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

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 Trim21-/-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

The ADAM17 sheddase complex regulator iTAP/Frmd8 modulates inflammation and tumor growth

The metalloprotease ADAM17 is a sheddase of key molecules, including TNF and epidermal growth factor receptor ligands. ADAM17 exists within an assemblage, the “sheddase complex,” containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology. The FERM domain–containing protein iTAP/Frmd8 is an iRhom-binding protein that prevents the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP/Frmd8 have not been addressed, we characterized the impact of iTAP/Frmd8 loss on ADAM17-associated phenotypes in mice. We show that iTAP/Frmd8 KO mice exhibit defects in inflammatory and intestinal epithelial barrier repair functions, but not the collateral defects associated with global ADAM17 loss. Furthermore, we show that iTAP/Frmd8 regulates cancer cell growth in a cell-autonomous manner and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP/Frmd8 may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, while avoiding the collateral impact on the vital functions associated with the widespread inhibition of ADAM17

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis

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

CD8+ T cells induce cachexia during chronic viral infection

Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8 T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8 T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8 T cell response and required T cell–intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8 T cells in IAC

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

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

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

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