Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, PCR analysis of the input HIV-1 gRNA stocks and viral variants obtained after the indicated days of passaging. The upper panel shows primary PCR data and the lower panel the position of the primer binding sites and the fragments obtained. b, Schematic structure of the HIV-1 genome and modifications at its 3′end to insert the U6-gRNA-scaffold expression cassette. Duplicated T-rich regions, poly-purine tract (PPT) and LTR sequences are highlighted in red. The arrow indicates the major recombination event. Mutations introduced to minimize recombination and the predicted Nef amino acid sequence are indicated. Numbers refer to nucleotide positions in the NL4-3 nef gene. c, PCR analysis was performed as in panel A but the optimized HIV-1 NL4-3 gRNA construct containing the changes shown in panel B were used for passaging. d, HEK293T cells were transfected with the parental HIV-1 NL4-3 construct, the original or optimized derivative containing the U6-gRNA-scaffold cassette or the gRNA library targeting 511 potential antiviral factors. Infectious virus yield was measured using the TZM-bl reporter cell infectivity assay and values were normalized to the infectious virus yield obtained for the parental NL4-3 construct (100%). Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, Representative Western blot of Nef and GAPDH expression levels in HEK293T cells transfected with the indicated HIV-1 NL4-3 constructs. f, NGS results showing the coverage of the HIV-1 NL4-3 gRNA libraries.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Passaging, Binding Assay, Expressing, Sequencing, Construct, Transfection, Virus, Infection, Western Blot

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Infected cells, infectious virus yields and p24 antigen production in CEM-M7 cells infected with the HIV-1 TC-NL4-3-CRF-gRNA library. Cells were infected and the virus passaged as indicated in . Every five days, the cell cultures were analyzed for the proportion of productively infected (p24+) cells by flow cytometry (left), infectious virus and p24 in the supernatant by TZM-bl infection assay (middle) and p24 antigen ELISA (right), respectively. b, Correlation between the enrichment of known RFs at the 15 and 20 day time-points and in the presence or absence of IFN-β in CEM-M7 Cas9 cells. c, Absolute counts of the indicated HIV-1 gRNA constructs during passage in CEM-M7 Cas9 cells. d, Correlation between MAGeCK score obtained in independent experiments in CEM-M7 vs SupT1 CCR5 high Cas9 cells.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Infection, Virus, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Construct

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Expression of cellular factors targeted by gRNAs selected during passage of HIV-1 in CEM-M7 and SupT1 CCR5 high Cas9 cells with or without the indicated IFNs (1000U/ml). Whole-cell lysates were immunoblotted and stained with antibodies against the indicated proteins. b, Percentage of eGFP positive cells indicating infected CEM-M7 Cas9 cells at 4 days post infection electroporated with either the NT or GRN gRNA and infected with WT NL4-3. Bars represent the mean of infected cells at 2dpi relative to the control (100%) of three independent experiments, ±SEM, *p<0.05, Student’s t-test Welch’s correction, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing PGRN KO efficiency. c, HEK293T cells were cotransfected with increasing amounts of GRN expression construct and proviral mutants of NL4-3 or CH077 lacking the accessory genes. Each point represents the average of three independent experiments ±SEM. In the lower panel a representative WB indicating expression of Env, p55, p24 and PGRN in virus supernatants or cell lysates. d, HEK293T cells were cotransfected with a luciferase reporter constructs under the control of the HIV-1 LTR and expression constructs for GRN in presence and absence of NL4-3 Tat or a vector control. Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, HEK293T cells were cotransfected with different amount of GRN expression plasmid with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/GRN+ population relative to vector control (100%). Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, Representative WB and quantification of PGRN KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. g, CD4 + T cells from 3 to 6 donors were electroporated with either the GRN gRNA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6dpi by TZM-bl infection assays. Values represent the mean of three to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison, *p<0.05, ** p<0.001, ***p<0.0001.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Staining, Infection, Construct, Virus, Luciferase, Plasmid Preparation, Flow Cytometry, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with increasing amount of either CIITA, CC2D1B or CEACAM3 expression constructs with and indicated proviral constructs. Values represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. b-d, CD4 + T cells from 3 to 4 donors were electroporated with either the gRNA targeting CIITA , CC2D1B , CEACAM3 or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Values represent the mean of two to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison *p<0.05, ** p<0.001, ***p<0.0001. Examples from primary data are shown in . e, Percentage of p24 antigen in the supernatants of CD4 + T cells from three to four donors electroporated with either the gRNA targeting CC2D1B or NT control at 3 days post infection with VSV-G pseudo-typed Δ env NL4-3 or CH077. Bars represent the mean of the infectious viral yield at two days post-infection relative to the control (100%) of three to four independent experiments, ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing CC2D1B KO efficiency.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Construct, Infection, Virus, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with different amount of CIITA expression plasmid with with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/AF647+ population relative to vector control (100%). Bars represent the mean of two independent experiments ±SD. b, Representative Western blot showing Env, p24 and CC2D1B in virus containing supernatants or cell lysates of HEN293Ts cotransfected with increasing amounts of CC2D1B and the indicated proviral constructs. c, d, Quantification the WB in panel (b) of the p24 release (c) and Env processing (d). Values represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, Representative western blot and quantification of CIITA, CC2D1B and CEACAM3 expression upon KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, CD4 + T cells were electroporated with gRNA targeting CIITA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Shown are representative results.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Plasmid Preparation, Infection, Flow Cytometry, Fluorescence, Western Blot, Virus, Construct

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Schematic structure of modifications at 3′end to insert the U6-gRNA-scaffold expression cassette. Duplicated T-rich regions, poly-purine tract (PPT) and LTR sequences are highlighted in red. b, Nef and GAPDH expression levels in HEK293T cells transfected with the HIV-1 NL4-3 constructs. c, Infection rate of parental HIV-1 CH077 construct, the original or optimized containing the U6-gRNA-scaffold cassette or the gRNA library. Bars represent the mean of four independent experiments ±SEM, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. d, Coverage of the HIV-1 NL4-3 gRNA libraries. e, CEM-M7 Cas9 cells infected with the HIV-1 TC-CH077-CRF-gRNA library. the cell cultures were analyzed by flow cytometry (left), infectious virus in the supernatant by TZM-bl infection assay (right), respectively.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Transfection, Construct, Infection, Flow Cytometry, Virus

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Infectious virus yields in CEM-M7 Cas9 cells infected with the TV-NL4-3-CRF-gRNA WT or Δ nef library. Cells were infected and the virus passaged as indicated in . Every five days, the cell cultures were analysed for infectious virus in the supernatant by TZM-bl infection. b, Correlation between the enrichment of genes at the 10, 15 and 20 day time-points between the WT and Δ nef kinetics. c, Read counts relative to input virus from the MAGeCK analysis showing the enrichment of gRNAs targeting GRN , CIITA , CC2D1B and IRF3 in Δ nef and WT kinetics.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Virus, Infection

Journal: Retrovirology

Article Title: The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

doi: 10.1186/1742-4690-10-29

Figure Lengend Snippet: NTD-CTD CA mutants with altered capsid stability are impaired in reverse transcription in target cells. HeLa-P4 cells were inoculated with equal quantities of WT or mutant viruses. Cultures were harvested 8 hr. after infection and DNA isolated and assayed for second-strand transfer products by qPCR. Shown are the mean values and error bars represent the standard deviations from three independent experiments.

Article Snippet: The 293T, HeLa-P4, HeLa, TZM-bl, and OMK cells used in this study were cultured in Dulbecco’s modification of Eagle’s medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS), penicillin (50 IU/ml), and streptomycin (50 μg/ml).

Techniques: Reverse Transcription, Mutagenesis, Infection, Isolation