Biochemical reconstitution of hemorrhagic-Fever arenavirus envelope glycoprotein-mediated membrane fusion.
ABSTRACT The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.
SourceAvailable from: Patrice Guillon[Show abstract] [Hide abstract]
ABSTRACT: Background: Over the past century dengue (DENV2), chikungunya (CHIKV) and human parainfluenza (hPiV3) viruses have profoundly impacted on human morbidity, mortality and the economy worldwide. Current therapy options to treat infections of these viruses have severe limitations leading to a continued search for novel drug candidates. Acrostichum aureum L. (Pteridaceae) is a mangrove fern, that has been used as a traditional medicine in Bangladesh and other various countries for a variety of diseases including infection. Objectives: Isolation and structural elucidation of novel antiviral secondary metabolites from the methanol extract of the aerial parts of A. aureum. Materials and methods: The novel phthalate acid ester was isolated (HPLC) and structurally elucidated using 1D and 2D NMR, MS and other spectroscopic methods. The compound was tested for antiviral activity against DENV2 andhPiV3in Vero cells using the fluorescent focus (FFA) assay and against CHIKV virus in LLC-MK2 cells using the plaque-forming unit assay (PFU). The activity of the isolated compound was further compared with its known derivative. Results: In this study, we report on the isolation of a novel phthalic acid ester, 2’’-(methoxycarbonyl)-5’’- methylpentyl 2’-methylhexyl phthalate from the aerial parts of the Bangladeshi mangrove fern Acrostichumaureum and its in vitro antiviral activity. The novel phthalate showed antiviral activity against dengue virus, human parainfluenza virus and chikungunya. The most potent activity was recorded against hPiV3 (EC50 29.4 μM) and was slightly higher than the activity determined for the positive control BCX 2798 (EC50 44 μM). Cellulose acetate phthalate was also evaluated for antiviral activity against these viruses for the first time and was found to be inactive. Both compounds were found to be non-toxic against Vero and LLC-MK2 cells. Conclusion: This study shows that some selected phthalates have potent antiviral activity and should be further investigated as potential novel antiviral agents.Journal of Antivirals and Antiretrovirals 10/2013; 5(6):139-144. DOI:10.4172/jaa.1000078