People of African ancestry carrying certain APOL1 mutant alleles are at elevated risk of developing renal diseases. However, the mechanisms underlying APOL1-associated renal diseases are unknown. Because the APOL1 gene is unique to humans and some primates, new animal models are needed to understand the function of APOL1 in vivo. We generated transgenic Drosophila fly lines expressing the human APOL1 wild type allele (G0) or the predominant APOL1 risk allele (G1) in different tissues. Ubiquitous expression of APOL1 G0 or G1 in Drosophila induced lethal phenotypes, and G1 was more toxic than was G0. Selective expression of the APOL1 G0 or G1 transgene in nephrocytes, fly cells homologous to mammalian podocytes, induced increased endocytic activity and accumulation of hemolymph proteins, dextran particles, and silver nitrate. As transgenic flies with either allele aged, nephrocyte function declined, cell size increased, and nephrocytes died prematurely. Compared with G0-expressing cells, however, G1-expressing cells showed more dramatic phenotypes, resembling those observed in cultured mammalian podocytes overexpressing APOL1-G1. Expressing the G0 or G1 APOL1 transgene in nephrocytes also impaired the acidification of organelles. We conclude that expression of an APOL1 transgene initially enhances nephrocyte function, causing hypertrophy and subsequent cell death. This new Drosophila model uncovers a novel mechanism by which upregulated expression of APOL1-G1 could precipitate renal disease in humans. Furthermore, this model may facilitate the identification of APOL1-interacting molecules that could serve as new drug targets to treat APOL1-associated renal diseases.

NK cell-mediated murine cytomegalovirus (MCMV) resistance ( Cmv r ) is under H-2 k control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmv r to the MHC class I (MHC-I) D k gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, D k itself is a candidate gene. A 10-kb genomic D k fragment was subcloned and microinjected into MCMV-susceptible ( Cmv s ) (MA/My.L- H2 b × C57L)F 1 or (B6 × DBA/2)F 2 embryos. Transgenic founders, which are competent for D k expression and germline transgene transmission, were identified and further backcrossed to MA/My.L- H2 b or C57L mice. Remarkably, D k expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I D k were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I D k expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I D k is the H-2 k -linked Cmv r locus; these findings suggest a role for NK cell interaction with D k -bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.

During virus infection, NK cells are needed in the body to provide early immune defense. Their activity is regulated by MHC class I-binding cell surface inhibitory and stimulatory receptors that include mouse Ly49, human killer Igrelated receptor (KIR), 3 and NKG2/CD94 receptors (1-3). NK cell-mediated virus control is subject to genetic factors that can influence viral replication and host mortality (4). For instance, the Ly49H activation receptor displayed on the surface of NK cells in C57BL/6 mice binds murine CMV (MCMV) m157 ligands at the surface of infected cells to impart MCMV resistance (5, 6). In New Zealand White, MA/My, and PWK strains, MCMV resistance also requires NK-mediated virus control, but Ly49H-independent defense mechanisms are key (7-11). It remains unclear which genetic factors are at work and how such factors mediate virus resistance through NK cells.Attempts have been made to identify genetic factors that underlie MCMV resistance/susceptibility traits in the offspring of genetic crosses between MCMV-resistant (Cmv r ) MA/My mice and strains that appear to be more MCMV susceptible (Cmv s ). To date, major MCMV control loci have been mapped to the MHC and NK gene complex (NKC) on chromosomes 17 and 6, respectively (9, 10). We have further shown that MHC polymorphism is respon- Materials and Methods Animalsmice were bred and maintained in a specificpathogen-free vivarium at the University of Virginia (Charlottesville, VA), which is fully certified by the American Association for Accreditation of Laboratory Animal Care. All recombinant congenic strains generated and used in this study were backcrossed to the progenitor strain C57L for at least 10 generations. All animal studies were approved by and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Virginia. Recombinant congenic strain generation, genetic markers, and genotyping (C57L.M-H2k ϫ C57L)F 1 or (MA/My.L-H2 b ϫ MA/My)F 1 were brother ϫ sister mated and screened for recombination crossovers within the D17Mit16-D17Uva09 interval using several simple sequence-length polymorphism (SSLP) markers to distinguish MA/My and C57L alleles (Table I). SSLP-amplified PCR products were resolved in POP7-filled capillaries and analyzed on a Genetic Analyzer 3130xl using Data Collection (version 3.0) and GeneMapper software (version 4.0; Applied Biosystems) as described previously (10). Some unlabeled SSLP amplicons were fractionated in 4% agarose gel electrophoresis and visualized on an UV transilluminator after ethidium bromide staining.For generation of novel SSLP markers, chromosome 17 sequence data available from the National Center for Biotechnology Information were manually inspected for microsatellite repeat sequences. Sequence-specific

Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions.