Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 2. Characterization of CD32-driven trogocytosis (A) 293T cells transiently expressing C-terminal GFP fusion proteins of FcgRs CD32A, CD32B, or CD32C or, as a control, the nucleocytoplasmic dNTPase SAMHD1 served as donors in co-cultures with CellTrace dye-stained SupT1 T target cells. All culture media contained IgG-depleted FCS. Shown are repre- sentative flow cytometry dot plots and the percentages of CD32+ and GFP+ target T cells. One experiment out of two is shown. (B) Schematic of topology determination of transferred CD32-GFP (top). Bottom: SupT1 T cells were co-cultured as described in (A) and stained with either an anti-GFP mAb or an isotype control antibody, both conjugated to Alexa 647, with or without prior cell permeabilization. One representative experiment is shown (n = 3). The illustration was created with BioRender.com. (C) 293T cells were co-transfected with plasmids encoding C-terminal GFP fusion proteins of CD32A, CD32B, or CD32C or, as a control, histone H2B-GFP, together with a plasmid encoding CCR5. After 2 days, cells were either left untreated or pre-treated with an anti-CD32 Ab or an isotype control Ab prior to co- cultivation with SupT1 T cells. One day later, the expression of GFP and CCR5 on the target T cells was determined by flow cytometry. Mean ± SEM are shown (n = 3). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). (D) Half-life of CD32 and CCR5 surface expression on SupT1 target cells following co-culture as in (A). Following 1 day of co-culture, SupT1 T cells positive for CD32-GFP were sorted by flow cytometry and kept in culture for an additional 9 days. The expression of CD32 (top) or CCR5 (bottom) on sorted cells was determined for up to 192 h of cultivation. One representative experiment is shown (n = 2). (E) Schematic of CD32B with important amino acids and motifs indicated. (F) Transfer of the indicated CD32B mutants, CD32A WT, CD32C WT, or H2B (GFP fusion proteins), assessed as in (A) (mean ± SEM; n = 4). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett). (G) Visualization of the material transfer from CD32B-GFP expressing 293T cells to LifeAct-mCherry-expressing SupT1 using live-cell imaging. 293T cells transiently expressing CD32B-GFP (green) were co-cultured with LifeAct-mCherry-expressing SupT1 cells (magenta), cultivated in IgG-depleted FCS and boosted with PGT151 antibody, and imaged using spinning disc microscopy for 4 h. The left panel shows the beginning of co-culture. (a) Labels the area with the first transfer event (middle panel). (b) Labels the area of the second transfer event (right panel). Dashed white box marks the area that is zoomed and depicted with individual time points before and after the transfer event (shown below). The time stamp (upper right corner, relative to the time frame which shows the transfer event (time 00:00) in zoom-ins). Scale bar, 10 mm. *p % 0.05; **p % 0.01; ***p % 0.001.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Expressing, Control, Staining, Cytometry, Cell Culture, Transfection, Plasmid Preparation, Comparison, Co-Culture Assay, Live Cell Imaging, Microscopy

Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 3. CD32-driven trogocytosis is boosted by T cell-autoreactive antibodies associated with chronic HIV-1 infection (A) CD32 expression on CD4 T cells from peripheral blood of healthy donors (HD) (n = 23) and chronic HIV-1 infected patients (CHI) (n = 39). Median with 95% CI are shown. Asterisks indicate statistical significance by Mann-Whitney test. (B) 293T cells transiently co-expressing CD32B-GFP and CCR5 were pre-treated with the indicated patient sera before 1 day of co-culture with SupT1 T cells. Shown are the percentage of CD32B-GFP+ and CCR5+ target cells (median with 95% CI, each dot represents a different patient; see also Figure S7C). CHI, chronic HIV-1 infection; ART, anti-retroviral therapy; AHI, acute HIV-1 infection. Fiebig stages II-III of acute HIV-1 infection42; HIV-2, HIV type 2; HTLV-1, human T cell lymphotropic virus type 1; HCV, hepatitis C virus; DENV, dengue virus; YFV, yellow fever virus-vaccinated; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2; EC, Echinococcus multilocularis; SCH, Schistosoma spp.; TB, Mycobacterium tuberculosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; CG, cryoglobulinemia. Asterisks indicate statistical significance by Mann-Whitney test. (C) Percentage of GFP+ target cells after 1 day of co-culture with 293T cells as in (B). IgG was depleted from the sera of two healthy donor (HD) and two HIV-1 patient (CHI) samples from (B, pink and red) and input (original sera), flowthrough and eluate of the IgG depletion were used for pre-treatment of cells prior to co- culture. Mean of two donors from each category is shown. (D) Correlation of antibody binding to SupT1 T cells and CD32B-GFP trogocytosis as in (B), with sera from HIV-1 patients. P, Pearson correlation coefficient. (E) Binding of sera with high or low trogocytotic activity (pink and red dots in B) to primary CD4 T cells as detected with fluorochrome-coupled anti-human IgG Ab (median with 95% CI, CD4 T cells; n = 3). Kruskal-Wallis test with Dunn’s multiple-testing correction. (F) A panel of bNAbs was analyzed for binding to uninfected resting CD4 T cells (top) or activated CD4 T cells (bottom). Mean ± SEM; n = 3. Asterisks indicate statistical significance by one-way ANOVA (top) or three-way ANOVA (bottom). p values were corrected for multiple comparison (Dunnett). (G) Purified, CMV-encoded, soluble Fc-binding proteins gp34 and gp68, or control proteins gp34 non-binding mutant (mtrp; W65F) and soluble ICOSL (inducible T cell co-stimulator ligand) were added to 293T donor cells as in (A), in the presence of PGT151 Ab, and subsequently co-cultured with SupT1 T cells. CD32 transfer was evaluated as in (B). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). *p % 0.05, **p % 0.01, ***p % 0.001; n.s., not significant. (H) Schematic of the determinants of antibodies for trogocytosis enhancement.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Infection, Expressing, MANN-WHITNEY, Co-Culture Assay, Retroviral, Virus, Binding Assay, Activity Assay, Comparison, Control, Mutagenesis, Cell Culture

Journal: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: Mutant frequency of HIV-1 among selected cell lines a

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Mutagenesis

Journal: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: Proportion of HIV-1 proviral sequences recovered per infected cell line analyzed possessing multiple G-to-A mutations in the HSA mutation target gene a

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Infection, Mutagenesis

Journal: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: G-to-A mutational load and mutation location in HIV-1 proviruses with multiple G-to-A mutations. (A) G-to-A mutational load in HIV-1 proviruses from CEM-GFP, U373-MAGI, and 293T cells. Each provirus with a mutant HSA sequence containing with multiple G-to-A mutations is indicated by a black circle (CEM-GFP), black square (MAGI), or black triangle (293T). The average G-to-A mutational load and standard deviation are indicated. (B) Location of G-to-A mutations in recovered proviruses harboring multiple G-to-A mutations. The red, green, and blue circles above G residues indicate the locations of G-to-A mutations (one circle per mutation identified) in proviruses recovered from CEM-GFP, U373-MAGI, and 293T, respectively. The start and stop codons of the HSA gene are identified by black rectangular boxes. (C) Quantitative RT-PCR was performed to determine the relative levels of APOBEC3 mRNA expression among the cell lines under investigation (i.e., CEM-GFP, SupT1, U373-MAGI, and 293T). The asterisk indicates that the APOBEC3C level from 293T cells was set to 1, and all other values are relative to this measurement. The mRNA expression levels were normalized to TATA-binding protein (TBP) mRNA levels. For these analyses, data with a difference in efficiencies between the APOBEC3 standard and TBP standard were ≤10% and R2 ≥0.98. Threshold cycle (CT) values were determined using the regression method, and data were analyzed by E−ΔCT. Experiments were conducted in triplicate with the standard deviation indicated.

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Mutagenesis, Sequencing, Standard Deviation, Quantitative RT-PCR, Expressing, Binding Assay

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Outline of the CRISPR/Cas9-based virus-guided discovery approach. Proviral HIV-1 constructs are engineered to contain the sgRNAs expression cassette between the nef gene and the 3’LTR. To produce virus stocks, HEK293T cells are transfected with libraries of HIV-1 constructs expressing various sgRNAs. The resulting swarms of HIV-1 sgRNA viruses are passaged every two days in Cas9-expressing cells in the presence or absence of IFN-β. Viral supernatants are harvested every five days and the frequencies of HIV-1 sgRNAs are determined by next-generation sequencing. Target genes of sgRNAs that are selected and hence are associated with an advantage for viral replication are cloned and examined for their antiviral activity and mechanism. Note that the U6-sgRNA-scaffold region is not to scale. b Enrichment of HIV-1 NL4-3 expressing sgRNAs targeting tetherin (red), GBP5 (blue) or non-targeting (NT, gray). The left panel provides a schematic showing tetherin trapping HIV-1 particles at the cell surface and inhibition of furin-mediated processing of the gp160 Env precursor to mature gp120 and gp41. The right panels show the enrichment of the indicated sgRNAs at different days post-infection in presence and absence of IFN-β. Relative enrichment was quantified using SYBR green qRT-PCR. Dots represent the mean of n = 3 ± SEM. c Flow cytometry analysis of tetherin and GBP5 expression levels in CEM-M7-Cas9 infected with HIV-1 NL4-3 expressing either Tetherin_1 (red), GBP5_2 (blue) or NT (gray) sgRNA as indicated.

Article Snippet: Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi).

Techniques: CRISPR, Virus, Construct, Expressing, Transfection, Next-Generation Sequencing, Clone Assay, Activity Assay, Inhibition, Infection, SYBR Green Assay, Quantitative RT-PCR, Flow Cytometry

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Scatter plot of individual sgRNA counts in CEM-M7-Cas9 cells supernatants 15 days post infection (red outlines) and in the input (blue outlines) versus gene names sorted by fold enrichment. NT and dotted line indicate the occurrence of the first non-targeting control sgRNA. Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi). Dashed lines indicate p value 0.05 and 2-fold change on Y and X axis, respectively. P value, negative binomial (NB) model with robust ranking aggregation (RRA). c Read counts relative to input virus from the MAGeCK analysis showing the enrichment of sgRNAs targeting GRN (red), CIITA (purple), CC2D1B (turquoise), CEACAM3 (brown), HMOX1 (blue) or EHMT2 (violet) in presence or absence of IFN-β in CEM-M7-Cas9 cells over time. NT, gray. Dots represent the mean of n = 3 ± SEM (independent experiments). d Correlation between MAGeCK score obtained in two independent experiments in CEM-M7 cells. Pearson’s correlation, r value and p -value are indicated. e Correlation between the enrichment of known RFs at the 15- and 20-day time-points in presence of IFN-β in CEM-M7-Cas9 cells. Pearson’s correlation, r value and p -value are indicated. f Venn diagram illustrating the sgRNAs that were enriched in the different conditions (e.g. different cell lines or in presence or absence of IFN-β). Genes were considered enriched when the -log 10 of the positive MAGeCK score was above or equal 1.5. g Read counts relative to input virus from the MAGeCK analysis showing the enrichment of sgRNAs targeting GRN (red), CIITA (purple), CC2D1B (turquoise), CEACAM3 (brown), HMOX1 (blue) or EHMT2 (violet) in SupT1-Cas9 cells over the course of 20 days. NT, gray. Dots represent n = 1. Error bars of the NT sample represents the mean of 11 NT sgRNAs ±SEM.

Article Snippet: Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi).

Techniques: Infection, Control, Virus

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Immunoblot showing expression of selected cellular factors in CEM-M7 and SupT1-Cas9 cells with or without the indicated IFNs (1000 U/ml). Whole-cell lysates were immunoblotted and stained with antibodies against the indicated proteins. Representative image of n = 2 (independent experiments). b Percentage of eGFP positive cells indicating infected CEM-M7-Cas9 cells at 4 days post infection electroporated with either the NT (gray) or GRN (red) sgRNA and infected with WT NL4-3. Bars represent the mean of infected cells at 2dpi relative to the control (100%) of n = 3 ± SEM (independent experiments). (lower panel) Representative WB showing GRN KO efficiency. c HEK293T cells were cotransfected with increasing amounts of GRN expression construct and proviral mutants of NL4-3 (black) or CH077 (red) lacking indicated accessory genes. Infectious virus yield was measured using the TZM-bl reporter cell infectivity. Each point represents the mean of n = 3 ± SEM (independent experiments). (lower panel) Representative WB indicating expression of Env, p55, p24 and GRN in virus supernatants or cell lysates. d HEK293T cells were transiently transfected with a luciferase reporter controlled by the HIV-1 LTR and expression constructs for GRN (red) in presence and absence of NL4-3 Tat or a vector control (black). Bars represent the mean of n = 3 ± SEM (independent experiments). e HEK293T cells were transiently transfected with different amount of GRN expression constructs with proviral constructs of NL4-3_eGFP (dark red), CH077_eGFP (red) or CH058_eGFP (light red) as indicated. At 48 h post transfection cells were analyzed by flow cytometry and mean fluorescence intensities (MFI) of eGFP in the eGFP+/GRN+ population relative to vector control (100%) quantified. Bars represent the mean of n = 3 ± SEM (independent experiments). f Representative WB and quantification of GRN KO (red) in primary CD4 + T cells. NT, gray. Bars represent the mean of n = 3 ± SD (independent donors). g CD4 + T cells were electroporated with sgRNA targeting GRN (red) or NT (gray), infected with the indicated WT HIV-1 strains and infectious virus yields determined by TZM-bl assays at 2–6 dpi. Dots represent the mean of n = 6 (NL4-3) or n = 3 (CH077) ± SEM (independent donors). b – f Student´s t test with Welch´s correction, two-sided. g Two-way Anova with Sidak´s multiple comparison. * p < 0.05, ** p < 0.001, *** p < 0.0001. Raw p values are provided in Supplementary Data .

Article Snippet: Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi).

Techniques: Western Blot, Expressing, Staining, Infection, Control, Construct, Virus, Transfection, Luciferase, Plasmid Preparation, Flow Cytometry, Fluorescence, Comparison

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Volcano plots indicating specific target genes of which the sgRNAs are enriched during passage in CEM-M7-Cas9 cells at different days post infection (dpi) after passaging of the TV- CH077-CRF-sgRNA in presence of IFN-β. Dashed lines indicate p -value 0.05 and 2-fold change on the Y and X axis, respectively. P value, negative binomial (NB) model with robust ranking aggregation (RRA). b Venn diagram illustrating the enriched sgRNAs with both viruses in presence of IFN-β. Genes were considered enriched when the MAGeCK score was equal to or above 1.5. c Read counts relative to input virus from the MAGeCK analysis showing the enrichment of sgRNAs targeting GRN (red), CIITA (purple), CEACAM3 (brown), HMOX1 (blue) or EHMT2 (violet) in absence (upper panel) or presence (lower panel) of IFN-β in CEM-M7-Cas9 cells after passaging of the CH077 library. NT, gray. Dots represent n = 1. Error bars of the NT sample represents the mean of 11 NT sgRNAs ±SEM. d Read counts relative to input virus from the MAGeCK analysis showing the enrichment of sgRNAs targeting CC2D1B (turquoise) after passaging the NL4-3 (left) or CH077 (right) library on CEM-M7-Cas9 cells in the presence of IFN-β. NT, gray. Dots represent n = 1. Error bars of the NT sample represents the mean of 11 NT sgRNAs ±SEM. e Read counts relative to input virus from the MAGeCK analysis showing the enrichment of sgRNAs targeting RHOA (green) after passaging the NL4-3 (left) or CH077 (right) library on CEM-M7-Cas9 cells in the presence of IFN-β. NT, gray. Dots represent n = 1. Error bars of the NT sample represents the mean of 11 NT sgRNAs ±SEM.

Article Snippet: Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi).

Techniques: Infection, Passaging, Virus

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Schematic representation of the nef -defective HIV-1 TV-NL4-3-CRF-sgRNA constructs. b Venn diagram illustrating sgRNAs enriched in presence and absence of Nef. Genes were considered enriched when the MAGeCK score was equal to or above 1.5. c Volcano plot indicating sgRNAs targeting SERINC5 enriched in Δ nef kinetics compared to WT kinetic during passage in CEM-M7-Cas9 cells at 15 dpi. Dashed lines indicate p value 0.05 and 2-fold change on the Y and X axis, respectively. P value, negative binomial (NB) model with robust ranking aggregation (RRA). d Read counts relative to input virus from the MAGeCK analysis showing enrichment of individual sgRNAs targeting SERINC5 in Δ nef (blue) and WT (orange) kinetics. e Read counts relative to input virus from the MAGeCK analysis showing enrichment of individual sgRNAs targeting SERINC5 comparing Δ nef (blue) and WT (orange) kinetic at 15 dpi. f – h sgRNAs targeting IFI16 as described for SERINC5 in panels c – e , except that in enrichment in CEM-M7-Cas9 was determined in the presence of IFN-β and at 20 dpi. i HEK293T transiently transfected with increasing amounts of IFI16 expression constructs and indicated proviral WT (orange) or Nef-deficient ( nef* , blue) constructs. Infectious virus yield was measured using the TZM-bl reporter cell infectivity. Dots represents the mean of n = 3 ± SD (independent experiments). Student´s t test with Welch´s correction, two-sided, * p < 0.05, ** p < 0.001, *** p < 0.0001. Raw p values are provided in Supplementary Data .

Article Snippet: Selected factors are highlighted by colors as indicated. b Volcano plots indicating specific target genes of which the sgRNAs are significantly enriched during passage in CEM-M7 (upper) or SupT1 (lower) Cas9 cells at indicated days post infection (dpi).

Techniques: Construct, Virus, Transfection, Expressing, Infection