Fig 1: TRIM2 decreases JUNV entry into cells.(A) TRIM2 knockdown does not affect virus binding to cells. U2OS cells were transfected with a TRIM2 siRNA and incubated with FITC-labeled Candid 1. Shown is a representative FACS plot. This experiment was performed twice with similar results. (B) Cells overexpressing hTRIM2 or mTRIM2 or GFP (Control) were incubated with Candid 1, and after a 1-hr incubation at 37°C, virus was stripped from cells, and RNA was isolated and analyzed for viral RNA by RT-qPCR. Shown are the averages ± SD of 6 independent experiments. P values were determined by unpaired t tests; ***P = 0.0002; ****P = 0.0001. See also S4 Fig. (C) The same experiment was performed with primary bone marrow–derived macrophages isolated from mice of the indicated genotype. Values represent the mean ± SD in 2 independent experiments with triplicate experimental replicates. FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; hTRIM2, human TRIM2; JUNV, Junín virus; mTRIM2, mouse TRIM2; RT-qPCR, real-time quantitative PCR; siRNA, small interfering RNA; TfR1, transferrin receptor 1; WT, wild type.
Fig 2: Role for SHP-2 in infection and TRIM2 in inhibition of phagocytosis.(A) U2OS cells were transfected with the indicated siRNAs and infected with Candid 1. Knockdowns of the RNAs are shown in S7B Fig. (B) Extracts from the brains of uninfected and Candid 1–infected mice were prepared and immunoprecipitated with anti-phosphotyrosine or anti-TRIM2 antisera and analyzed by WB with the indicated antibodies. (C) BMDMs isolated from 3 mice of each genotype were incubated with apoptotic phrodo Red–labeled thymocytes. Shown is the average percent internalization in CD11b+ cells for 3 experiments, normalized to WT in each experiment. Statistical significance was determined by unpaired t test. **P = 0.005. Representative FACS plots are in S8 Fig. No difference was seen when BMDMs from either genotype were incubated with live thymocytes (S8 Fig). BMDM, bone marrow–derived macrophage; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; JUNV, Junín virus; NEFL, neurofilament light chain; NP, nucleoprotein; Phospho-ERK1/2, phosphorylated extracellular regulated kinase 1/2; siRNA, small interfering RNA; SIRPA, signal regulatory protein a; WB, western blot; WT, wild type.
Fig 3: Model for TRIM2–SIRPA inhibition of New World arenavirus infection.TRIM2 and phosphorylated SIRPA form a complex that limits virus endocytosis. Dephosphorylation of SIRPA, possibly by SHP-2, leads to dissociation of the complex and downstream signaling, thereby allowing virus entry to proceed, similar to what is thought to occur when SIRPA-mediated inhibition of phagocytosis is relieved. PTPase, protein phosphatase; SIRPA, signal regulatory protein a.
Fig 4: Overexpression of TRIM2 decreases Junín virus but not OWA infection.(A) U2OS cells were transfected with TRIM2 or TRIM5a expression vectors and 24 hr later were infected with Junín virus, Lassa virus, or LCMV GP-pseudoviruses containing the luciferase gene. The data shown are the average and SD of 3 independent experiments. Western blot is from U2OS cells and those transfected with the TRIM2 expression vector or siRNA. Blots were probed with anti-TRIM2 and anti-ß-tubulin antisera. (B) U2OS cells were transfected with the indicated siRNAs or TRIM expression vectors and 24 hr later were infected with Candid 1 (MOI 0.1). Reverse-transcribed RT-qPCR for the nucleoprotein RNA was analyzed. Values represent the mean ± SD in 2 independent experiments with triplicate experimental replicates. Control refers to cells treated with a control siRNA. (C) U2OS cells were transfected with the indicated siRNAs and expression vectors and infected with Tacaribe virus; the panel on the right shows the knockdown of each gene. The data shown represent the average and SD of 3 independent experiments. One-way ANOVA was used to determine significance. **P = 0.005; ***P = 0.0005. A2D2, calcium channel subunit a2d2; LCMV, lymphocytic choriomeningitis virus; MOI, multiplicity of infection; OWA, Old World arenavirus; RT-qPCR, real-time quantitative PCR; siRNA, short interfering RNA; TfR1, transferrin receptor 1.
Fig 5: The TRIM2 FIL domain is required for antiviral activity.(A) Diagram of the different deletion constructs. The ?RBCC, ?NHL, and NHL constructs were previously described [25]. All constructs were c-myc-tagged. (B) Western blot showing expression of the different deletion constructs. * denotes the monomeric FIL domain. (C) TRIM2 and deletion constructs were transfected into U2OS cells for 24 hr and then infected with Candid 1 (MOI 1). RNA was isolated at 24 hpi and analyzed by RT-qPCR for viral RNA levels. Shown are the averages ± SD of 3 independent experiments. One-way ANOVA was used to determine significance. *P = 0.05; ***P = 0.001; ****P = 0.0001. (D) The same experiment was done with the B and C expression constructs. The average ± SD of 3 independent experiments are shown. One-way ANOVA was used to determine significance. **P = 0.005. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hpi, hours post infection; JUNV, Junín virus; MOI, multiplicity of infection; ns, not significant; RT-qPCR, real-time quantitative PCR; WT, wild type.
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