West Nile virus and other arboviruses have the remarkable ability to replicate and assemble virus particles despite activating innate immune responses. While animals have evolved highly complex and powerful immune signals, viruses have adopted mechanisms to evade, subvert, disrupt, or inhibit them. Hence the relationship between cellular agonists and viral antagonists of the innate immune response is a driving force in viral pathogenesis.
WNV has an approximately 11
kb-long positive-sense RNA genome encoding a single polyprotein, which is then proteolytically processed into 10 individual proteins, including three structural proteins (the capsid C, membrane M, and envelope E), and seven nonstructural proteins (glycoprotein NS1, NS2A, protease cofactor NS2B, protease and helicase NS3, NS4A, NS4B, and the polymerase NS5) (reviewed in 5
). The virus is maintained in an enzootic cycle between mosquitoes and birds, but can infect mammals, including horses and humans. In humans, WNV infection typically presents as a febrile illness that can generally be resolved in healthy individuals (49
). However, in some cases WNV infection can progress to more serious CNS-associated sequelae, including lethal encephalitis (46
). Since its introduction into North America in 1999, WNV has spread rapidly across the continental United States, and subsequently into Mexico and South America, and has emerged as the major cause of viral encephalitis in the Western hemisphere (18
). So far, antiviral therapies or vaccines are not available to treat or prevent WNV infections.
WNV has evolved the ability to block the interferon (IFN) signal transduction pathway (19
). In WNV-infected cells, IFN exposure fails to induce phosphorylation of the Janus kinases JAK1 and Tyk2, and as a consequence, the STAT transcription factors remain latent, preventing transcriptional activation of interferon-stimulated genes (ISGs) (19
). Mutagenesis experiments revealed that NS4B is a determinant for blocking IFN signaling in cells harboring a replicating genome. However, homologous mutations in infectious virus did not phenocopy these results, indicating that additional viral factors contribute to IFN antagonism during virus infections (13
). Results derived from transfection studies with individual proteins remained inconclusive, invoking roles for NS4B and several other NS proteins in inhibition of the IFN response (31
). Recently, various studies with WNV and dengue virus (DENV), a closely related flavivirus, have suggested a number of potential mechanisms employed to block IFN-stimulated signals and antiviral responses (1
The IFN-α receptor (IFNAR) has two major subunits, IFNAR1 and IFNAR2c, which tightly dimerize upon IFN-α binding (58
). IFNAR1 is a highly glycosylated protein with an apparent molecular weight of ~110
). Tyk2 is associated with IFNAR1, while JAK1, STAT1, and STAT2 are associated with IFNAR2 (8
). Exposure to IFN induces dimerization of the receptor components and leads to the activation of the associated kinases (8
). Following activation of Jak1 and Tyk2 kinases, the STAT proteins are phosphorylated, which leads to their translocation to the nucleus, resulting in transcription of ISGs. In response to IFN, IFNAR1 is phosphorylated on serines 535 (S535) and 539 (S539), leading to ubiquitination on several lysines (K501, K525, and K526) by the E3 ligase complex SCF-Trcp
). Ubiquitinated IFNAR1, together with IFNAR2, translocate into endosomes. While IFNAR2 is recycled to the plasma membrane, IFNAR1 is proteolytically degraded in lysosomes (25
). IFN-induced downregulation of IFNARs represents one of several negative feedback loops used by cells to counter potential cytotoxic effects associated with the expression of ISGs.
Since WNV infection inhibits IFN responses at a receptor-proximal point in the pathway, we envisioned a scenario in which WNV infection alters the biochemical composition of the IFN receptor complex. To this end, we predicted that WNV activates or deregulates natural mechanisms for control of IFNAR complex components that play a role in negative feedback regulation of the IFN response. The results presented in this study demonstrate that WNV infections lead to a reduction in IFNAR1 expression.