Journal: bioRxiv

Article Title: A modified cyclosporine enhances lentivector transduction ex vivo and in vivo by degrading IFITM3

doi: 10.1101/2025.08.26.669098

Figure Lengend Snippet: (A) IFITM3 levels at 6 and 24 h post treatment with 5 µM TEs/DMSO in THP-1 pretreated with +/- 10 ng/ml IFNβ. (B) IFITM3 protein quantification from (A), normalised to actin and expressed as a percentage of DMSO + IFNβ. n=2. (C-E), Titration of TEs on THP-1 pretreated with +/- 10 ng/ml IFNβ, or in THP-1 over-expressing IFITM3, with LV-GFP for 48 h, n = 3, +/-SEM. (F) HSPC treated with 1% LB and 4 μg/ml PS, or novel TEs, with LV-GFP at an MOI of 10 genome copies (GC) /cell, % GFP positive cells measured at 48 hpi, n=2, +/- SEM. One-way ANOVA with Tukey’s test compared to DMSO with LV. (G) Unmodified and TRIM5-KO U87 cells treated with 5 μM TEs, with LV-GFP 48 h, n=3, +/- SEM. Statistical significance compared to DMSO (control or TRIM5 KO as appropriate) two-way ANOVA with Tukey’s test. p ≤ 0.0001, ****; p ≤ 0.0002, ***; p ≤ 0.0021, **; p < 0.0332, *; p < 0.1234 ns. h, Model of TE mechanism.

Article Snippet: TaqMan qPCR set up using 2x TaqMan gene expression master mix (4369016, Applied Biosystems) and FAM/TAMRA probes for OAZ1 (Hs00427923_m1), TRIM5 (Hs01552559_m1), and IFITM3(Hs03057129_s1).

Techniques: Titration, Expressing, Control

Journal: mBio

Article Title: Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration

doi: 10.1128/mbio.00169-25

Figure Lengend Snippet: Increased CypA binding to HIV-1 capsid inhibits infection in a CPSF6-dependent manner. ( A ) OMK cells were infected with different amounts of WT HIV-1 and CA mutants (0.1–100 ng p24). Average luciferase values are shown for two independent experiments. ( B–E ) Infectivity of WT and mutant HIV-1 (10 ng p24) was determined after 48 h by luciferase activity in DMSO or 5–10 μM CsA in HeLa ( n = 3) ( B ), Jurkat ( n = 3) ( C ), primary CD4+ T cells ( n = 2) ( D ), and Jurkat PPIA −/ − ( n = 3) ( E ). ( F ) Primary CD4+ T cells were transduced with lentiviruses expressing control or TRIM5α miRNA prior to infection with WT and mutant HIV-1 (10 ng p24) in DMSO or 10 μM CsA. Infections were determined after 48 h by luciferase activity ( n = 2). Error bars represent standard errors of the mean (SEM). Comparisons between infection conditions were analyzed by unpaired t tests. P values of <0.05 were considered significant and significant values are denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. ns, P > 0.05.

Article Snippet: Lentiviral vectors encoding control or TRIM5α miRNAs (pAPM-D4-miR30-L1221 or pAPM-D4-miR30-TRIM5α; Addgene) were produced by co-transfection with the packaging plasmid pcHelp ( ) and pCMV-VSV-G ( ).

Techniques: Binding Assay, Infection, Luciferase, Mutagenesis, Activity Assay, Transduction, Expressing, Control

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Schematic overview of the domain swap strategy, which replaces the TRIM5 SPRY domain with the TRIM37 TRAF domain to generate a chimeric TRIM5-TRAF protein. (B) Immunoblot showing total protein expression levels of indicated HA-tagged TRIM5 constructs in RPE-1 tet-on TRIM5 cells. Actin, loading control. Representative data; n = 3 biological replicates. (C) Representative images of the localization and effect of indicated HA-tagged TRIM5 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM5 cells. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated constructs in RPE-1 tet-on TRIM5 cells from (C), with TRIM37 included as a benchmark. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (E) Representative images of RPE-1 TRIM37 −/− cells expressing the indicated HA-tagged TRIM5 constructs. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (F) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM5 constructs from (E). n = 3 biological replicates, each with >100 cells.

Article Snippet: To generate TRIM5-WT or TRIM5 chimera expressing RPE-1 cell lines, the TRIM5 ORF (#79066; Addgene) was PCR amplified and cloned into a tet-inducible lentiviral vector containing a C-terminal 3xHA tag.

Techniques: Western Blot, Expressing, Construct, Control, Staining

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Left, diagram illustrating the B-box trimerization interface of TRIM5 dimers on the HIV capsid. Trimers are stabilized by W117 residues within the hydrophobic core, as shown in the magnified top-down view of the TRIM5 B-box crystal structure (PDB 5VA4). Right, analogous diagram representing a putative oligomer formed by TRIM37 dimers at the centrosome, where B-box domain trimerization is hypothesized to be stabilized by W120 residues, the synonymous counterpart to TRIM5’s W117. A magnified top-down view shows the putative TRIM37 B-box trimer modelled by fitting AlphaFold-predicted TRIM37 monomers onto the TRIM5 crystal structure. (B) Comparative alignment of the B-box domains from human TRIM37 and human and rhesus macaque ( Macaca mulatta ) TRIM5. Residue Cys109, mutated in MUL disease, is pivotal for zinc (Zn) coordination. The highlighted region in grey denotes the sequence alignment where W115/W117 residues in TRIM5 correspond to the W120 residue in TRIM37, signifying a conserved structural motif critical for higher-order assembly. (C) Immunoblot showing total protein expression levels of TRIM37 variants in RPE-1 tet-on TRIM37 cells from (D). Vinculin, loading control. Representative data; n = 3 biological replicates. (D) Representative images of RPE-1 tet-on TRIM37 cells expressing the RING domain mutant TRIM37(C18R) or RING-B-box double mutants (C18R-C109S and C18R-W120E). Cells were treated with DMSO (control) or nocodazole (3.3 μM) 30 min before doxycycline induction to depolymerize microtubules. n = 3 biological replicates. Scale bars, 5 μm. (E) Quantification of centrosomal TRIM37 signal in DMSO-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m. (F) Left, immunoblot showing detection of higher molecular weight (HMW) species of indicated TRIM37 variants upon in vivo DSG crosslinking. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with graph depicting normalized HMW TRIM37 intensity with DSG crosslinker (+) relative to DMSO control (−DSG). Mean ± s.e.m. (G) Evaluation of cytoplasmic TRIM37 puncta prevalence in nocodazole (Noc)-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m.

Article Snippet: To generate TRIM5-WT or TRIM5 chimera expressing RPE-1 cell lines, the TRIM5 ORF (#79066; Addgene) was PCR amplified and cloned into a tet-inducible lentiviral vector containing a C-terminal 3xHA tag.

Techniques: Residue, Sequencing, Western Blot, Expressing, Control, Mutagenesis, Molecular Weight, In Vivo