The lipid-sensor TREM2 aggravates disease in a model of LCMV-induced hepatitis

textabstractLipid metabolism is increasingly being appreciated to affect immunoregulation, inflammation and pathology. In this study we found that mice infected with lymphocytic choriomeningitis virus (LCMV) exhibit global perturbations of circulating serum lipids. Mice lacking the lipid-sensing surface receptor triggering receptor expressed on myeloid cells 2 (Trem2 -/-) were protected from LCMV-induced hepatitis and showed improved virus control despite comparable virus-specific T cell responses. Non-hematopoietic expression of TREM2 was found to be responsible for aggravated hepatitis, indicating a novel role for TREM2 in the non-myeloid compartment. These results suggest a link between virus-perturbed lipids and TREM2 that modulates liver pathogenesis upon viral infection. Targeted interventions of this immunoregulatory axis may ameliorate tissue pathology in hepatitis

Non-Smc element 5 (Nse5) of the Smc5/6 complex interacts with SUMO pathway components

The Smc5/6 complex in Saccharomyces cerevisiae contains six essential non-Smc elements, Nse1-6. With the exception of Nse2 (also known as Mms21), which is an E3 small ubiquitin-like modifier (SUMO) ligase, very little is understood about the role of these components or their contribution to Smc5/6 functionality. Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway. Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO. SUMO also mediates the interactions between Nse5 and the two PIAS family E3 SUMO ligases, Siz1 and Siz2. Cells carrying the nse5-ts1 allele or lacking either SIZ1 or SIZ2 exhibit a reduction in Smc5 sumoylation upon MMS treatment and demonstrate functional redundancy for SUMO mediated events in the presence of DNA damage. Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein

<div><p>RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected <i>Trim21</i>^-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.</p></div

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein

<div><p>RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected <i>Trim21</i>^-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.</p></div

Functional screening for L protein interactors involved in LCMV life cycle.

<p>(<b>A</b>) 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>B</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. (<b>C</b>-<b>D</b>) C57BL/6 and <i>Trim21</i>^-/- mice were infected with 2x10⁶ FFU of the indicated viruses. Viral titers were determined in (<b>C</b>) blood at indicated time points and in (<b>D</b>) organs at 21 days post infection. The data shown in (<b>C</b>) 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 (<b>A, B, D</b>) or by Two-way ANOVA (<b>C</b>). Significant p values were indicated as follows: ns—non significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.</p

Generation and characterization of LCMV strains expressing a tagged L protein.

<p><b>(A)</b> 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>B</b>) HEK293T cells were infected at a MOI of 0.01 with either Cl13_L-HA or with untagged Cl13. Supernatant was harvested and viral loads were measured at the indicated time points by focus forming assay. <b>(C</b> and <b>D)</b> C57BL/6J mice were infected with 2x10⁶ FFU of the indicated viruses. Viral titers were determined in <b>(C)</b> blood at indicated time points and in <b>(D)</b> organs 20 days post infection. <b>(E)</b> 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-<b>C</b>) or unpaired t-test (<b>D-E</b>). Significant p values were indicated as follows: ns—non significant, * p≤0.05,: ** p≤0.01.</p

Viral RNA-dependent RNA-polymerases target host proteome by common and virus-specific strategies.

<p><b>(A)</b> 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>(B)</b> Largest connected component (LCC) analyses for global RdRps and LCMV only datasets. <b>(C)</b> Functional protein modules targeted by RdRps based on the community detection method. <b>(D)</b> Heat map representing virus-specific targeting of protein functional modules.</p

Identification of L protein interactome.

<p><b>(A)</b> 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>(B)</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.</p

Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage

Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways in the liver, including downregulation of superoxide dismutase 1 (Sod1). Sod1(-/-) mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was independent of T and NK cells and could be ameliorated by antioxidant treatment. Type I interferon (IFN-I) led to a downregulation of Sod1 and caused oxidative liver damage in Sod1(-/-) and wild-type mice. Genetic and pharmacological ablation of the IFN-I signaling pathway protected against virus-induced liver damage. These results delineate IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage and describe a mechanism of innate-immunity-driven pathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage. VIDEO ABSTRACT