[show abstract][hide abstract] ABSTRACT: Given the poor immunogenicity of current H5N1 influenza vaccines, additives and adjuvants remain a viable solution for increasing efficacy. Here, we demonstrate that a 20-amino acid peptide (EB) possessing influenza antiviral activity also enhances the immune response to H5N1 vaccination in mice. The addition of EB to formalin-inactivated whole-virus vaccine induced virion aggregation and these aggregates were readily engulfed by phagocytic cells in vitro. In vivo, mice vaccinated with a suboptimal dose of inactivated vaccine containing EB peptide had reduced morbidity, improved viral clearance, and faster recovery than mice receiving vaccine alone. This phenomenon was not accompanied by an increase in virus-specific antibodies. Instead, cell-mediated immunity was enhanced as demonstrated by increased interferon-γ production from splenocytes. This data demonstrates that the EB peptide may a useful adjuvant for boosting the efficacy of poorly immunogenic influenza vaccines.
[show abstract][hide abstract] ABSTRACT: The antiviral peptide, entry blocker (EB), inhibits influenza virus replication by preventing attachment to cells. Here, we identified the minimal and optimal EB sequence that retained antiviral activity with a 50% inhibitory concentration (IC(50)) and 50% effective concentration (EC(50)) similar to those of the full-length EB peptide and several truncated variants that possessed up to 10-fold lower IC(50)s. These data have implications for improving the antiviral efficacy of EB-derived peptides while decreasing production costs and easing synthesis.
Antimicrobial Agents and Chemotherapy 01/2011; 55(4):1810-3. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ∼50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only "zippered" together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.
Journal of Virology 10/2010; 84(24):12492-503. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family, Nodaviridae), induces robust apoptosis of permissive Drosophila Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of Drosophila apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone (z-VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal Drosophila effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in Drosophila melanogaster, was dramatically reduced. DIAP1 was depleted despite z-VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable diap1-transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.
Journal of Virology 03/2008; 82(3):1378-88. · 5.08 Impact Factor