Journal: Cell

Article Title: Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

doi: 10.1016/j.cell.2023.03.033

Figure Lengend Snippet: (A) Schematic showing production of lentiviruses (LV) from Myomaker- and Myomerger-expressing HEK293T cells. (B) Western blot for Myomaker, Myomerger, and GAPDH on HEK293T cell lysates after various times of doxycycline treatment. (C) Western blot on concentrated viral particles for Myomaker, Myomaker, and VSV-G. (D) Western blot on Bald-LVs and Mymk+Mymg-LVs for viral components and the muscle fusogens. (E) Representative images from transmission electron microscopy on VSV-G-LVs and Mymk+Mymg-LVs. Scale bar, 100 nm. (F) Images from immunogold electron microscopy using the indicated antibodies on the pseudotyped lentiviruses. Myomaker and Myomerger antibodies were detected in the same sample using gold particles of different sizes. Scale bar, 200 nm. (G) Representative images of empty and Mymk+Mymg target cells after application of Bald GFP-encoding LVs or LVs pseudotyped with VSV-G or Myomaker and Myomerger. Nuclei were stained with Hoechst. Scale bar, 100 μm. (H) Quantification of titers for VSV-G-LVs and Mymk+Mymk-LVs prior to concentration of supernatants. Each sample is an average of 3–4 replicates from independent viral preparations. Titers were determined on Mymk+Mymg BHK21 cells. (I) Representative images from cultures of differentiated myotubes, proliferating myoblasts, and fibroblasts that were treated with Bald-LV-GFP or Mymk+Mymg-LV-GFP. Cells were stained with Phalloidin and Hoechst. Scale bar, 100 μm. GFP+ cells were quantified for each cell type and histograms are shown on the right. Data are presented as mean ± standard deviation. Statistical test used was an unpaired t-test with Welch’s correction; ***p<0.001, ****p<0.0001.

Article Snippet: Mouse anti-VSV-G , Kerafast , Cat #8G5F11.

Techniques: Expressing, Western Blot, Transmission Assay, Electron Microscopy, Staining, Concentration Assay, Standard Deviation

Journal: Cell

Article Title: Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

doi: 10.1016/j.cell.2023.03.033

Figure Lengend Snippet: (A) Top panel displays experimental design for analysis of muscle progenitors. Middle panels show representative FACS plots and quantification for α7-Integrin+ myogenic cells (y-axis) and tdTomato+ cells (x-axis) from mdx4cv; RosatdTom muscle that received Bald-LV-Cre or Mymk+Mymg-LV-Cre. Bottom panels show representative FACS plots and quantification for tdTomato+ non-myogenic interstitial cells. (B) Top panel is the experimental design using lentivirus encoding for luciferase that was pseudotyped with Bald, VSV-G, or Myomaker and Myomerger. After cardiotoxin injury, lentiviruses were injected intramuscularly and bioluminesence measured through IVIS imaging multiple time points after viral delivery. Representative pseudocolored images show a progressive increase in bioluminescence in the muscles treated with Myomaker and Myomerger-pseudotyped virus. Bottom panel is quantification of bioluminescence for muscles transduced with lentivirus containing Myomaker and Myomerger. (C) Mdx4cv; RosatdTom mice received an intramuscular injection of Bald-LV-Cre or Mymk+Mymg-LV-Cre, then 2 weeks later some mice were injured with cardiotoxin. Representative images for tdTomato+ myofibers are shown to the right. Nuclei were stained with DAPI. Scale bar, 100 μm. Bottom, left panel shows quantification of the percentage of tdTomato+ myofibers. Data are presented as mean ± standard deviation; data in (A, B) combined from at least two independent lentiviral preparations; data in (C) is from one lentiviral preparation. Statistical tests used were (A) unpaired t-test with Welch’s correction; (B) Friedman test with Dunn’s correction for multiple comparisons; (C) Two-way ANOVA with Tukey’s correction for multiple comparisons; **p< 0.01, ****p< 0.0001.

Article Snippet: Mouse anti-VSV-G , Kerafast , Cat #8G5F11.

Techniques: Luciferase, Injection, Imaging, Muscles, Virus, Transduction, Staining, Standard Deviation

Journal: Cell

Article Title: Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

doi: 10.1016/j.cell.2023.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-VSV-G , Kerafast , Cat #8G5F11.

Techniques: Virus, Recombinant, Membrane, Plasmid Preparation, Electron Microscopy, Protease Inhibitor, Clone Assay, Modification, Software, Transmission Assay, Microscopy, Inverted Microscopy, In Vivo Imaging

Journal: bioRxiv

Article Title: Sarbecovirus RBD indels and specific residues dictating ACE2 multi-species adaptiveness

doi: 10.1101/2024.02.11.579781

Figure Lengend Snippet: a, Flow diagram illustrating the retrieval of 265 non-redundant spike sequences from non-human sarbecovirus, and three additional human sarbecoviruses. b, The RBD clade information of the 268 sarbecoviruses. c, RBM sequence logo illustrating the three high variable regions (numbering based on SARS-CoV-2). d, Phylogenetic tree based on RBD amino acids for the 268 sarbecoviruses (details in ) and multi-sequence alignment of 23 representative sarbecoviruses with four RBM indel types. e, Summary of the deleted residue numbers in Region1 and Region2 compared with SARS-CoV-2 sequence. The numbers of each deletion length are indicated in the parentheses. f, The sequence numbers of the four RBM indel types. g, Distribution of RBD clades in different indel types. h, Analysis of the reduced length of Region1 and Region2 indels in each RBM indel types. i, Structural display of the two interaction patches in the SARS-CoV-2 RBD/hACE2 complex (6M0J). Residues involved in receptor recognition are indicated in the close-up views of the two interaction patches. Region 1,2 and 3 in RBD (Region 1 and 3 comprise Patch 2, while Region 2 consists of Patch 1. j, Structure superimposing of SARS-CoV-2 RBD (6M0J) with RBDs from representative viruses belonging to each indel types. k, Schematic illustration of the VSV-based pseudovirus entry assay. l, Western Blot detecting the level of spike protein of 14 selected sarbecoviruses in lysate or supernatant, with VSV-M and β-tubulin serving as loading controls.

Article Snippet: Primary antibodies targeting HA (MBL, MBL-M180-3), β-tubulin (Immunoway, YM3030), or VSV-M (Kerafast, EB0011) were applied at a 1:10,000 dilution in TBST with 1% milk.

Techniques: Sequencing, Residue, Western Blot

Journal:

Article Title: A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding

doi:

Figure Lengend Snippet: VSV M budding assay. (A) Radiolabeled lysates from CV-1 cells receiving no DNA (mock, lane 2), T7VSVMWT DNA (MWT, lane 3), and T7VSVMY-A DNA (MY>A, lane 4) were immunoprecipitated with polyclonal antiserum against the M protein of VSV and fractioned by SDS-PAGE. The position of the M protein of VSV is indicated. MW, 14C-labeled protein standards. (B) Radiolabeled proteins released into the media covering cells transfected with no DNA (mock, lane 1), T7VSVMWT DNA (lane 2), and T7VSVMY-A DNA (lane 3) were immunoprecipitated with polyclonal antiserum against the M protein of VSV and fractionated by SDS-PAGE. The position of the M protein of VSV is indicated. (C) Radiolabeled lysates from CV-1 cells receiving no DNA (mock, lane 1), T7VSVMA4 DNA (lane 2), and T7VSVMWT DNA (lane 3) were immunoprecipitated with polyclonal antiserum raised against VSV virions (ATCC) and fractionated by SDS-PAGE. (D) Radiolabeled proteins released into the media covering cells transfected with no DNA (mock, lane 1), T7VSVMA4 (lane 2), and T7VSVMWT (lane 3) were immunoprecipitated with polyclonal antiserum raised against VSV virions (ATCC) and fractionated by SDS-PAGE.

Article Snippet: The primary antibody was polyclonal anti-VSV M, while the secondary antibody was affinity-purified goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (Boehringer Mannheim Corporation).

Techniques: Immunoprecipitation, SDS Page, Labeling, Transfection

Journal:

Article Title: A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding

doi:

Figure Lengend Snippet: Indirect immunofluorescence and confocal microscopy of transfected CV-1 cells. (A) CV-1 cells expressing wild-type VSV M protein at 8 h posttransfection. (B) CV-1 cells expressing the VSV M protein containing a tyrosine (Y) to alanine (A) mutation within the PY motif at 8 h posttransfection. (C) Untransfected CV-1 cells. Primary polyclonal antiserum (identical to that used in the experiment shown in Fig. ​Fig.7A7A and B) was directed against the M protein of VSV.

Article Snippet: The primary antibody was polyclonal anti-VSV M, while the secondary antibody was affinity-purified goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (Boehringer Mannheim Corporation).

Techniques: Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Mutagenesis