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...