**1. Introduction**

The genus *Flavivirus* comprises several important human pathogens including West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), and *Zika virus* (ZIKV). These (+)-sense, single-stranded RNA viruses are transmitted to humans by mosquito bite and represent (re)emerging viruses. Roughly half of the world's population is at risk of DENV infection, causing ≈390 million infections and 21,000 deaths per year [1]. Since emergence in the USA, WNV has spread to all contiguous states causing ≈2340 deaths (The US Centers for Disease Control and Prevention (CDC) records as of August 2019), and is a model example of emerging infectious disease [2]. Recently, ZIKV emerged with outbreaks in Oceania and Latin America [3,4]. ZIKV infection causes symptoms ranging from flu-like illness to Gullain–Barré syndrome [4]. ZIKV can undergo maternal–fetal transmission, causing congenital Zika syndrome (CZS) marked by microencephaly and post-natal cognitive disorders [5–9]. ZIKV is of global concern but the virus–host interactions of ZIKV infection linked with CZS are not fully understood. Defining the processes in which flaviviruses employ to evade and antagonize host immune responses is paramount to informing vaccine and therapeutic strategies to combat infection. No approved vaccines or specific antivirals are currently available to prevent or treat human infections with DENV, WNV, or ZIKV.

The immune response to flavivirus infection initiates upon viral recognition by host cell pattern recognition receptors (PRRs) [10,11]. PRR signaling in the infected cell leads to activation of interferon regulatory factor (IRF) 3 and induction of interferons (IFNs) α, β, and λ. IFN secretion and binding to the cognate receptor then triggers the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade for rapid induction of IFN-stimulated genes (ISGs). Janus kinases JAK1, JAK2, and Tyk2 phosphorylate STAT1 and STAT2 on specific tyrosine residues (pY) in response to IFN/receptor interaction. pY-STATs heterodimerize and move to the nucleus to bind target promoters inducing ISG expression [12]. ISG products establish an antiviral state in responding cells to restrict virus replication and spread. The innate immune response is essential for control of flavivirus infection [13]. Flavivirus antagonism of IFN-responsive JAK/STAT signaling is a feature associated with virulence [14–19]. Flavivirus nonstructural protein 5 (NS5) has been identified as the primary mediator of IFN signaling inhibition [14]. Roles in JAK/STAT antagonism have also been attributed to other NS proteins and subgenomic flavivirus RNA [16,20–23].

In addition to IFN signaling, the JAK/STAT pathway is utilized by several distinct cytokines that activate various Janus kinases (JAKs; including JAK1, JAK2, JAK3, and Tyk2) upon receptor interaction [24,25]. However, flaviviral inhibition of JAK/STAT signaling by cytokines other than IFN remains unexplored. We examined flavivirus regulation of JAK/STAT signaling induced by IFNs β, γ, and λ3 as well as inflammatory (IL6), and also immune-regulatory (IL4, IL10) cytokines that utilize this pathway. We revealed the fact that flaviviruses broadly inhibit JAK/STAT signaling across STAT1-6 to disrupt responses to IFNs, as well as pro-inflammatory and immune-regulatory cytokines. Mechanistically, this broad antagonism of JAK/STAT signaling was mediated by flavivirus NS5 binding to host heat shock protein 90 (HSP90), leading to dysregulation of chaperone stabilization (and eventual subsequent degradation) of JAKs, as well as abrogation of cytokine-induced pY-STAT. Unlike previous reports of flavivirus interaction with other heat shock proteins [26–28], we showed that flaviviruses including ZIKV do not usurp chaperone activity to promote viral protein function and genome replication. Rather, flaviviruses appear to specifically target and disrupt HSP90 chaperone activity to suppress host JAK/STAT signaling, thus controlling the actions of a broad range of immunoregulatory and antiviral cytokines. Importantly, this broad-acting innate immune evasion strategy has never before been identified.

Thus, HSP90 is a key virus-targeted host factor, wherein interaction with viral NS5 prevents its interaction with client kinases, resulting in suppression of JAK/STAT-dependent cytokine signaling. NS5 targeting of HSP90 is thus the mechanism that underlies the disruption of broad cytokine signaling by flaviviruses.
