeting cyclin T1 were obtained from Genscript ( https://www.genscript.com/gRNA-detail/904/CCNT1-CRISPR-guide-RNA.html ).

Techniques: CRISPR, Infection, Construct, Transduction, Plasmid Preparation, Electroporation, Transfection, Control

Journal: Nature Communications

Article Title: Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation

doi: 10.1038/s41467-023-36116-9

Figure Lengend Snippet: a Schematic representation of the experimental plan to characterise the functional relevance of MEK1/2, JNK and p38 pathways in endoderm differentiation. hPSCs were grown for 3 days in culture conditions inducing endoderm differentiation in the presence of small-molecule inhibitors. b QPCR, c FACS and d immunostaining were performed after 3 days for pluripotency markers (POU5F1/OCT4, NANOG and SOX2), mesendoderm/mesoderm markers (BRACHYURY/T, NKX2.5, CDX2 and MIXL1) and endoderm markers (SOX17, EOMES, GATA4 and FOXA2). For QPCR, the boxes show the interquartile range, with the median marked as a heavy horizontal band. Whiskers represent the highest (lowest) datapoint within 1.5 times the interquartile range of the 75th (25th) percentile. The diamonds represent each datapoint. For FACS, one-way ANOVA was performed, followed by Dunnett’s multiple comparisons test where each of the three treatment conditions was compared against the control in n = 5 experiments. The statistical significance for FACS marks adjusted P values: ** (0.0096), **** (<0.0001). For immunostaining, the scale bar represents 200 µm. Experiments represent three replicates. Statistical analysis was performed by two-way ANOVA with multiple comparisons and **** marks adjusted P value <0.0001. e Consensus binding motifs for AP-1 transcription factors (JUN and FOSL2), SMAD2/3 and SMAD4 were obtained from ATAC-seq analyses at 36 h time point with corresponding P values. f The switching of transcription factor complexes during hPSC differentiation to definitive endoderm. SMAD2/3 was immunoprecipitated from nuclear extracts of undifferentiated hPSCs, at 36 and at 72 h after initiating endoderm differentiation and analysed for the co-immunoprecipitation of NANOG, EOMES, GATA4, FOSL2 and JUN. g Schematic representation of the experimental outline for analysing the impact of p38-MAPK and TGFβ/Activin A signalling on SMAD2/3 and FOSL2/JUN binding and H3K27ac enrichment on mesendoderm at 24 h, and endoderm or mesoderm loci at 36 h by ChIP-qPCR. Cells were treated with p38-MAPK and TGFβ/Activin A inhibitors for 12 h in the presence of differentiation signals before sample collection. h FOSL2/JUN and SMAD2/3 cooperative binding to MIXL1 and EOMES regulatory regions at mesendoderm differentiation stage during hPSC differentiation. Early G1 phase synchronised cells were differentiated to endoderm for 12 h to receive the pluripotency exit signalling, and then treated with p38-MAPK inhibitor SB203580 or TGFβ/Activin signalling inhibitor SB431542 for 12 h, followed by cell fixation for ChIP-qPCR (before first cell division). Analyses reveal that p38-MAPK and TGFβ/Activin signalling regulate the cooperative binding of AP-1 TFs FOSL2/JUN and SMAD2/3 to mesendoderm genes at 24 h time point; n = 3 biologically independent experiments. i FOSL2/JUN and SMAD2/3 cooperative binding to CER1 , FOXA2 , LZTS1 , GATA4 and GSC regulatory regions at endoderm differentiation stage during hPSC differentiation. Early G1 phase synchronised cells were differentiated to endoderm for 24 h, and then treated with p38-MAPK inhibitor SB203580 or TGFβ/Activin signalling inhibitor for 12 h, followed by cell fixation for ChIP-qPCR (before second cell division). p38-MAPK and TGFβ/Activin signalling regulate the cooperative binding of AP-1 TFs FOSL2/JUN and SMAD2/3 to definitive endoderm genes at 36 h time point. n = 3 biologically independent experiments. j – m Activation of p38-MAPK signalling improves definitive endoderm and pancreatic differentiation. Cells were treated with 200 mM Sorbitol during 24 to 72 h of endoderm differentiation and analysed by j qPCR of FOXA2 and GSC expression at day 3, k , l FACS of PDX1, SOX9 and CD142 co-expression at day 12, m qPCR of NGN3 , SST , GSG and Insulin expression at day 18 of pancreatic differentiation. Experiments represent three replicates. n Activation of p38-MAPK signalling by Sorbitol improves definitive endoderm differentiation and the formation of endoderm-derived pancreatic cell types. Schematic representation of hESC and hiPSC differentiation to definitive endoderm, pancreatic progenitors and pancreatic islets of the Langerhans that contain ɑ, β, δ and F cells expressing their corresponding secreted factors such as insulin by β cells. Activation of p38-MAPK by Sorbitol improved definitive endoderm differentiation and the formation of subsequent pancreatic cells. Adapted from 'Pancreatic Islet of Langerhans', by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates . Statistical analyses in ( h , i , j , m ) were performed by two-way ANOVA with Tukey’s multiple comparisons tests and * marks adjusted P value <0.05 and ** marks adjusted P value <0.01, *** marks adjusted P value <0.001, **** marks adjusted P value <0.0001. Data were presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Inducible knockdown of EOMES was performed by using a dox-inducible CRISPR interference (CRISPRi) knock-in construct that was first targeted to the AAVS1 locus to create a stable hESC line as described in ref. . gRNA sequences for EOMES knockdown were obtained from the GenScript gRNA database and cloned into the gRNA construct according to the protocol from ref. . 2 mM Doxycyclin (Dox) was used for dCas9-KRAB mediated inducible knockdown of EOMES during endoderm differentiation.

Techniques: Functional Assay, Immunostaining, Control, Binding Assay, Immunoprecipitation, ChIP-qPCR, Activation Assay, Expressing, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Retromer has a selective function in cargo sorting via endosome transport carriers

doi: 10.1083/jcb.201806153

Figure Lengend Snippet: Ultrastructural alteration of lysosomal structures and elevated autophagy in Vps35 KO cells. (A) Generation of CRISPR/Cas9-mediated Vps35 KO HeLa cells and Vps35-GFP rescue cells. Equal amounts of cell lysates from HeLa, Vps35 KO, and Vps35-GFP rescue cells were subjected to SDS-PAGE and immunoblotted with antibodies against Vps35, Vps26A, Vps29, and tubulin. (B) Electron micrographs of HeLa, Vps35 KO, and Vps35-GFP rescue cells. Enlarged circular structures are indicated as late endosomal/lysosomal structures. Scale bars, 2,000 nm; in zoomed images, 500 nm. Graph represents the percentage volume density of lysosomal compartments relative to the cytoplasm in HeLa, Vps35 KO, and Vps35-GFP rescue cells (means ± SEM). Two-tailed Student’s t test was used to determine the statistical significance. **, P < 0.01; ***, P < 0.001. n = two independent experiments with 10 images each. (C) Flow cytometric analysis of cellular acidification based on LysoTracker fluorescence in HeLa and Vps35 KO cells. Graph represents the mean fluorescent intensity within HeLa and Vps35 KO cells (means ± SEM). Two-tailed Student’s t test was used to determine the statistical significance ( n = 3). (D) HeLa, Vps35 KO, and Vps35-GFP rescue cells were fixed and coimmunolabeled with antibodies against LC3-II and LAMP1, followed by Alexa Fluor–conjugated fluorescent secondary antibodies. Scale bars, 5 µm. The colocalization between LC3-II and LAMP1 was quantified by the Pearson’s correlation coefficient (means ± SEM). Two-tailed Student’s t test was used to determine the statistical significance among HeLa, Vps35 KO, and Vps35-GFP rescue cells upon amino acid stimulation ( n = 3). ***, P < 0.001; ****, P < 0.0001. (E) HeLa and Vps35 KO cells were treated with chloroquine (CQ, 50 µM) for 6 h. Cells were harvested, and equal amounts of protein samples were used for SDS-PAGE and immunoblotting with antibodies against LC3-II, Vps35, and GAPDH. Graph represents the level of LC3-II normalized to GAPDH (mean ± SEM). Two-tailed Student’s t test was used to determine the statistical significance ( n = 3). *, P < 0.05; **, P < 0.01. (F) Amino acid–starved HeLa, Vps35 KO, and Vps35-GFP rescue cells were treated with 2× essential amino acid solution for 30 min, fixed with ice-cold methanol, and coimmunolabeled with antibodies against mTORC1 and LAMP1, followed by Alexa Fluor–conjugated fluorescent secondary antibodies (means ± SEM). Scale bars, 5 µm. The colocalization of mTORC1 with LAMP1 was quantified by Pearson’s correlation coefficient. Two-tailed Student’s t test indicates the difference between HeLa and Vps35 KO cells upon amino acid stimulation ( n = 3). ***, P < 0.001; ****, P < 0.0001. (G) HeLa and Vps35 KO cells were treated with AZD8055 (1 µM) for 25 h before being subjected to SDS-PAGE and immunoblotted with antibodies against LC3-II, Vps35, and GAPDH. Graph represents the expression level of LC3-II normalized to GAPDH (mean ± SEM). Two-tailed Student’s t test was used to determine the statistical significance ( n = 3). *, P < 0.05.

Article Snippet: The SNX3 CRISPR guide RNA (gRNA) plasmid (gRNA targeting sequence: 5′-CGGCCGACCCCCACCGTTTG-3′), SNX1 CRISPR gRNA plasmid (gRNA targeting sequence: 5′-AAATCATCCTACCATGTTAC-3′), and the SNX2 CRISPR gRNA plasmid (gRNA targeting sequence: 5′-TGATGGCATGAATGCCTATA-3′) were synthesized by Genscript.

Techniques: CRISPR, SDS Page, Two Tailed Test, Fluorescence, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: Retromer has a selective function in cargo sorting via endosome transport carriers

doi: 10.1083/jcb.201806153

Figure Lengend Snippet: SNX3 is required for the retrograde transport of CI-M6PR GCC88-tethered ETCs. (A) Equal amounts of cell lysates from HeLa, SNX1/2 dKO, SNX3 KO, and SNX27 KO cells were subjected to SDS-PAGE and immunoblotted with antibodies against Vps35, Vps26A, SNX1, SNX2, SNX5, SNX6, SNX27, SNX3, and tubulin. (B and C) HeLa, SNX3 KO, SNX1/2 dKO, and SNX27 cells were transiently transfected with HA-tagged mitochondria-targeting golgin constructs GCC88-MAO, Golgin-97-MAO, Golgin-245-MAO, or GM130-MAO; fixed; and coimmunolabeled with antibodies against HA and endogenous CI-M6PR (B) or CD-M6PR (C), followed by Alexa Fluor–conjugated secondary antibodies. The intensity plots of the fluorescent intensity (y-axis) against distance (x-axis) represent the overlap between channels. The colocalization of CI-M6PR (B) or CD-M6PR (C) with HA-tagged golgin-mito proteins was quantified by Pearson’s correlation coefficient (means ± SEM). Two-tailed Student’s t test was used to determine the statistical significance ( n = 3). **, P < 0.01; ****, P < 0.0001.

Article Snippet: The SNX3 CRISPR guide RNA (gRNA) plasmid (gRNA targeting sequence: 5′-CGGCCGACCCCCACCGTTTG-3′), SNX1 CRISPR gRNA plasmid (gRNA targeting sequence: 5′-AAATCATCCTACCATGTTAC-3′), and the SNX2 CRISPR gRNA plasmid (gRNA targeting sequence: 5′-TGATGGCATGAATGCCTATA-3′) were synthesized by Genscript.

Techniques: SDS Page, Transfection, Construct, Two Tailed Test

Journal: STAR Protocols

Article Title: Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells

doi: 10.1016/j.xpro.2021.100744

Figure Lengend Snippet: Restriction digest to linearize the SpCas9:sgRNA dual plasmid backbone

Article Snippet: Use a commercial manufacturer to generate the SpCas9:sgRNA dual plasmid Vectors for expressing sgRNAs and SpCas9 can be commercially generated. (e.g., https://en.vectorbuilder.com/resources/vector-system/pRP_gRNA_parent.html )

Techniques: Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts

doi: 10.1371/journal.ppat.1009146

Figure Lengend Snippet: (A) Proposed enterovirus capsid assembly pathways. The viral polyprotein is cleaved by the virally-encoded 3CD protease forming protomers that rapidly self-assemble into pentamers, a proportion of which in turn self-assemble into RNA-free ECs in which VP0 subunits remain intact. It has been proposed that pentamers can interact with genomic RNA and assemble directly or enter preformed ECs, both pathways generating a procapsid. Maturation to infectious virion occurs following cleavage of VP0 into VP2 and VP4. These species are distinguishable by centrifugation and their respective sedimentation coefficients are shown. (B) Bernoulli Plot of Sequence Identities of anti-EV-E CP pentamer aptamers to the gRNA (7,414 nts long NC_001859). Each aptamer selected region was assessed for sequence matches to the genome, equivalent to 12 continuous identities, by sliding the sequence across the gRNA 5′ to 3′ incrementing 1 nt at a time. At each nucleotide position fulfilling this requirement a counter was incremented by 1 for each match, resulting in the peaks seen in red. In grey is the equivalent for the unselected starting library. Peaks 7, 9, 21, 24 have matching frequencies ~ 20, 37, 11 and 15 x10 3 , respectively, i.e. are well off the scale of the y-axis. The gene map is shown below the Bernoulli plot. (C) Mfold structure of the SL that can form from the gRNA sequence in Peak #9, the highest peak in (B). (D) Alignment of loop motif regions identified from SELEX peaks. A common RAR motif is found.

Article Snippet: The EV-E gRNA sequence (NCBI, NC_001859) was supplied by Genscript Biotech (Netherlands) in a pUC57 plasmid vector, which encompasses ampicillin-resistance.

Techniques: Centrifugation, Sedimentation, Sequencing

Journal: PLoS Pathogens

Article Title: Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts

doi: 10.1371/journal.ppat.1009146

Figure Lengend Snippet: (A) Radially colour-coded slab of the cryo-EM unsharpened density map of EV-E capsid shown at 1.6 σ and viewed along a two-fold axis. Dashed circles indicate contacts between CP (dark red) and gRNA (white and blue). Symbols indicate icosahedral symmetry axes. Bar = 50 Å. (B) Atomic model of the front half capsid of EV-E shown as ribbon diagrams, colour-coded as in and viewed along a two-fold axis. Colour-coded dotted circles indicate location of contacts between CP and gRNA on the capsid. (C-H) Detail of the contacts between CP and gRNA indicated in (A, B). The cryo-EM density maps are shown as grey mesh with the density corresponding to RNA facing the bottom part. The atomic model is shown as ribbon diagrams and sticks coloured in orange for the RNA, and sticks colour-coded as previously for the CP. Residues involved in the contact are indicated and coloured by heteroatom. (C) Base stacking contact close to two-fold axis (green dotted circle in A, B) between VP2 W38 and A19-U20 bases of the RNA model generated for the most frequent PS in EV-E , fitted into a local resolution low-pass filtered density map obtained after symmetry expansion and focused classification on two-fold axes shown at 1.8 σ. (D) Zoom out view of (C) showing fitting of the RNA model into lower resolution density adjacent to the contact. (E) Contact between VP1 R50 and RNA close to two-fold axis (blue dashed circle in A, B) fitted into the same map as in (C) but low-pass filtered to 5 Å resolution shown at 1.3 σ. (F) Contact between VP3 F35 and RNA close to two-fold axis (red dashed circle in A, B) fitted into the icosahedrally averaged map low-pass filtered to 5 Å resolution shown at 1.5 σ. (G) Contact located on the three-fold axis (green dashed circle in A, B) involving VP2 Y9 from three different asymmetric units fitted into the icosahedrally-averaged unsharpened map shown at 1.5 σ. (H) Contact between VP4 G21 and RNA close to five-fold axis (yellow dashed circle in A, B) fitted into the same map as in (G). (I) 3D model of the lowest energy fold for Peak #9 of the Bernoulli Plot obtained by structure prediction in RNA composer shown as in (C, D).

Article Snippet: The EV-E gRNA sequence (NCBI, NC_001859) was supplied by Genscript Biotech (Netherlands) in a pUC57 plasmid vector, which encompasses ampicillin-resistance.

Techniques: Cryo-EM Sample Prep, Generated

Journal: PLoS Pathogens

Article Title: Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts

doi: 10.1371/journal.ppat.1009146

Figure Lengend Snippet: (A) Radially colour-coded cryo-EM density map of EV-E capsid shown at 2 σ and viewed along a two-fold axis. One of the pentamers is coloured in orange and the asymmetric unit is coloured following the “classic” colour-code for picornaviruses, VP1 in blue, VP2 in green, VP3 in red and VP4 in yellow. Bar = 50 Å. (B) Atomic model of the asymmetric unit of EV-E shown as ribbon diagrams (top view, left; side view, right) colour-coded as in (A). Symbols and arrows indicate icosahedral symmetry axes. (C) Atomic model of EV-E viral proteins shown as ribbon diagrams and colour-coded as previously. First and last residues at N- and C- termini are indicated. Amino acid side chains for residues involved in contact with gRNA are shown as heteroatom stick diagrams and indicated by dotted circles. Updated atomic coordinates for VP1, VP2 and VP4 are coloured in pink. (D) VP1 pocket factor and (E) VP4 N-terminal domain densities filled by myristic acid molecules shown as stick diagrams and colour-coded as previously, fitted into the 2.2 Å resolution cryo-EM density map shown as grey mesh. Residues are indicated and coloured by heteroatom.

Article Snippet: The EV-E gRNA sequence (NCBI, NC_001859) was supplied by Genscript Biotech (Netherlands) in a pUC57 plasmid vector, which encompasses ampicillin-resistance.

Techniques: Cryo-EM Sample Prep