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.

Efficient RNA polyuridylation by noncanonical poly(A) polymerases.

Nuclear poly(A) polymerase (PAP) polyadenylates nascent mRNAs, promoting their nuclear export, stability, and translation, while the related cytoplasmic polymerase GLD-2 activates translation of deadenylated mRNAs. Here we characterize the biochemical activity of fission yeast Schizosaccharomyces pombe Cid1, a putative cytoplasmic PAP implicated in cell cycle checkpoint controls. Surprisingly, Cid1 has robust poly(U) polymerase activity in vitro, especially when isolated in native multiprotein complexes. Furthermore, we found that upon S-phase arrest, the 3' ends of actin mRNAs were posttranscriptionally uridylated in a Cid1-dependent manner. Finally, Hs2 (ZCCHC6), a human ortholog of Cid1, shows similar activity. These data suggest that uridylation of mRNA forms the basis of an evolutionarily conserved mechanism of gene regulation.

The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3.

The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-beta) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-alpha/beta receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-alpha4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-beta gene, by blocking IRF-3 activation.

Existing antivirals are effective against influenza viruses with genes from the 1918 pandemic virus.

The 1918 influenza pandemic caused more than 20 million deaths worldwide. Thus, the potential impact of a re-emergent 1918 or 1918-like influenza virus, whether through natural means or as a result of bioterrorism, is of significant concern. The genetic determinants of the virulence of the 1918 virus have not been defined yet, nor have specific clinical prophylaxis and/or treatment interventions that would be effective against a re-emergent 1918 or 1918-like virus been identified. Based on the reported nucleotide sequences, we have reconstructed the hemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of the 1918 virus. Under biosafety level 3 (agricultural) conditions, we have generated recombinant influenza viruses bearing the 1918 HA, NA, or M segments. Strikingly, recombinant viruses possessing both the 1918 HA and 1918 NA were virulent in mice. In contrast, a control virus with the HA and NA from a more recent human isolate was unable to kill mice at any dose tested. The recombinant viruses were also tested for their sensitivity to U.S. Food and Drug Administration-approved antiinfluenza virus drugs in vitro and in vivo. Recombinant viruses possessing the 1918 NA or both the 1918 HA and 1918 NA were inhibited effectively in both tissue culture and mice by the NA inhibitors, zanamivir and oseltamivir. A recombinant virus possessing the 1918 M segment was inhibited effectively both in tissue culture and in vivo by the M2 ion-channel inhibitors amantadine and rimantadine. These data suggest that current antiviral strategies would be effective in curbing the dangers of a re-emergent 1918 or 1918-like virus.