Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Although many workers have investigated the maturation and processing of the flavivirus nonstructural glycoprotein NS1 in infected cells, these studies have provided little insight into a possible function for NS1. In this study we investigated the subcellular localization of NS1 both by immunofluorescence and cryo-immuno electron microscopy of infected Vero and C6/36 cells. NS1 was found to be tightly associated with intracellular membranes, in particular with vesicle packets and large cytoplasmic vacuoles. Surprisingly, NS1 did not associate with mature virus particles, a finding that is inconsistent with the postulated role for this protein in virion assembly and/or maturation. However, dual-labeling experiments did reveal in both the confocal immunofluorescence and cryo-immuno EM studies the colocalization of NS1 with the viral dsRNA replicative form. Furthermore, localization of the dsRNA to the vesicle packets and cytoplasmic vacuoles seen in infected Vero and C6/36 cells, respectively, suggests that these structures may comprise the flavivirus replication complex. These findings provide further insights into the maturation pathway of NS1 and suggest a possible role for this protein in viral RNA replication.

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...

In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immunogold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.

Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically hampered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0-and 2.35-Å resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.The Flaviviridae form a large family of single-stranded positive-sense RNA viruses comprising the three genera Hepacivirus, Pestivirus, and Flavivirus. The genus Flavivirus contains more than 80 known arthropod-borne viruses, including major human and animal pathogens such as dengue virus (DENV), 3 yellow fever virus, Japanese encephalitis virus, and West Nile virus (WNV). Both DENV and WNV are considered as emerging pathogens. Dengue fever is one of the most important mosquito-borne viral diseases in the world, with more than 3 billion people at risk in endemic tropical areas (1). Dengue outbreaks are increasingly severe in terms of cases and fatalities in many regions of the world (2). WNV was discovered in the West Nile district in Uganda in 1937 and was subsequently shown to have an extensive worldwide distribution with the exception of the Americas (1). In 1999, WNV was introduced into the Americas in the New York City area and has since spread throughout the mainland United States, southern Canada, and Mexico. WNV epidemics in the United States have resulted in a total of 23,925 cases of human disease and 946 deaths reported to the Centers for Disease Control (CDC) from 1999 to 2006. WNV consists of 2 lineages (I and II). The North American WNV isolates belong to lineage I, which also includes the Australian subtype Kunjin (3). In contrast to other lineage I WNV strains (4), infections with the Kunjin subtype of WNV do not cause fatal disease in humans (5).The Flavivirus positive sense RNA genome contains a single open reading frame encoding a polyprotein that is processed into three structural and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Signature-sequence analysis suggests that the ...

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.