The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein.

Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNVKUN) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNVKUN particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNVKUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNVKUN assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNVKUN assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system.

Unusual tubulin-clustering ability of specifically c7-modified colchicine analogues.

Highly cytotoxic C7-modified colchicine analogues, exemplified by tubuloclustin, promote microtubule disassembly followed by the formation of very stable tubulin clusters, both in vitro and in cells. The proposed mechanism of action of tubuloclustin and its analogues, beyond that of colchicine, includes additional specific interactions with the α-tubulin subunit.

The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex.

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV(KUN)) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV(KUN) replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV(KUN) replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.

West Nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral MxA protein.

The human MxA protein is a type I and III interferon (IFN)-induced protein with proven antiviral activity against RNA viruses. In this study, we investigated the effect of MxA expression on the replication of West Nile Virus strain Kunjin (WNV(KUN)). Pretreatment of A549 cells with IFN-alpha lead to increased expression of MxA, which contributed to inhibition of WNV(KUN) replication and secretion. However, in Vero cells stably expressing the MxA protein, WNV(KUN) replication, maturation and secretion was not inhibited. Biochemical and subcellular localization studies of WNV(KUN) proteins and MxA suggest that the MxA activity was not compromised by a flavivirus-encoded antagonist. Instead, we show that characteristic membranous structures induced during WNV(KUN) replication provide partial protection from MxA, possibly by 'hiding' WNV(KUN) replication components. This distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can hide from host surveillance.