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 replicontransfected 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 (WNV) belongs to the Flaviviridae, which is a large family of enveloped, positive-strand RNA viral pathogens that are responsible for causing severe disease and mortality in humans and animals each year. The Australian WNV strain Kunjin virus (WNV KUN ) is a relatively low-pathogenic virus that is closely related to the pathogenic WNV strain New York 99 (WNV NY99 ), the causative agent of the 1999 epidemic of encephalitis in New York City (11).It has become increasingly known that the replication of most, if not all, positive-sense RNA viruses, whether they infect plants, insects, or humans, is associated with dramatic membrane alterations resulting in the formation of membranous microenvironments that facilitate efficient virus replication. In most cases the induced membrane structures house the actively replicating viral RNA and comprise 70-to 100-nm membrane "vesicles" (sometimes referred to as spherules). Although this distinct morphology is shared across virus families, the cellular origins of these membranes is diverse: the endoplasmic reticulum (ER), mitochondria, peroxisomes, and trans-Golgi membranes have been implicated in different viral systems (1,8,13,23,31,38,41,45). This diversity implies that the processes involved in inducing the membrane vesicles/ spherules are shared, rather than the composition of the membrane itself, although the exact purpose for utilizing membranes derived from different cellular compartments is still not completely resolved or understood.The replication of the flavivirus WNV KUN is associated with the induction of morphologically distinct membrane structures that have defined roles during the WNV KUN replication cycle. Three well-defined structures can be seen as large convoluted membranes (CM), paracrystalline arrays (PC), or membrane sacs containing small vesicles, termed vesicle packets (VP) (18,20,48). Based on localization studies with viral proteins of specific functions, we observed that components of the virus protease complex (namely, nonstructural 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-a 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. KUN ) is a member of the Flaviviridae, a family of mosquito-borne, enveloped, positive-strand RNA viruses that cause many thousands of deaths each year. WNV KUN is endemic within Australia and is a causative agent of Australian encephalitis, but is seldom associated with severe disease (Mackenzie et al., 1994). Sequencing analyses have revealed that WNV KUN is closely related to WNV strain New York 99 (WNV NY99 ), the causative agent of the 1999 epidemic of encephalitis in New York City. Comparison between the nucleotide and amino acid sequences of WNV KUN and WNV NY99 revealed a very close phylogenetic relationship (88 and 98.1 %, respectively) (Lanciotti et al., 1999;Liu et al., 2003;Shi et al., 2002). Kunjin strain of West Nile virus (WNVThe interferon (IFN)-dependent immune response appears essential for the protection and clearance of most viral infections, including flaviviral infections (Diamond & Harris, 2001;Lobigs et al., 2003;Samuel & Diamond, 2005). IFNs are inducible cytokines and their antiviral effect is mediated by several IFN-induced proteins; some prime examples of these are double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), 29-59 oligoadenylate synthetases (OAS) and MxA (Goodbourn et al., 2000).The Mx proteins belong to the dynamin superfamily of large GTPases, which are involved in different functions within the cell, including host defence (Haller & Kochs, 2002). MxA is induced by type I (IFN-a and IFN-b) and III (IFN-l) IFNs via an autocrine feedback mechanism that initiates upregulation of MxA at the level of RNA transcription. MxA functions as a potent antiviral protein both in vitro and in vivo (Haller & Kochs, 2002;Holzinger et al., 2007). The antiviral activity of MxA appears to be based on recognition of pre-formed or forming viral nucleocapsids (NC) or at early steps of viral RNA transcription, perhaps uncoating (Marschall et al., 2000;Pavlovic et al., 1990Pavlovic et al., , 1992Reichelt et al., 2004; Schwemmle et al., 1995;Zhao et...

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.