MOESM3 of The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion

Additional file 3. Sequences of primer ODNs used in this study

MOESM2 of The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion

Additional file 2. Western blot analysis of hPML isoforms overexpressed in PML-KO MEFs

Design of the gRNA and donor ssODN for the HDR-mediated editing of <i>TRIM5</i>.

<p>(A) <i>TRIM5</i> localization on chromosome 11 (top), and Arg332-Arg335 localization in exon 8 of the gene (bottom). (B) Top panel: position of the three gRNAs (gRNA1, 9 and 19) designed to target the Arg332-Arg335 region. The two arginine codons are underlined and in bold. Bottom panel: Surveyor assay following the transfection of HEK293T cells with CRISPR-Cas9 plasmids expressing one of the three gRNAs. WT DNA from untransfected cells was used as a control. (C) HDR donor DNA mutagenesis strategy. 8 substitutions were present, including three nonsilent substitutions to mutate Arg332 and Arg335 into Gly (green), one silent mutation to disrupt the PAM sequence (pink), and four silent mutations in the sequence targeted by gRNA1 (orange). The HaeIII restriction site created as a result of one of the silent substitutions is indicated, as is the position of the primer used in specific PCR screening.</p

Pre-screening of isolated clones by specific PCR.

<p>Following 20 days of growth, 161 isolated HEK293T clones were screened for HDR-edited TRIM5 gene by PCR using a primer specific for the mutated <i>TRIM5</i>. 14 clones that passed this pre-screen step are indicated by their names. MWM, molecular weight marker.</p

Identification of HDR-edited clones.

<p>(A) Mutation-specific PCR was performed on 13 clones showing a positive signal in the pre-screen. Untransfected HEK293T cells were used as a control. M, molecular weight marker. (B) Non-specific PCR of the targeted region followed by HaeIII digestion. The expected sizes of the digested PCR products are shown on the right. The full-length gels are available on the FigShare public repository (see “Availability of data” section).</p

Restriction by exogenous or endogenous TRIM5α is inefficient in HEK293T cells.

<p>(A) HEK293T and THP-1 cells were retrovirally transduced with WT huTRIM5α, R332G-R335G huTRIM5α or with the “empty” vector as indicated. Untransduced cells were eliminated by hygromycin treatment, and the cell populations were then challenged with increasing amounts of the HIV-1_NL-GFP vector. The percentage of cells expressing GFP was then determined by FACS.(B) HEK293T Jurkat cells were infected with increasing amounts of N-MLV_GFP and B-MLV_GFP. The amounts of virus used are expressed as multiplicities of infection (MOI) as calculated in the non-restrictive CRFK cells. The percentage of infected cells was determined by FACS. For N-MLV in Jurkat cells, only the 3 virus doses that yielded detectable infections are shown. (C) HEK293T cells were treated with IFN-α, IFN-β or IFN-ω for 16 h prior to a single-dose infection with N-MLV_GFP and B-MLV_GFP, designed to yield 10–20% infected cells in the absence of IFN-I. The percentage of infected cells was determined by FACS.</p

Deep sequencing analysis of <i>TRIM5</i> editing in 10 screened clones.

<p>The ~200-nt HDR-targeted <i>TRIM5</i> region was amplified by PCR and the PCR products were then analyzed by Illumina MiSeq sequencing. The alignment shown includes the targeted locus for each allele of the 10 clones, in comparison with the WT sequence and with the expected HDR-mutated sequence (top 2 lines). The codons for aa 332 and 335 are in bold. The Cas9 cleavage site on the WT sequence is shown at the top (arrow). Expected substitutions are shown in green (R332G/R335G), in orange (silent mutations in the gRNA target sequence) and in pink (silent mutation in the PAM). Duplications/insertions are underlined whereas deletions are represented by dashes. The star indicates the position of an unexpected substitution within a duplicated region in one allele of F2X. On the left is a table summarizing the results obtained for each clone: presence of the two therapeutic mutations R332G/R335G, proportion of <i>TRIM5</i> alleles modified by HDR and the proportion of the expected substitution mutations in the HDR-edited alleles. Note that only one clone (D11) has an allele containing all the desired mutations and that most of the non-HDR-edited alleles contain indels at the cleavage site.</p