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 the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

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 , Electropherogram representation (left panels) of the CRISPR-Cas9-introduced compound-heterozygous TMEFF1 mutations confirmed by Sanger sequencing on genomic DNA from a gene-edited TMEFF1 KO hPSC line (TMEFF1 KO #1). Sequencing results for the parental line (Ctrl parental, BJ1) are also shown. The relative abundance of WT or mutated TMEFF1 cDNA generated from mRNA extracted from the control parental and TMEFF1 KO hPSCs was assessed by TOPO-TA cloning and is shown in the right panels. b , Representative images of cortical neurons from controls (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 KO hPSCs. Cells were fixed and stained with DAPI (blue) and for a neuron-specific marker, microtubule-associated protein 2 (MAP2, green). c , FOXG1 and PAX6 mRNA levels, as measured by RT-qPCR, in cortical neurons from control and TMEFF1 KO hPSCs. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 1, Fibros ctrl 2) were used as a negative control in this assay. d , Representative images of cortical neurons from control (H9) and various patient-specific hPSC lines (P1, P2, IFNAR1 −/− , TLR3 −/− ). Cells were fixed and stained with DAPI (blue), and for a neuron-specific marker MAP2 (green). e , FOXG1 and PAX6 mRNA levels were measured by RT-qPCR in cortical neurons derived from control and patient-specific hPSC lines. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 3, Fibros ctrl 4) were used as a negative control in this assay. f , Electropherogram representation of the three TMEFF1 mutant isoforms, as detected by TOPO-cloning of cDNA from P2’s hPSC-derived cortical neurons.

Article Snippet: For the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

Techniques: CRISPR, Sequencing, Generated, Control, TA Cloning, Staining, Marker, Quantitative RT-PCR, Transformation Assay, Negative Control, Derivative Assay, Mutagenesis, Cloning

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 the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

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

Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles in regulating IFN response by poly(I:C) in fish cells (A–C) DrLGP2 and HsLGP2 downregulated fish IFN and human IFN promoter activation by poly(I:C) at a high concentration of 1 μg/ml in EPC cells. EPC cells seeded in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (A, B) , or HsIFNβpro-luc (200 ng each) (C) , together with DrLGP2 or HsLGP2 at increasing doses (0, 10, 50, 100, 200 ng). 24 h later, cells were transfected with 1 μg/ml poly(I:C) for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01, *P < 0.05. (D–G) DrLGP2 and HsLGP2 switched a first positive role to a following negative one in regulating IFN response by increasing concentrations of poly(I:C) in EPC cells. EPC cells seeded overnight in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (D, E) , or HsIFNβpro-luc (F, G) , together with DrLGP2 (D, F) or HsLGP2 (E, G) (200 ng each). 24 h later, cells were transfected with poly(I:C) at increasing doses for another 24 h, followed by luciferase assays. P values were calculated using Student’s t-test. ** P <0.01. (H) Agarose electrophoresis showed the molecular weight spanning of MMW poly(I:C) and HMW poly(I:C). (I) RNA pull-down assays verified the binding of poly(I:C) to DrLGP2 and HsLGP2 in fish cells. CO cells seeded in 10 cm dishes were transfected with DrLGP2-HA, HsLGP2-HA or GFP-HA as control. 24 h later, cells were lysed. One-tenth of cell lysates were taken as input, the remaining was incubated with 100 ng biotinylated poly(I:C), followed by western blots with anti-HA antibody.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Activation Assay, Concentration Assay, Transfection, Luciferase, Electrophoresis, Molecular Weight, Binding Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles under low concentrations of poly(I:C) in mammalian cells and do so alone in fish cells. (A–E) Zebrafish and human LGP2s played antithetic roles under low concentrations of poly(I:C) in mammalian cells. HEK293T cells seeded in 24-wells plates were co-transfected with HsIFNβpro-luc (200ng), together with HsLGP2 (A–D) or DrLGP2 (E) (200 ng each). 24h later, cells were transfected again with MMW poly(I:C) [indicated as poly(I:C) in the text or all Figures] at 2 μg/ml (B, E) or at 4 ng/ml (C) , or with HMW poly(I:C) at 4 ng/ml (D) . Renilla vector (pRL-TK, 0.2 ng) was transfected as internal control. Another 24 h later, cells were collected for luciferase assays. P values were calculated using ANOVA. ** P < 0.01. (F) Overexpression of zebrafish or human LGP2s alone revealed antithetic roles in mammalian cells. EPC cells seeded in 24-well plates were transfected with DrLGP2 at the indicated increasing doses for 48 h. Or at 24 h post transfection, cells were transfected again with poly(I:C) at a high concentration of 1 μg/ml for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Transfection, Plasmid Preparation, Luciferase, Over Expression, Concentration Assay