West Nile virus (WNV) is a neurotropic ffavivirus that has emerged globally as a significant cause of viral encephalitis. WNV is maintained in an enzootic cycle between mosquitoes and birds (reviewed in reference 75) but can also infect and cause disease in horses and other vertebrate animals. Infection of humans is associated with a febrile illness that can progress to a lethal encephalitis with symptoms including cognitive dysfunction and ffaccid paralysis (30, 148, 167). Since the mid-1990s, outbreaks of WNV fever and encephalitis have occurred in regions throughout the world where WNV is endemic, including the Middle East, Europe, and Africa (43). Following its introduction into the United States in 1999, WNV rapidly disseminated across North America and more recently has been reported in Mexico, South America, and the Caribbean (45, 95, 102). Annual outbreaks of WNV fever and neuroinvasive disease occur in the United States (67), with 19,000 diagnosed human cases between 1999 and 2005 (http://www. cdc. gov/ncidod/dvbid/westnile/surv&control. htm# maps) and an estimated 750,000 undiagnosed infections in 2003 (25). Although vaccines are available for animal use, no vaccines or specific therapies for WNV are currently approved for humans. WNV is a member of the Flaviviridae family of RNA viruses and is related to other important human pathogens, including dengue viruses, yellow fever viruses, and Japanese encephalitis viruses. Similar to other ffaviviruses, WNV is an enveloped virus with a single-stranded, positive sense, 11-kb RNA genome. The genome is transcribed as a single polyprotein that is cleaved by host and viral proteases into three structural and seven nonstructural proteins (32). The structural proteins include a capsid protein (C) that binds viral RNA, a premembrane (prM) protein that blocks premature viral fusion and may chaperone envelope (E)-protein folding, and an E protein that mediates viral attachment, membrane fusion, and viral assembly (131). The viral nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A NS4B, and NS5) regulate viral transcription and replication and attenuate host antiviral responses (17, 71, 87, 110, 111, 115, 132). NS1 has cofactor activity for the viral replicase and is secreted from infected cells, NS2A inhibits interferon (IFN) responses and may participate in virus assembly, and NS3 has protease, NTPase, and helicase activities (87, 113–115, 118). NS2B is a cofactor required for NS3 proteolytic activity, NS4A and NS4B modulate IFN signaling, and NS5 encodes the RNA-dependent RNA polymerase and a methyltransferase (49, 86, 115, 202).

Several molecules have been implicated in the attachment of WNV to cells in vitro, including DC-SIGN, DC-SIGN-R, and the integrin v 3 (37, 44). However, attachment receptors in vivo for physiologically important cell types, such as neurons, remain uncharacterized. After binding, WNV enters cells via receptor-mediated endocytosis that may involve clathrincoated pits (36, 63). Following a pH-dependent conformational change in the E protein, the viral and endosomal membranes fuse, releasing the viral nucleocapsid into the cytoplasm (2, 23, 65). WNV is believed to replicate on endoplasmic reticulum-associated membranes to generate a negative-strand RNA intermediate that serves as a template for nascent positive-strand RNA synthesis (119). Positive-strand RNA is either packaged within progeny virions or used to translate additional viral proteins. WNV assembles and buds into the endoplasmic reticulum to form immature particles that contain the prM protein. Following transport through the trans-Golgi network, furin-mediated cleavage …