Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse. (Journal of Biomolecular Screening 2010:798-805)

Borna disease virus (BDV), the prototypic member of the Bornaviridae family, within the order Mononegavirales, is highly neurotropic and constitutes an important model system for the study of viral persistence in the central nervous system (CNS) and associated disorders. The virus surface glycoprotein (G) has been shown to direct BDV cell entry via receptor-mediated endocytosis, but the mechanisms governing cell tropism and propagation of BDV within the CNS are unknown. We developed a small interfering RNA (siRNA)-based screening to identify cellular genes and pathways that specifically contribute to BDV G-mediated cell entry. Our screen relied on silencing-mediated increased survival of cells infected with rVSV⌬G*/BDVG, a cytolytic recombinant vesicular stomatitis virus expressing BDV G that mimics the cell tropism and entry pathway of bona fide BDV. We identified 24 cellular genes involved in BDV G-mediated cell entry. Identified genes are known to participate in a broad range of distinct cellular functions, revealing a complex process associated with BDV cell entry. The siRNA-based screening strategy we have developed should be applicable to identify cellular genes contributing to cell entry mediated by surface G proteins of other viruses. (12,49,51). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (13, 49). BDV is highly neurotropic and causes central nervous system (CNS) disease in a variety of vertebrate species that is frequently manifested by behavioral abnormalities (25,53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (18,20,32,44,46). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby viruses induce neurodevelopmental and behavioral disturbances in the absence of the hallmarks of cytolysis and inflammation (19,24,42). Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand (NNS) RNA genome whose organization (3Ј-N-p10/P-M-G-L-5Ј) is characteristic of mononegaviruses (MNV)Serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans, and it might be associated with certain neuropsychiatric disorders (23,29). Moreover, the recent identification of an avian bornavirus (22, 45) underscores the prospect of finding other BDV-like emerging viruses of potential relevance to human health. The identification of host cell factors involved in BDV cell entry will contribute to a better understanding of BDV cell tropism and propagation within the CNS, a first and necessary step to elucidate the mechanisms by which BDV disrupts normal brain functions. Likewise, the development of effective antiviral strategies to combat bornavirus infections may be facilitated by targeting host cell fact...