atcc 17 978 wt strain  (ATCC)

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Levels of TMEFF1 mRNA, as determined by RT–qPCR, in various human cell lines or primary cells. b , TMEFF1 mRNA levels were determined by RT–qPCR in cortical neurons from control and TMEFF1 -KO hPSCs. c , TMEFF1 protein expression was studied by confocal microscopy on cortical neurons derived from healthy control and TMEFF1 -KO hPSCs. Cells were fixed and stained for TMEFF1 (anti-TMEFF1 antibody, green), cell membrane (wheat germ agglutinin (WGA), white) and chromosomes (DAPI, blue). Scale bar, 10 μm. d , Cortical neurons derived from hPSCs from healthy controls, TMEFF1 -KO hPSCs and TLR3 −/− hPSCs were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated. TCID 50 , 50% tissue culture infectious dose. e , TMEFF1 mRNA levels were determined by RT–qPCR on hPSC-derived cortical neurons for healthy controls and the two patients with TMEFF1 mutations (P1 and P2). f , Relative abundance of TMEFF1 cDNA isoforms generated from mRNA extracted from hPSC-derived cortical neurons for healthy controls and P2, as assessed by TOPO-TA cloning. g , h , hPSC-derived cortical neurons from a healthy control (H9), the patients with TMEFF1 mutations (P1 and P2) and other TLR3 −/− and IFNAR1 −/− HSE patients were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated, without ( g ) or with ( h ) IFNβ pretreatment for 18 h. The data shown in a , b , d , e , g and h are mean ± s.e.m. of three independent experiments. Statistical analysis: for b and e , two-tailed Mann-Whitney U -tests; for d , g and h , mean log-transformed relative values were compared between control cells and TMEFF1 -mutated cells in one-way analysis of variance (ANOVA) with Tukey tests for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Quantitative RT-PCR, Control, Expressing, Confocal Microscopy, Derivative Assay, Staining, Membrane, Infection, Generated, TA Cloning, Two Tailed Test, MANN-WHITNEY, Transformation Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 mRNA levels were determined by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls treated with poly(I:C) for 2 or 4 h or left untreated (NS). b , TMEFF1 mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls treated with IFN-α2b for 8 h or left untreated. c , TMEFF1 mRNA levels were measured by RT-qPCR in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from a TLR3 −/− HSE patient, after treatment with poly(I:C) for 6 h, or without treatment. d , TMEFF1 mRNA levels were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from an IFNAR1 −/− HSE patient treated with IFN-β for 8 h or left untreated. In a - d , two probes, targeting exons 1-2 (upper panels) and exons 9-10 (lower panels) of TMEFF1 were used. The data shown are the means ± SEM from three ( a , b ) or two ( c , d ) independent experiments. e , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy control neurons (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8), TLR3 −/− ( n = 2) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons treated with poly(I:C) or IFN-α2b, or left unstimulated (NS). Data are presented as mean ± SD. f , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy controls (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons infected with HSV-1 for 24 h, or left unstimulated (NS). g , IFNB1 (upper panel) or IFNL1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls, after treatment with poly(I:C) for 2 or 4 h or without treatment. h , MX1 (upper panel) or IFIT1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls, after treatment with IFN-α2b for 8 h, or without treatment. The data shown in g , and h are the means ± SEM from three independent experiments. i , Basal levels of IFNAR1 (top panel), IFNAR2 (middle panel), and TLR3 (lower panel) mRNA were measured by RT-qPCR, in hPSC-derived cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1), TMEFF1 KO hPSCs, or hPSCs from TMEFF1-mutated patients. j , Levels of MX1 (upper panels) or IFIT1 (lower panels) mRNA were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, hPSCs from a TLR3 −/− HSE patient, and an IFNAR1 −/− HSE patient, with and without treatment with poly(I:C) for 6 h (left panels), or with IFN-β for 8 h (right panels). Statistical analysis was performed with two-tailed Mann-Whitney U tests. ns: not significant. k , Scatterplots of the mean log 2 fold-changes in RNAseq-quantified gene induction following stimulation with 100 IU/ml IFN-β for 8 h (upper panel) or HSV-1 (MOI 1) for 24 h (lower panel) in hPSC-derived CNS cortical neurons from two healthy controls (Ctrl1-H9, Ctrl2 parental-BJ1), TMEFF1-mutated patients (TMEFF1 Pts) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient. Each point represents a single gene. Genes with an absolute fold-change in expression > 2 in response to IFN-β or HSV-1 treatment relative to NS samples in the Ctrl group are plotted. l , Heatmaps of RNA-Seq-quantified gene expression (z-score-scaled DESeq2 vst-normalization) in hPSC-derived CNS cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient, a TLR3 −/− HSE patient and TMEFF1-mutated P1 and P2 (TMEFF1 Pts), not stimulated (NS), stimulated with HSV-1 for 24 h, or stimulated with IFN-β for 8 h. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses. The heatmap includes genes with a relative fold-change in expression > 2 in response to HSV-1 or IFN-β treatment relative to NS samples in the Ctrl group.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Quantitative RT-PCR, Transformation Assay, Derivative Assay, Control, Infection, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Sequencing Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , RFP intensity in HeLa and HEK293T cells, as measured in an HSV-1-RFP nuclear translocation reporter assay 10 h after infection with an RFP-reporter HSV-1, on parental WT cells or IFNAR1 KO cells transfected with an empty vector (EV) or WT TMEFF1 expression construct. HeLa or HEK293T cells were labeled with an anti-TMEFF1 antibody. RFP intensity was assessed under a confocal microscope. Statistical analysis was performed with two-tailed Mann-Whitney U tests. *** p -value < 0.001. The data shown are representative of three independent experiments. b , TMEFF1 mRNA levels in HeLa (left panel) and HEK293T cells (right panel), either parental WT (top) or IFNAR1 KO (bottom), transfected with an EV or a WT TMEFF1 expression plasmid, as measured by RT-qPCR. A probe targeting exons 1-2 of TMEFF1 was used. c , TMEFF1 mRNA levels in HeLa cells transfected with an EV, or a WT or mutant TMEFF1 expression plasmid, as measured by RT-qPCR. A probe targeting exons 1-2 of TMEFF1 was used. d , Representative images of HeLa cells transfected with an EV, or a WT or mutant TMEFF1 expression plasmid, in an HSV-1-RFP nuclear translocation reporter assay. The cells were fixed and identified by DAPI staining (blue). HSV-1-RFP infection results in the expression of RFP, the levels of which were assessed in the nucleus at 10 hpi. e , TMEFF1 mRNA levels in HeLa cells transfected with an EV, or a WT full-length (FL) TMEFF1 or different domains of TMEFF1 in an expression plasmid (EX: extracellular domain; TM + IN: transmembrane and intracellular domains), as measured by RT-qPCR. Two probes, targeting exons 1-2 (upper panel) and exons 9-10 (lower panel) of TMEFF1 , were used. The data shown are representative of three independent experiments. f , TMEFF1 protein levels, as assessed by western blotting in HeLa cells transfected with an EV, or a WT FL or different domains of TMEFF1 in an expression plasmid. g , TMEFF1 immunostaining in HeLa cells transfected with an EV, or a WT FL or different domains of TMEFF1 in an expression plasmid. HeLa cells were labeled with anti-TMEFF1 antibody (green) and DAPI (blue) and TMEFF1 overexpression was assessed under a confocal microscope. h , TMEFF1 mRNA levels in HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs, as measured by RT-qPCR. Two probes, targeting exons 1-2 (upper panel) and exons 9-10 (lower panel) of TMEFF1 , were used. i , TMEFF1 immunostaining in HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs. HeLa cells were labeled with anti-TMEFF1 antibody (green), membrane stain (MemBrite, white), and DAPI (blue) and TMEFF1 overexpression was assessed under a confocal microscope. The data shown in c - i are representative of three independent experiments. j , TMEFF1 protein levels, as assessed by western blotting on HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs. k , Measurement of RFP intensity in an HSV-1-RFP nuclear translocation reporter assay, 10 h after infection, in HeLa cells cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, I344V) TMEFF1 cDNAs. HeLa cells were labeled with anti-TMEFF1 antibody and DAPI. RFP intensity was assessed under a confocal microscope. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ns: not significant, * p -value < 0.05; **** p -value < 0.0001. The data shown in j - k are representative of three independent experiments.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Translocation Assay, Reporter Assay, Infection, Transfection, Plasmid Preparation, Expressing, Construct, Labeling, Microscopy, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Mutagenesis, Staining, Western Blot, Immunostaining, Over Expression, Membrane

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , b , Measurement of HSV-1–RFP intensity in HeLa cells overexpressing an EV, WT or various patient-specific TMEFF1 mutants ( a ) or WT full-length TMEFF1 , TMEFF1 extracellular domain (EX) or transmembrane and intracellular domains (TM + IN) ( b ) at 10 h post-infection (hpi). Statistical analysis: Kruskal–Wallis tests with Dunn’s test for multiple comparisons; *** P < 0.001. a.u., arbitrary units. c , d , HEK293T cells were cotransfected with Flag-tagged NECTIN-1 and EV or WT TMEFF1 plasmids ( c ) or with WT TMEFF1 and EV or Flag-tagged NECTIN-1 plasmids ( d ), and subjected to immunoprecipitation (IP) with anti-TMEFF1 antibodies or anti-Flag antibody-conjugated agarose beads, and immunoblotting with anti-Flag or anti-TMEFF1 antibodies. e , HEK293T cells were infected with HSV-1 (MOI 1) and subjected to immunoprecipitation with mouse IgG isotype control or anti-NECTIN-1 antibody and immunoblotting with anti-NECTIN-1 or anti-TMEFF1 antibody. f , HEK293T cells were cotransfected with C-terminal Myc-tagged WT full-length or truncated (EX, TM + IN) TMEFF1 or N-terminal Myc-tagged WT full-length TMEFF1 plasmids with EV or Flag-tagged NECTIN-1 plasmids, and subjected to immunoprecipitation with anti-Myc antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. g , HEK293T cells were cotransfected with the N-terminal Flag–GFP-tagged full-length or truncated (EX, TM + IN) NECTIN-1 plasmids with N-terminal Myc-tagged TMEFF1 plasmids, and subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. h , HEK293T cells were cotransfected with the N-terminal Flag-tagged WT full-length or EX NECTIN-1 plasmids and N-terminal Myc-tagged WT full-length or EX TMEFF1 plasmids, and subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. i , HEK293T cells were cotransfected with the N-terminal Flag-tagged NECTIN-1 and EV, WT or various patient-specific mutant TMEFF1 plasmids, and subjected to immunoprecipitation with anti-TMEFF1 antibody and immunoblotting with anti-TMEFF1 or anti-Flag antibody. The data shown in a – i are representative of three independent experiments.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Infection, Immunoprecipitation, Western Blot, Control, Mutagenesis

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 and NECTIN-1 localization in HeLa cells after cotransfection or single transfection with the TMEFF1 and NECTIN-1 plasmids. Green, TMEFF1; purple, NECTIN-1; blue, DAPI; white, MemBrite. Scale bars, 20 μm. b , HeLa cells were cotransfected with CFP-tagged TMEFF1 and YFP-tagged NECTIN-1 or HVEM, and subjected to FRET imaging. Scale bars, 20 μm. CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; Ex, excitation; Em, emission. c , Bleed-through-corrected FRET at the cell surface was quantified. d – f , Histograms of the surface His-tagged gD signal ( d ), the MFI of surface gD binding ( e ) and the percentage of surface gD-positive cells ( f ) after incubation with a His-tagged gD for 150 min in WT or TMEFF1 -KO HEK293T cells stably expressing NECTIN-1. g , HEK293T cells were cotransfected with Flag-tagged gD, Myc-tagged NECTIN-1 and EV or TMEFF1 plasmids, then subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads, and immunoblotting with anti-Flag, anti-Myc and anti-TMEFF1 antibodies. The data shown in a – g are representative of three independent experiments. h, i , MFI of surface NECTIN-1 after gD treatment ( h ) or HSV-1 infection ( i ) relative to untreated cells (left) and MFI of total NECTIN-1 in the presence or absence of gD treatment or HSV-1 infection (right) on WT or TMEFF1 -KO HEK293T cells. j , HSV-1–RFP infection rates in WT and TMEFF1 -KO HEK293T cells 8 hours after infection. k , HSV-1–RFP intensity in WT and TMEFF1 -KO HEK293T cell nucleus 8 hours after infection. Data are shown as median ± interquartile range and are representative of five independent experiments. l , HSV-1–RFP infection rates in WT, TMEFF1 KO, NECTIN-1 KO and TMEFF1 -and- NECTIN-1 double-KO HEK293T cells, as assessed by flow cytometry 8 hours after infection. Data are shown as mean ± s.e.m. from three ( b , c , e and f ), six ( h and i ), five ( j ) or four ( l ) independent experiments. Statistical analysis was done for c , h , i , j , k and l using two-tailed Mann–Whitney U tests; * P < 0.05; *** P < 0.001.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Cotransfection, Transfection, Imaging, Binding Assay, Incubation, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Infection, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Representative gating strategy for HEK293T cells stably expressing NECTIN-1, not treated or treated with recombinant His-tagged HSV-1 gD for 150 min. b , Mean fluorescence intensity (MFI) of surface gD binding in NECTIN-1-positive or NECTIN-1-negative fractions of HEK293T cells stably expressing NECTIN-1, following incubation with different concentrations of His-tagged gD (gD-His-Tag) for 150 min. c , Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells incubated with recombinant His-tagged HSV-1 gD (5 µg/ml) for 150 min. The data shown are representative of three independent experiments. d , Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells infected with HSV-1 (MOI 10) for 45 min. The data shown are representative of three independent experiments. e , NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells upon gD treatment for 150 min or without treatment. f , NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells after infection with HSV-1 for 45 min, or without infection. g , Representative contour plots of the RFP signal in WT, TMEFF1 KO, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells infected with HSV-1-RFP (MOI 10) at 8 hpi. h , Basal mRNA levels for NECTIN-1 , HVEM , and PILRa , as assessed by RT-qPCR, in WT parental or TMEFF1 KO hPSC-derived cortical neurons and HEK293T cells (left panel), and WT HeLa cells (right panel). The data shown are the mean ± SEM from three independent experiments. i , MFI of N-ter YFP-tagged HVEM on the cell surface in NECTIN-1 KO and TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV), or N-ter YFP-tagged HVEM-expressing plasmid. The data shown are the mean ± SEM from four independent experiments. j , Percentage of HSV-1-positive cells, as assessed in an assay of HSV-1 translocation to the cell nucleus 10 h after infection with an RFP-reporter HSV-1, in NECTIN-1 KO or TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV) or HVEM-expressing plasmid. The data are presented as the mean ± SEM from four independent experiments. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ns: not significant.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Stable Transfection, Expressing, Recombinant, Fluorescence, Binding Assay, Incubation, Infection, Quantitative RT-PCR, Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Translocation Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Representative images of healthy control (Ctrl 2 parental-BJ1) and TMEFF1 -KO hPSC-derived cortical neurons, stained for endogenous TMEFF1 (green), NECTIN-1 (purple), chromosomes (DAPI, blue) and cell membrane (WGA, white) before and 10 h after infection (hpi) with an RFP reporter HSV-1 (red). The dashed grey line is located immediately beneath the WGA-stained cell membrane. The areas in white squares are enlarged in the image on the right. Scale bar, 10 μm. The images are representative of three independent experiments. NI, non-infected. b , Comparison of HSV-1 entry into heathy control (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 -KO hPSC-derived cortical neurons in a β-lactamase assay (449/520 nm). Data are shown as median ± interquartile range and are representative of three independent experiments. c , Representative images of hPSC-derived cortical neurons in an HSV-1–RFP cell nuclear translocation reporter assay 10 h after infection. Neurons were identified by staining for microtubule-associated protein 2 (MAP2, green) and chromosomes (DAPI, blue). Scale bar, 10 μm. d , e , Percentage of HSV-1-positive ( d ) and cell nuclear RFP intensity ( e ) of healthy control and TMEFF1 -KO hPSC-derived cortical neurons 10 h after infection. f , Comparison of HSV-1 entry into healthy control (H9), IFNAR1 −/− , P1 and P2 hPSC-derived cortical neurons in the β-lactamase assay. g , h , Percentage of HSV-1-positive ( g ) and cell nuclear RFP intensity ( h ) of healthy control, IFNAR1 −/− , P1 and P2 hPSC-derived cortical neurons 10 h after infection. i , Percentage of HSV-1-positive TMEFF1 -KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific TMEFF1 variant cDNA 10 h after infection. Data are shown as mean ± s.e.m. ( d , g and i ) or median ± interquartile range ( e , f and h ) from four ( d and e ) or three ( f , g , h and i ) independent experiments. Statistical analysis was done for d , g and i using two-tailed Mann–Whitney U tests, and for b , e , f and h using Kruskal–Wallis tests with Dunn’s test for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Control, Derivative Assay, Staining, Membrane, Infection, Comparison, Lactamase Assay, Translocation Assay, Reporter Assay, Transduction, Variant Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Immunostaining of hPSC-derived cortical neurons from healthy control (H9) and TMEFF1 KO hPSCs (TMEFF1 KO #2) for endogenous TMEFF1 (green), NECTIN-1 (purple), AT-rich DNA (DAPI, blue) and the cell membrane (WGA, white) before and 10 h after infection with an RFP-reporter HSV-1 (red). The dashed white line is immediately beneath the WGA-stained cell membrane. b , Abundance of NECTIN1 mRNA, as assessed by RNAseq, in WT controls (Ctrl1-H9, Ctrl2 parental-BJ1) and TMEFF1 KO hPSC-derived cortical neurons infected with HSV-1 for 24 h or left unstimulated (NS). c , Immunostaining of hPSC-derived cortical neurons from a healthy control (Ctrl1-H9), an IFNAR1 −/− patient, and P1 and P2 with TMEFF1 mutations, in a reporter assay for the nuclear translocation of HSV-1, 10 h after infection with an RFP-reporter HSV-1. Neurons were fixed and identified by staining for a neuron-specific microtubule-associated protein 2 (MAP2, green) and with DAPI (blue). HSV-1-RFP infection results in the expression of RFP, which was detected in the nucleus 10 hpi. d , Replication levels for HSV-2, measles virus (MeV), or EMCV, following infection at various time points as indicated, in hPSC-derived cortical neurons from healthy controls (Ctrl1-H9, Ctrl2 parental-BJ1), or TMEFF1 KO hPSCs. RT-qPCR was performed with the SYBR green assay, to assess HSV-2 polymerase ( Pol ), MeV nucleocapsid ( N ) or EMCV 3D gene expression indicative of viral replication levels. The data are presented as the mean ± SD and are representative of four independent experiments.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Immunostaining, Derivative Assay, Control, Membrane, Infection, Staining, Reporter Assay, Translocation Assay, Expressing, Virus, Quantitative RT-PCR, SYBR Green Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 and NECTIN1 mRNA levels, as measured by RT-qPCR, in TMEFF1 KO cortical neurons transduced with an EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentivirus. Two probes, targeting exons 1-2 (left) and exons 9-10 (center) of TMEFF1 , were used. The data shown are representative of two independent experiments. b , Immunostaining of exogenous TMEFF1 (green), endogenous NECTIN-1 (purple), AT-rich DNA (DAPI, blue) and the cell membrane (WGA, white), for hPSC-derived TMEFF1 KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentiviruses. The data shown are representative of three independent experiments. c , Measurement of RFP intensity, 10 h after infection with HSV-1-RFP, in TMEFF1 KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentiviruses. Cortical neurons were labeled with anti-TMEFF1 antibody (green) and DAPI (blue). RFP intensity was assessed under a confocal microscope. HSV-1 infection results in the expression of RFP, which is detected in the nucleus at 10 hpi. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ** p -value < 0.01; **** p -value < 0.0001. The data are presented as the mean ± SEM from three independent experiments.

Article Snippet: For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 10 5 cortical neurons per well were used to seed 48-well plates.

Techniques: Quantitative RT-PCR, Transduction, Mutagenesis, Expressing, Immunostaining, Membrane, Derivative Assay, Infection, Labeling, Microscopy

Journal: Virulence

Article Title: The Salmonella virulence protein PagN contributes to the advent of a hyper-replicating cytosolic bacterial population

doi: 10.1080/21505594.2024.2357670

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: Chromosomal deletion of pagN was performed in S . Typhimurium ATCC 14028 STm WT strain by the λ-Red recombinant method using primers P1-PagN and P2-PagN , as previously described [ ].

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Clone Assay