In this study, we characterized the antiviral mechanism of action of AZD0530 and dasatinib, two pharmacological inhibitors of host kinases, that also inhibit dengue virus (DV) infection. Using Northern blot and reporter replicon assays, we demonstrated that both small molecules inhibit the DV2 infectious cycle at the step of steady-state RNA replication. In order to identify the cellular target of AZD0530 and dasatinib mediating this anti-DV2 activity, we examined the effects of RNA interference (RNAi)-mediated depletion of the major kinases known to be inhibited by these small molecules. We determined that Fyn kinase, a target of both AZD0530 and dasatinib, is involved in DV2 RNA replication and is probably a major mediator of the anti-DV activity of these compounds. Furthermore, serial passaging of DV2 in the presence of dasatinib led to the identification of a mutation in the transmembrane domain 3 of the NS4B protein that overcomes the inhibition of RNA replication by AZD0530, dasatinib, and Fyn RNAi. Although we observed that dasatinib also inhibits DV2 particle assembly and/or secretion, this activity does not appear to be mediated by Src-family kinases. Together, our results suggest that AZD0530 and dasatinib inhibit DV at the step of viral RNA replication and demonstrate a critical role for Fyn kinase in this viral process. The antiviral activity of these compounds in vitro makes them useful pharmacological tools to validate Fyn or other host kinases as anti-DV targets in vivo.
Dengue virus (DV) is a significant human pathogen and the cause of dengue fever and dengue hemorrhagic fever. The four DV serotypes (DV1, DV2, DV3, and DV4) are members of the Flaviviridae family and have a positive-sense RNA genome encoding a single polyprotein. This polyprotein is processed by host-and DV-encoded proteases into 10 proteins: three structural proteins (core [C], premembrane [prM], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Replication of the DV genome occurs in close association with the cytosolic-faced membranes of the endoplasmic reticulum (ER) (1) and requires the enzymatic activities of NS3 (RNA helicase and nucleotide triphosphatase [1][2][3][4]) and NS5 (RNA-dependent RNA polymerase [5][6][7] and RNA capping [8]). The NS1 protein has also been demonstrated to modulate viral RNA replication (9), and study of related flavivirus systems has indicated that interactions of NS1 with Yellow Fever virus NS4A (10) and West Nile virus (WNV) NS4B (11) are important for the replication of their respective genomes. The NS4A and NS4B proteins are thought to anchor the RNA replication complex to the ER membrane (9, 10, 12). After RNA replication and translation, the viral RNA is encapsidated by C to form the nucleocapsid that buds at the ER membrane to associate with the prM and E proteins and form an immature DV virion (1). This immature virion then transits through the secretory pathway, where the virion matures through the glycosylation of prM and E proteins (11,...