Journal: PLoS Pathogens

Article Title: TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner

doi: 10.1371/journal.ppat.1008507

Figure Lengend Snippet: A : HIV-CRISPR screens were performed after overnight IFN induction in ZAP-KO THP-1 cells transduced with the PIKA HIV (ISG-specific) library. HIV-CRISPR cells were infected with either WT HIV-1 LAI , the HIV-1 N74D capsid mutant (HIV-1 LAI CA-N74D) or the HIV-1 P90A capsid mutant (HIV-1 LAI CA-P90A). All of these viruses are variants of an HIV-1 LAI provirus with GFP cloned in place of Nef (pBru3GFP3). B and C : Two replicates of the WT and N74D screens (C) were performed while the P90A screen (B) includes only a single replicate. At 3 days post-infection cells and viral supernatants were collected, genomic DNA and viral RNA was extracted and 20bp sgRNA cassettes amplified by PCR or RT-PCR, respectively. MAGeCK Analysis of the enrichment of 20bp sgRNA sequences in viral RNA as compared to the genomic DNA was performed to calculate a MAGeCK Gene Score. Magenta: IFN pathway genes. Cyan: Gene hits shared across screens. Green: TRIM34. Yellow: TRIM5α. Dark Blue: MxB. Gray: Non-Targeting Controls (NTCs). X-Axis: inverse log MAGeCK Gene Score (-log 10 MAGeCK Gene Score) for the WT HIV-1 screen. Y-Axis: inverse log MAGeCK Gene Score (-log 10 MAGeCK Gene Score) for the HIV-1 P90A CA mutant screen (B) or the HIV-1 N74D CA mutant screen (C). The Top 200 Gene hits for each capsid mutant virus (CA-P90A or CA-N74D) along with corresponding scores for the WT virus are shown (the same data is used for WT in both B and C). D : A schematic of the human TRIM5α and human TRIM34 domain structures, including the RING, B-Box, Coiled-Coil and B30.2 PRY/SPRY domains. An alignment of the v1 region (yellow star) of the SPRY domains of human TRIM5α and human TRIM34 is shown below the protein schematic. Identical residues are shown with an asterisk, similarity is indicated with a colon. Gray shading indicates residues in the v1 region identified to be evolving under positive selection in primate TRIM5 by Sawyer et al .

Article Snippet: Multiplexed Gene Knockout Kits targeting TRIM34 and TRIM5 were purchased from Synthego.

Techniques: CRISPR, Transduction, Infection, Mutagenesis, Clone Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Virus, Selection

Journal: PLoS Pathogens

Article Title: TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner

doi: 10.1371/journal.ppat.1008507

Figure Lengend Snippet: A and B: THP-1 cells were transduced with a lentiviral vector encoding Cas9 and an sgRNA targeting either TRIM34 (Gray Bars: n = 2 pools, each an independent sgRNA) or a Non-Targeting Control (NTC) sgRNA (White Bars: n = 2 pools, each an independent sgRNA). TRIM34 KO in each cell pool was determined by ICE Analysis (TRIM34-KO_1 = 63% ICE KO-Score, TRIM34_2 = 83% ICE KO-Score). Edited cells were infected with either WT or N74D HIV and the amount of HIV replication assayed by analysis of GFP+ cells 2 days post-infection after overnight IFN treatment (A) or in untreated cells (no IFN) (B). P values were calculated by combining all replicates from triplicate infections in both edited populations for either the NTC control or TRIM34 KO. The fold increase in infection in the TRIM34-KO cells as compared to the NTC cells is shown for each virus in each condition. Data are represented as the mean +/- s.d. C: An HIV-CRISPR screen (with the ISG-enriched PIKA HIV library) was performed with a single replicate in THP-1 cells without any IFN treatment for the N74D capsid mutant virus. MAGeCK Gene Scores (-log 10 MAGeCK Gene Scores) for the Top 10 gene hits are shown. Black bar: TRIM34. Gray bar: Non-Targeting Control (NTC). White bars: other gene hits. D: Primary, activated CD4+ T cells were electroporated with Cas9-RNP complexes targeting TRIM34 (gray bars) or control complexes (white bars: control). TRIM34 KO in this pool was determined to be edited at 54% (ICE KO-Score). 2 days post-electroporation edited CD4+ T cell pools were infected with GFP reporter HIV-1 viruses (WT or N74D) and infection levels assayed 2 days later by flow cytometry. Data are represented as the mean +/- s.d. from triplicate infections. E: THP-1 cells were transduced with a lentiviral vector (pHIV-dTomato) encoding TRIM34 (gray bars) or empty vector (white bars: control). Cell populations were sorted for dTomato expression and, following recovery, were infected with VSV-G pseudotyped WT or N74D Luciferase reporter viruses (HIV-1 LAI CA-WT LUC or HIV-1 LAI CA-N74D LUC). Two days later levels of infection were assayed through a Luciferase Assay. Data are represented as the mean +/- s.d. from duplicate infections. F: TRIM34-overexpressing cells (gray bars) or control cells (white bars) were infected with VSV/G-pseudotyped HIV-1 LAI CA-WT LUC or HIV-1 LAI CA-N74D LUC viruses with or without Nevirapine (NVP) to inhibit HIV reverse transcription. 16 hours later viral cDNA was collected and levels of HIV reverse transcription products were assayed by qPCR. Data are represented as the mean +/- s.d. from triplicate (no NVP) or duplicate (+NVP) infections. P values were determined using two-sided unpaired t -tests (ns = not significant, *P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Multiplexed Gene Knockout Kits targeting TRIM34 and TRIM5 were purchased from Synthego.

Techniques: Transduction, Plasmid Preparation, Control, Infection, Virus, CRISPR, Mutagenesis, Electroporation, Flow Cytometry, Expressing, Luciferase, Reverse Transcription

Journal: PLoS Pathogens

Article Title: TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner

doi: 10.1371/journal.ppat.1008507

Figure Lengend Snippet: A: THP-1 cells stably-overexpressing TRIM34 (gray bars) or control cells (white bars) were infected with WT, N74D or A77V HIV-1 and levels of infection assayed 2 days post-infection by flow cytometry. The relative infection is normalized to the average infection in the control cells for each virus. Data are represented as the mean+/- s.d. from triplicate infections. B: Primary, activated CD4+ T cells were electroporated with Cas9-RNP complexes targeting TRIM34 (gray bars: TRIM34-KO) or Non-Targeting Control crRNAs (white bars: NTC). 2 days later edited CD4+ T cell pools were infected with GFP reporter HIV viruses (WT, N74D or A77V) and infection levels assayed 2 days later by flow cytometry. Data is shown as infection levels relative to the Non-Targeting Control infections for each virus (relative infection). TRIM34-KO pool edited at 52% (ICE KO-score). Data are represented as the mean +/- s.d. from triplicate infections. C: THP-1 cells stably-overexpressing TRIM34 (gray bars) or control cells (white bars) were infected with WT, N74D or N57A HIV-1 and levels of infection assayed 2 days post-infection by flow cytometry. The relative infection is normalized to the average infection in the control cells for each virus. Data are represented as the mean+/- s.d. from triplicate infections. D and E: THP-1 cells were transduced with lentiviral vectors encoding rhesus TRIM5α (gray diamonds), human TRIM5α (gray circles), human TRIM34 (black squares) or a control vector (white squares). Each cell pool was infected with VSV-G pseudotyped SIVmacLUC (D) or SIVagmLUC (E) Luciferase reporter viruses at 3 viral doses as indicated. Levels of infectivity were assayed 2 days later by luciferase assay (RLU = Relative Luciferase Units). Data are represented as the mean +/- s.d. from triplicate infections. F and G: THP-1 cells stably-overexpressing rhesus macaque TRIM34 (mac TRIM34—gray bars) or control cells (white bars) were infected with WT (F) or N74D (G) HIV-1 and levels of infection assayed 2 days post-infection by luciferase assay. Data are represented as the mean+/- s.d. from triplicate infections. P values were determined using two-sided unpaired t -tests (ns = not significant, *P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Multiplexed Gene Knockout Kits targeting TRIM34 and TRIM5 were purchased from Synthego.

Techniques: Stable Transfection, Control, Infection, Flow Cytometry, Virus, Transduction, Plasmid Preparation, Luciferase

Journal: PLoS Pathogens

Article Title: TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner

doi: 10.1371/journal.ppat.1008507

Figure Lengend Snippet: A and B: THP-1 cells stably-overexpressing TRIM34 (gray bars) or control cells (white bars) were transduced with sgRNA-encoding lentiviral vectors targeting TRIM5α (TRIM5KO) or a control. TRIM5 alleles were edited at 70% for both control and TRIM34-overexpressing cells as determined by ICE analysis. Cell pools were infected with the N74D-LUC virus (A) or WT-LUC virus (B) and levels of infection assayed 2 days post-infection by luciferase assay (Relative Luciferase Units). Data are represented as the mean +/- s.d. from triplicate infections. C: Primary, activated CD4+ T cells were electroporated with Cas9-RNP complexes targeting TRIM34 (gray bars), TRIM5α (hatched, dark gray bars) or NTC crRNAs (white bars). 2 days later edited CD4+ T cell pools were infected with GFP reporter HIV-1 viruses (WT, N74D or P90A) and infection levels assayed 2 days later by flow cytometry. TRIM34-KO pools were edited at 69% and TRIM5-KO pools were edited at 87% (ICE KO-Scores). The relative infection is normalized to the average infection in the control cells for each virus. Data are represented as the mean +/- s.d. from triplicate infections. D, E and F: Monocyte-derived dendritic cells were simultaneously transduced with a lentiviral vector encoding shRNAs targeting TRIM34 or luciferase control (Luc) and the other shRNA vector specific for TRIM5α or Luc, for the knockdown as indicated. The pooled cells were challenged with VSV-G pseudotyped HIV-1 vectors expressing GFP reporter and containing CA-N74D (D), WT CA (E), or CA-P90A (F), across a range of viral inputs normalized for RT activity (RTU: Reverse Transcriptase Units/mL). The percentage of GFP-positive cells was determined 2 days later by flow cytometry. P values were determined using two-sided unpaired t -tests (ns = not significant, *P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Multiplexed Gene Knockout Kits targeting TRIM34 and TRIM5 were purchased from Synthego.

Techniques: Stable Transfection, Control, Transduction, Infection, Virus, Luciferase, Flow Cytometry, Derivative Assay, Plasmid Preparation, shRNA, Knockdown, Expressing, Activity Assay, Reverse Transcription

Journal: PLoS Pathogens

Article Title: TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner

doi: 10.1371/journal.ppat.1008507

Figure Lengend Snippet: HeLa cells were transduced to express YFP-TRIM5α (green) and HA-TRIM34 stably. They were plated on coverslips and synchronously infected with VSV-G pseudotyped HIV-1 with WT or N74D viruses as indicated. At 2 hpi, cells were fixed and stained for viral capsid protein p24 gag + (blue) and HA-TRIM34 tag (red). Images from 15–20 cells were collected per condition in three independent biological replicates. A: Representative images for mock-infected cells (top row), WT-infected cells (middle row), and N74D-infected cells (bottom row). White triangles indicate colocalization of TRIM34 and TRIM5α in the zoomed in images for each channel. Triple colocalization between TRIM34, TRIM5α and p24 gag + are indicated by arrowheads in the zoomed in images for each channel. B: Quantification of percent p24 gag + colocalizing with both TRIM34 and TRIM5α for the WT and N74D virus. Error bars represent s.e.m. of all events collected across three biological replicates for each condition. P value was determined by a two-sided unpaired t -test.

Article Snippet: Multiplexed Gene Knockout Kits targeting TRIM34 and TRIM5 were purchased from Synthego.

Techniques: Stable Transfection, Infection, Staining, Virus

Journal: Viruses

Article Title: The Disassociation of A3G-Related HIV-1 cDNA G-to-A Hypermutation to Viral Infectivity

doi: 10.3390/v16050728

Figure Lengend Snippet: UNG2 knockout increases G-to-A hypermutation frequency without altering viral infectivity. The CRISPR Cas9 method was used to knockout the UNG2 gene in Sup-T1 and H9 cells. Synthego ICE was used to estimate the knockout score for Sup-T1 ( A ) and H9 ( D ). The knockout efficiency of UNG2 protein expression was analyzed via Western blot ( B , E ). The IIIB/H9 virus was used to infect Sup-T1 and Sup-T1ΔUNG2, or H9 and H9ΔUNG2 cells, and the G-to-A hypermutation frequency was analyzed ( C , F ). A Student’s t -test showed significant changes in hypermutation frequency between Sup-T1 and Sup-T1ΔUNG2 cells ( p = 0.0003) ( C ) and H9 and H9ΔUNG2 cells ( p = 0.005) ( F ). The infectivity of the IIIB virus harvested from H9 and H9ΔUNG was analyzed via MAGI assay ( G ). A Student’s t -test showed no significant difference in infectivity between the two viruses ( p = 0.1857). The results shown are representative of three independent experiments.

Article Snippet: The guide RNA sequences were submitted to Synthego to synthesize sgRNA Kits for A3G and UNG2 knockout.

Techniques: Knock-Out, Infection, CRISPR, Expressing, Western Blot, Virus