Journal: Nature microbiology

Article Title: Centrosomal protein TRIM43 restricts herpesvirus infection by regulating nuclear lamina integrity

doi: 10.1038/s41564-018-0285-5

Figure Lengend Snippet: a, Schematic representation of the TRIM shRNA screen in 293.rKSHV.219 cells. b, rKSHV.219 reactivation from a following lentiviral transduction of non-targeting control shRNA (sh.C) or TRIM-specific shRNAs (x axis) by analyzing RFP-positive cells using fluorescence-activated cell sorting (FACS). c, TRIM transcripts in iSLK.219 cells treated with doxycycline (1 μ g ml-1) for 3 days to induce rKSHV.219 reactivation, determined by qRT-PCR. d, Left, TRIM43 transcripts in EBV-infected AKBM or EBV-negative BJAB cells that were mock-treated or treated with 100 μ g ml-1 anti-human immunoglobulin G (IgG) for 24 h to induce EBV reactivation, determined by qRT-PCR. Right, EBV reactivation determined by analyzing BMRF1 transcripts by qRT-PCR. e, TRIM43 transcripts in human foreskin fibroblast (HFF) cells infected with HCMV (MOI of 3) for 24 h, determined by qRT-PCR. f, TRIM43 transcripts in Huh7 cells infected with VSV (MOI 0.01), DV (MOI 1) or HSV 1 (MOI 1) for 18 h, assessed by qRT-PCR. g, TRIM43 mRNA expression in BAL samples from patients with acute pulmonary infection, assessed by qRT-PCR. h, Heatmap summarizing the results from the siRNA mini-screen to test the effect of TRIM knockdown on the replication of HSV-1, Ad, EMCV and VSV (Supplementary Fig. 2d–g). i, Left, EBV BMRF1 transcripts in AGS-EBV cells transfected with non-targeting control siRNA (si.C), si.TRIM43 or si.TRIM4 (negative control), determined by qRT-PCR at 96 h post-transfection. Middle and right, knockdown of TRIM43 and TRIM4, determined by qRT-PCR at 48 h after siRNA transfection. j, EBV Zta-protein abundance of AGS-EBV cells from i, determined by immunoblot (IB) using anti-Zta at 120 h after siRNA transfection. k, HCMV IE-1 protein-positive HFF cells following transfection with si.TRIM43 or si.C and subsequent infection with HCMV (50 IE-1 units) for 24 h, determined by immunofluorescence (IF). Data represent mean and s.d. of n = 3 (biological replicates) (b,c–f,i,k), or mean and s.d. of 18 HSV-1-positive or 16 HSV-1-negative BAL samples (g). Statistical significance was calculated by unpaired two-tailed t-test (b,d,i,k), or two-tailed Mann-Whitney U test (g). *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values for b are provided in Supplementary Table 1. Results are representative of one screen (b) or three independent experiments (c–f,h–k). nd, not detectable.

Article Snippet: EMCV (strain EMC) was purchased from ATCC.

Techniques: shRNA, Transduction, Control, Fluorescence, FACS, Quantitative RT-PCR, Infection, Expressing, Knockdown, Transfection, Negative Control, Quantitative Proteomics, Western Blot, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: A . Typical structure for the genomic RNA of EMCV. The magenta double head arrow indicates the PCR target region to detect a reverse transcribed EMCV genome. B . The illustration shows a strategy of the testis culture and viral infection. A reverse-transcription of the viral genomic RNA is evaluated by PCR and electrophoresis. C . A model for the pathway from viral infection to endogenization. A genomic sequence of an RNA virus is considered to be reverse-transcribed by host-derived reverse-transcriptase. Subsequently, the cDNA might be integrated into the host genome by non-homologous end joining. D . The photographs show typical images for transverse section of seminiferous tubule in the cultured testes at 21 dpi and the uninfected control. Arrowheads indicate spermatogonia. Scale bars, 20 µm. E . The electrophoresis shows a result of PCR to detect DNA fragment that is identical to the EMCV genome. The samples were harvested from the EMCV-infected testes at 1-, 3-, 5-, 7-, 14-, and 21-dpi. Expected size of the amplicon is 315 bp. M, size marker. EI, EMCV-infected. UI, uninfected. PC, positive control (EMCV cDNA). NC, negative control (empty vector).

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Reverse Transcription, Infection, Electrophoresis, Sequencing, Virus, Derivative Assay, Non-Homologous End Joining, Cell Culture, Control, Amplification, Marker, Positive Control, Negative Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: The electrophoresis shows a result of PCR to detect DNA fragment that is identical to the EMCV genome. The samples were harvested from the EMCV-infected TERT (+/+) or TERT (−/−) littermate testes at 5- and 7-dpi. Expected size of the amplicon is 315 bp. M, size marker. NC, negative control (uninfected testis).

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Electrophoresis, Infection, Amplification, Marker, Negative Control

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: A . The illustration shows a strategy of the viral infection to live animal and a molecular hypothesis for subsequent reverse-transcription and endogenization. B . The line graph indicates a survival rate of the EMCV-infected adult male mice. All infected mice were dead within 4 dpi except lowest viral titer (1.0 × 10 −1 pfu). C . The electrophoresis shows a result of PCR to detect DNA fragment that is identical to the EMCV genome. The DNA samples were harvested from testes extracted from the male mice deceased after EMCV injection at the titer of 10 × 10 1 , 10 3 , and 10 5 pfu. Expected size of the amplicon is 315 bp. M, size marker. D . The electrophoresis shows a result of PCR to detect DNA fragment that is identical to the EMCV genome. The DNA samples were harvested from mating plugs derived from the male mice survived after EMCV injection at the titer of 10 × 10 −1 pfu. Expected size of the amplicon is 315 bp. M, size marker. NC, negative control (empty vector). PC, positive control (EMCV cDNA).

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Infection, Reverse Transcription, Electrophoresis, Injection, Amplification, Marker, Derivative Assay, Negative Control, Plasmid Preparation, Positive Control

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: The illustration shows the time course of the EMCV infection to mice and the actual timing for the important events and sampling in this study. The events described above are about the infection and mating. The others are samplings mentioned in the main text and figures.

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Infection, Sampling

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: A . The illustration shows a strategy to validate a heredity of the endogenized EMCV sequence from the infected male to the offspring. B . The line graph indicates a survival rate of F1 offspring obtained from the mating between the survived EMCV-infected male and uninfected female. Each color means the littermate derived from respective male mice. C . The electrophoresis shows a result of PCR to detect an endogenized EMCV fragment in the lethal offspring. The DNA samples were harvested from livers extracted from the F1 neonate deceased within 3 d after birth. The numbers were assigned for the parental male (#1∼#3) and littermate recognition. Expected size of the amplicon is 315 bp. M, size marker. NC, negative control (empty vector). PC, positive control (EMCV cDNA). D . The electrophoresis shows a result of PCR to detect an endogenized EMCV fragment in the survived offspring. The DNA samples were harvested from earlobes cut from the F1 offspring and their parent males. The numbers were assigned for the parental male (#1∼#4) and littermate recognition. Expected size of the amplicon is 315 bp. NC, negative control (empty vector). PC, positive control (EMCV cDNA). M, size marker.

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Sequencing, Infection, Derivative Assay, Electrophoresis, Amplification, Marker, Negative Control, Plasmid Preparation, Positive Control

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: The table shows the numerical data for survival rate of the F1 generation mice obtained from the mating between the EMCV-infected male and uninfected female. This figure is related to . Survival number was recorded every day until 15 days after the birth.

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Infection

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: The alignments illustration shows the raw sequence reads mapped to the EMCV and mouse genome. The genomic DNA extracted from the EMCV-infected testes was used for the NGS. The above read contains a small insertion in the border of the EMCV and mouse sequences. The other is considered to have undergone a precise non-homologous end joining, while an extra sequence was observed in the terminals of the read.

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Sequencing, Infection, Non-Homologous End Joining

Journal: bioRxiv

Article Title: Heritable endogenization of an RNA virus in a mammalian species

doi: 10.1101/2020.01.19.911933

Figure Lengend Snippet: A-C. The table and graphs show the body weight of the survived F1 generation mice measured at 6-weeks. The offspring of the EMCV-infected male mice tended to exhibit smaller weight than the average of the C57BL/6J strain (Male, 21.9 ± 1.8. Female, 18.5 ± 0.9.). The dotted lines in B and C indicate the averages in each sex. The data were obtained from the Jackson Laboratory website ( https://www.jax.org/jax-mice-and-services/strain-data-sheet-pages/body-weight-chart-000664 ).

Article Snippet: The EMCV strain NIID-NU1 (DDBJ, #LC508268) was propagated in Vero cells (JCRB9013) purchased from the Japanese Collection of Research Bioresources (JCRB; Ibaraki, Japan).

Techniques: Infection