Evolutionary and functional characterization of lagomorph guanylate binding proteins (GBPs): a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

Luca Schelle¹ João V. Côrte-Real^{1, 2, 3, 4} Sharmeen Fayyaz^{5, 6} Augusto Del Pozo Ben¹ Margarita Shnipova¹ Moritz Petersen⁵ Rishikesh Lotke⁵ Bhavna Menon¹ Dana Matzek⁷ Lena Pfaff⁷ Ana Pinheiro^{2, 4} João P. Marques^{2, 4, 8} José Melo-Ferreira^{2, 3, 4} Bastian Popper⁷ Pedro J. Esteves^{2, 3, 4, 9} Daniel Sauter⁵ Joana Abrantes^{2, 3, 4*} Hanna-Mari Baldauf^1*

• ¹Department of Virology, Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, Germany
• ²Centro de Investigacao em Biodiversidade e Recursos Geneticos (CIBIO-InBIO), Portugal
• ³Departament of Biology, Faculty of Sciences, University of Porto, Portugal
• ⁴BIOPOLIS Program in Genomics, Portugal
• ⁵Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital and Faculty of Medicine, University of Tübingen, Germany
• ⁶International Center for Chemical and Biological Sciences, University of Karachi, Pakistan
• ⁷Biomedical center (BMC), Core facility Animal Models (CAM), LMU München, Faculty of Medicine, Germany
• ⁸IRD UMR226 Institut des sciences de l’évolution de Montpellier (ISE-M), France
• ⁹Center of Investigation in Health Technologies, CESPU, Portugal

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family. From an evolutionary perspective, lagomorph GBPs also experienced multiple gain and loss events. For example, lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or in the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, our work provides valuable information on the evolution of lagomorph GBPs, their characteristics and suggests a role for ocGBPs in innate immunity.