Date Published: February 13, 2014
Publisher: Public Library of Science
Author(s): Ari Yasunaga, Sheri L. Hanna, Jianqing Li, Hyelim Cho, Patrick P. Rose, Anna Spiridigliozzi, Beth Gold, Michael S. Diamond, Sara Cherry, Abraham L. Brass.
Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.
Historically, West Nile virus (WNV) epidemics were observed in Africa, the Middle East, Europe, India, Australia, and parts of Asia, In 1999, WNV entered into the North America as part of an outbreak of neuroinvasive disease in New York City , and since then has become endemic in the United States with large numbers of cases occurring annually in different regions of the country. Indeed, the occurrence, size, and severity of outbreaks in humans overall have increased worldwide since the mid 1990s , with a large outbreak in Texas in 2012 leading to many fatalities , . Different strains of WNV, with variable worldwide distributions, exhibit significant differences in pathogenesis. In humans infected with North American WNV strains, approximately 80% of infections are asymptomatic, with 20% developing WNV fever and other relatively mild symptoms, and 1% progressing to encephalitis, meningitis, or flaccid paralysis . In contrast, WNV-Kunjin, endemic in Australia, has not been associated with any human fatalities or severe disease . The natural transmission cycle of WNV is between mosquitoes and birds, with humans, horses, and other vertebrates being incidental dead-end hosts . WNV is a member of the Flavivirus genus, which includes many globally important vector-borne pathogens, such as Dengue (DENV), yellow fever (YFV), tick-borne encephalitis (TBEV), and Japanese encephalitis viruses (JEV) . DENV is endemic in more than 110 countries with 3.6 billion people at risk, and 390 million people infected yearly , . At present, there are no specific antiviral therapies against any flavivirus, and only three insect-borne flaviviruses have approved vaccines for humans (YFV, TBEV, and JEV) .
Genome-wide RNAi screens have been employed to identify cellular factors required by viruses to successfully infect cells as well as factors that, if left unmodulated by the virus, serve to suppress infection. In addition, this screening approach can identify pathways that regulate the expression and activity of direct antiviral factors, orchestrating a robust antiviral response. Since our goal was to identify conserved inhibitory pathways that span insects and mammals with a particular interest in those having broad antiviral activity against disparate viruses, we performed a genome-wide RNAi screen in Drosophila in which we deliberately set a low infection rate, thereby sensitizing our assay to detect factors that when suppressed result in higher levels of infection. This is in contrast to previous genome-wide flavivirus RNAi screens, which targeted a higher level of infection and so led to the identification of a larger number of genes that promote infection , . Nonetheless, our screen was sufficiently sensitive and robust to enable us to identify 96 genes that promoted WNV infection. Enriched gene ontology categories included pathways such as clathrin-mediated endocytosis and endosomal acidification that are required for flavivirus entry and were identified by earlier RNAi screens.