Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Photo of a Myotis yumanensis bat at a sampling site (Credit: Elise Lauterbur). B, Primary cells derived from a biopsy wing punch. C, Schematic of the workflow from bat sampling to RNA sequencing analyses. Bat primary cell lines were similarly derived from three M. yumanensis individuals and treated with, or without, universal type I IFN to trigger ISG expression. Total RNA was extracted and sequenced, followed by analyses to identify differentially expressed genes between stimulated cells compared to control cells. D, Volcano plot representing the differential gene expression between untreated and IFN-stimulated cells from three M. yumanensis individuals. Genes significantly ( p < 0.05) differentially expressed (DE) are colored in red. Several known ISGs are highlighted on the graphic. GBP5 is the most upregulated ISG. E, Expression of ISGs from panel D in M. yumanensis primary cells derived from three individuals in control (light gray) and I-IFN (dark gray) conditions. TPM, Transcripts per million counts (log10 scale; complete table in ). F, Comparative analysis of differential gene expression (Log2 Fold Change) of GBP5 upon universal type I IFN treatment M. yumanensis cells as compared to six other mammalian species cells in a similar experimental setup (; details in Materials and methods). Pictos from https://www.phylopic.org/ and Illustrations from NIAID NIH BioArt Source ( https://www.bioart.niaid.nih.gov/ ). The data underlying this Figure can be found in S1 and . See Data availability for access to the code.

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Sampling, Derivative Assay, RNA Sequencing, Expressing, Control, Gene Expression

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Phylogenetic analysis of GBP5 across mammals. The phylogeny was built from a PRANK codon alignment of 348 GBP5 homologous sequences. Maximum likelihood phylogenetic tree was built with IQ-TREE with 1000-bootstrap replicates for statistical support (see Data availability). Branches under significant positive selection ( p -value < 0.05) assigned by aBSREL are thickened and in red. The scale bar indicates the number of substitutions per site. B, Evidence of positive selection in GBP5 from several mammalian orders. Positive selection analyses were performed with BUSTED using a PRANK codon alignment for each mammalian order. OWM, Old world monkeys. Species silhouettes are from https://www.phylopic.org . The data underlying this Figure can be found in , the alignments for phylogenetic analyses are all openly available in FigShare Dataset ( https://doi.org/10.6084/m9.figshare.26180785.v1 , https://doi.org/10.6084/m9.figshare.26180764.v1 , https://doi.org/10.6084/m9.figshare.26180803.v1 , https://doi.org/10.6084/m9.figshare.26180761.v1 , https://doi.org/10.6084/m9.figshare.26180767.v1 , https://doi.org/10.6084/m9.figshare.26180776.v1 , https://doi.org/10.6084/m9.figshare.26180794.v1 , https://doi.org/10.6084/m9.figshare.26180797.v1 ).

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Selection

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Phylogenetic and positive selection analyses of bat GBP5. Maximum likelihood phylogenetic tree of bat GBP5 was built with IQ-TREE and statistical support is from 1,000 bootstrap replicates (values are shown below branches). Branches under significant positive selection ( p -value < 0.05) assigned by aBSREL are in red and the corresponding estimated values of ω are reported in panel B. The scale bar indicates the number of substitutions per site. The black arrows identify species with GBP5 genes that were functionally tested in this study. 1.1 and 1.2 identify GBP5 duplicates within a given bat species. B, Evidence of lineage-specific positive selection during bat GBP5 evolution. aBSREL identified at least six branches under significant positive selection. ω1 and ω2, estimation of ω in the rate class not allowing positive selection, and allowing positive selection (ω > 1), with % of sites in this class in parentheses, respectively. C, Sites under positive selection (PS) in bat GBP5. Site-specific positive selection analyses were performed using FUBAR , MEME , and FEL from HYPHY/Datamonkey [ , ]. Only the sites above the indicated “statistically significant cut-off” are shown (PP, posterior probabilities for FUBAR; p -value for MEME and FEL). In bold are the sites identified by several methods. Nb of PSS, number of positively selected sites. “PS sites” numbering is according to the codon numbering in the PRANK codon alignment. D, Schematic representation of GBP5 with its functional domains and the herein identified sites under positive selection (red arrows at the top). LG domain, large GTPase domain. MD, Middle domain. GED, GTPase effector domain. The size of the domains is not to scale. Residues identified by at least two positive selection methods are highlighted in bold. In blue, sites or motifs involved in known functions in human GBP5. The bat phylogenetic tree from can be found in https://doi.org/10.6084/m9.figshare.30924512 and all results from positive selection analyses in https://doi.org/10.6084/m9.figshare.30924551 .

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Selection, Functional Assay

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: TZM-bl cells were transfected with a plasmid coding for indicated HA-GBP5 species proteins: 10 bat orthologs, and 2 human GBP5s: wt and mutant C583A. Two days post-transfection, GBP5 localization was analyzed by confocal fluorescence microscopy with the indicated marker. Nuclei and trans- Golgi-network (TGN) were stained with DAPI and anti-TGN46, respectively. A, All the channels and a zoom are shown for Homo sapiens , the mutant Homo sapiens-C583A, Myotis yumanensis and Eptesicus fuscus . B, Only the merge is shown for the remaining bat species. The complete panel is shown in . The pictures present representative results observed in 3 independent experiments. Scale bar indicates 15 μm.

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Fluorescence, Microscopy, Marker, Staining

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Experimental setup. Briefly, HEK-293T cells were transfected with plasmids coding for HA-GBP5 or the control (EV “empty vector” expressing E2-Crimson), and for HIV-1 LAI genome and Luciferase reporter (Bru∂EnvLuc2 vector), NL4.3 Envelope and Rev. 48 hour post-transfection, cells were harvested for western blot analysis, and supernatant for western blot analysis after ultracentrifugation, titration of virus by RT-qPCR (RT activity), and infection of the RT-normalized viruses in HeLaP4P5 cells. Infection was quantified 72 hour later by luminescence from the viral-encoded Luciferase reporter. B, HIV-1 intrinsic infectivity (RLU, Relative light units) normalized to RT activity in the supernatant, in the context of control (EV, encoding solely E2-crimson) or a dose of HA-GBP5 (1, 2, or 4 µg; total DNA identical across conditions). The corresponding species of GBP5 is shown (name follows the UCSC nomenclature, three letters from genus followed by three letters from species). Results from three independent experiments, bars correspond to SD. Statistics vs. the corresponding control EV condition, by one-way ANOVA, Dunnett’s test: *, p value < 0.05, **, p value < 0.01. C and D , Corresponding western blot analyses of HA-GBP5, HIV-1 Env, and HIV-1 Gag, and Tubulin from the total cell lysates of the HIV-1 producer cells in the context of a GBP5 dose (constant total transfected DNA). The cladogram at the top shows the phylogenetic relationships of the tested species. The RT activity titers and a western blot of purified supernatants are in . Panel C shows one representative experiment and panel D the quantification of western blots from three independent experiments (Tubulin is in ). Illustrations from NIAID NIH BioArt Source ( https://www.bioart.niaid.nih.gov/ ). The data underlying this Figure can be found in .

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Luciferase, Western Blot, Titration, Virus, Quantitative RT-PCR, Activity Assay, Infection, Purification

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Experimental setup similar to but with viral pseudotyping with VSVg (VSV condition) or EBLV-1 envelope (EBLV-1 condition). B, Infectivity of RT-normalized pseudotyped-viruses produced in the presence of GBP5, normalized to the condition without GBP5 (EV, Empty vector E2-Crimson control) at 100%. The cladogram on the left shows the phylogenetic relationships of the tested GBP5 species. RLU, Relative light units. Results from three independent experiments, bars correspond to SD. Statistics vs. the corresponding control condition, One-way ANOVA, Dunnett’s test: *, p value < 0.05, **, p value < 0.01, ***, p value < 0,001. Illustrations from NIAID NIH BioArt Source ( https://www.bioart.niaid.nih.gov/ ). The data underlying this Figure can be found in .

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Infection, Produced, Plasmid Preparation, Control

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Ancestral state sequence reconstruction upstream of the Eptesicus fuscus- CaaX prenylation motif. C-terminal end of the protein alignment of the 10 bat GBP5s tested in functional assays (asterisk, stop codon). Phylogenetic tree was used to infer the ancestral sequence of the C-terminal region, the branch where the prenylation motif was lost by a premature stop codon is annotated on the tree. The site of mutagenesis for reconstruction is indicated by the blue arrow. B, Reconstruction of the C-ter relocalizes Eptesicus fuscus GBP5-CaaX to the trans- Golgi network (TGN). Briefly, TZM-bl cells were transfected with plasmids encoding HA-GBP5s and, 48 hour later, were analyzed by confocal fluorescence microscopy. GBP5, nuclei and TGN were stained with anti-HA, DAPI and anti-TGN46 antibodies, respectively. Scale bar indicates 15 μm. C, GBP5 mean intensity at the Golgi vs. the total cell was quantified for the wild-type eptFus and the mutant eptFus-CaaX . Each dot corresponds to one cell. Two independent replicates are identified by different dot colors. D, Pearson coefficient correlation per cell calculated between GBP5 and TGN signals for the wild-type eptFus and the mutant eptFus-CaaX. Data are represented as a mean ± SD. Statistics vs. the corresponding control condition, Nested t test: **, p -value < 0.01 ( n = 2). E–G, Ancestral reconstruction of the prenylation CaaX did not increase Eptesicus fuscus GBP5 restriction of intrinsic viral infectivity. E, Infectivity of RT-normalized HIV-1 pseudotyped-viruses in the presence of GBP5, normalized to the condition without GBP5 (EV control) at 100%. Dose of GBP5 plasmids: 1, 2, and 4 µg with constant total DNA transfected across conditions. Experimental setup as in . RLU, Relative light units. Viral titers (RT activity) are shown in . F, Corresponding western blot showing the expression of HA-GBP5, HIV-1 Env and Gag in the viral producer Tzm-bl cells with beta-actin as loading control (kDa, on the right). Quantification of three independent experiments is shown in . G, Intrinsic infectivity of (RT-normalized) VSVg or EBLV-1g pseudotyped retroviruses in the presence of GBP5s, normalized to EV control at 100%. Experimental setup as in .**, p -value < 0.01 (versus control). The data underlying this Figure can be found in .

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Sequencing, Functional Assay, Mutagenesis, Transfection, Fluorescence, Microscopy, Staining, Control, Infection, Activity Assay, Western Blot, Expressing

Journal: PLOS Biology

Article Title: Genomic and functional adaptations in the guanylate-binding protein GBP5 highlight specificities of bat antiviral innate immunity

doi: 10.1371/journal.pbio.3003760

Figure Lengend Snippet: A, Eptesicus fuscus cells were transfected with plasmids encoding HA-GBP5s and, 48 hours later, were analyzed by confocal fluorescence microscopy with anti-HA antibody. Nuclei were stained with DAPI. Of note, anti-TGN46 antibody did not cross-react in bat cells. MyoYum GBP5 was also transfected as a control of TGN subcellular localization. B and C, VSV-GFP infections of eptFus bat cells expressing or not GBP5s: total % of cell death (B) and % of VSV-GFP infected live cells as measured by flow-cytometry. Each point corresponds to an independent replicate. D, 3D protein structure prediction (AlphaFold) of the reconstructed Eptesicus fuscus - CaaX GBP5 dimer. Colored and gray chains each correspond to a monomer. Blue, GTPase domain. Green, hinge domain. Yellow, middle domain. Orange, catalytic domain. Red, residues different from Myotis yumanensis . Credit: https://www.phylopic.org/ . The data underlying this Figure can be found in .

Article Snippet: A total of 3 × 10 4 TZM-bl cells or 2.5 × 10 4 eptFus bat cells were seeded on glass coverslips and were transfected with 1 μg of GBP5 plasmid with jetPRIME (Polyplus) or Lipofectamine 3,000, respectively, according to the manufacturers’ instructions.

Techniques: Transfection, Fluorescence, Microscopy, Staining, Control, Expressing, Infection, Flow Cytometry