Fig 1.
Generation and characterization of LCMV strains expressing a tagged L protein.
(A) Viral titer of N- and C-terminal L protein-tagged Cl13 LCMV and WT Cl13 LCMV measured by focus forming assay at 72 hours post infection after reverse genetic rescue on BHK21 cells. (B) HEK293T cells were infected at a MOI of 0.01 with either Cl13L-HA or with untagged Cl13. Supernatant was harvested and viral loads were measured at the indicated time points by focus forming assay. (C and D) C57BL/6J mice were infected with 2x106 FFU of the indicated viruses. Viral titers were determined in (C) blood at indicated time points and in (D) organs 20 days post infection. (E) C57BL/6J mice were infected with 2x106 FFU of the indicated viruses and the percentage of GP33-specific-tetramer+ CD8+ T cells was quantified in the spleen at 8 days post infection. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by Two-way ANOVA (B-C) or unpaired t-test (D-E). Significant p values were indicated as follows: ns—non significant, * p≤0.05,: ** p≤0.01.
Fig 2.
Identification of L protein interactome.
(A) GO enrichment analyses for the L protein interactome based on the molecular functions (light grey) and biological processes (dark grey) of interactors followed by visualization with ReviGO. (B) Overview of L protein interactomes classified based on the protein functions and visualized in Cytoscape. The data is based on the mass-spectrometry derived list of proteins identified in L protein pulldowns after filtration using Top3 quantitation and SAINTexpress software as detailed in Materials and Methods.
Fig 3.
Viral RNA-dependent RNA-polymerases target host proteome by common and virus-specific strategies.
(A) Integrated interactome of viral RdRp targets. Host proteins interacting with viral RdRps are highlighted in blue, the rest of the human proteome—in grey. (B) Largest connected component (LCC) analyses for global RdRps and LCMV only datasets. (C) Functional protein modules targeted by RdRps based on the community detection method. (D) Heat map representing virus-specific targeting of protein functional modules.
Fig 4.
Functional screening for L protein interactors involved in LCMV life cycle.
(A) Two independently generated HeLa S3 CRISPR-Cas9 targeted cell pools per gene of interest for 5 genes were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01 and viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. (B) Two HeLa S3 CRISPR-Cas9 TRIM21-targeted cell pools were reconstituted either with TRIM21-expressing plasmid or with non-target control and 36 hour post transfection were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01. Viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. (C-D) C57BL/6 and Trim21-/- mice were infected with 2x106 FFU of the indicated viruses. Viral titers were determined in (C) blood at indicated time points and in (D) organs at 21 days post infection. The data shown in (C) is representative of two similar experiments. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by unpaired t-test (A, B, D) or by Two-way ANOVA (C). Significant p values were indicated as follows: ns—non significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.