A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous "organelles" that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus.

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.

Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center.

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Although Human noroviruses are significant enteric pathogens, there exists no reliable vaccine or therapy to treat infected individuals. To date, attempts to cultivate Human noroviruses within the laboratory have met with little success; however, the related murine norovirus mouse norovirus 1 (MNV-1) has provided an ideal model system to study norovirus replication due to the ease with which the virus is cultivated and the ability to infect a small animal model with this virus. Previously we have identified the association between MNV-1 and components of the host secretory pathway and proposed a role for the viral open reading frame 1 proteins in the replication cycle. Here we describe for the first time a role for cytoskeletal components in early MNV-1 replication events. We show that the MNV-1 utilizes microtubules to position the replication complex adjacent to the microtubule organizing center. Chemical disruption of the microtubule network disperses the sites of MNV-1 replication throughout the cell and impairs production of viral protein and infectious virus. Furthermore, we demonstrate the ability of MNV-1 to redistribute acetylated tubulin to the replication complex and that this association is potentially mediated via the MNV-1 major structural protein, VP1. Transient expression of MNV-1 VP1 exhibited extensive colocalization with both α-tubulin and acetylated tubulin and was observed to alter the distribution of acetylated tubulin in transfected cells. This study highlights the role of the cytoskeleton in early virus replication events and demonstrates the importance of this interaction in establishing the intracellular location of MNV-1 replication complexes.

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.