Antiviral Activity of Flexibilane and Tigliane Diterpenoids from Stillingia lineata
Florent OlivonHéliciane PalenzuelaEmmanuelle Girard-ValenciennesJohan NeytsChristophe PannecouqueFanny RoussiIsabelle GrondinPieter LeyssenMarc Litaudon
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Abstract:
In an effort to identify new potent and selective inhibitors of chikungunya virus and HIV-1 and HIV-2 virus replication, the endemic Mascarene species Stillingia lineata was investigated. LC/MS and bioassay-guided purification of the EtOAc leaf extract using a chikungunya virus-cell-based assay led to the isolation of six new (4–9) and three known (1–3) tonantzitlolones possessing the rare C20-flexibilane skeleton, along with tonantzitloic acid (10), a new linear diterpenoid, and three new (11, 13, and 15) and two known (12 and 14) tigliane-type diterpenoids. The planar structures of the new compounds and their relative configurations were determined by spectroscopic analysis, and their absolute configurations were determined through comparison with literature data and from biogenetic considerations. These compounds were investigated for selective antiviral activity against chikungunya virus (CHIKV), Semliki Forest virus, Sindbis virus, and, for compounds 11–15, the HIV-1 and HIV-2 viruses. Compounds 12–15 were found to be the most potent and are selective inhibitors of CHIKV, HIV-1, and HIV-2 replication. In particular, compound 14 inhibited CHIKV replication with an EC50 value of 1.2 μM on CHIKV and a selectivity index of >240, while compound 15 inhibited HIV-1 and HIV-2 with EC50 values of 0.043 and 0.018 μM, respectively. It was demonstrated further that potency and selectivity are sensitive to the substitution pattern on the tigliane skeleton. The cytotoxic activities of compounds 1–10 were evaluated against the HCT-116, MCF-7, and PC3 cancer cell lines.Keywords:
Sindbis virus
Semliki Forest virus
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AbstractAlphaviruses are RNA enveloped viruses that are proving their value as expression vectors. They are particularly well-suited for this role as they are easily and quickly engineered and can be used to produce high levels of proteins of interest. A promising and important use is as vaccines against disease-causing agents such as HIV. The three alphaviruses now serving as vectors are Sindbis virus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEE) virus. Sindbis virus and SFV are well-known models for studies in molecular and cell biology; VEE virus is a human pathogen and had received some previous notoriety as a potential biological weapon. It is now becoming a potentially valuable vaccine vector. All three viruses are being tested as vaccines but, at present, only Sindbis virus and SFV have been considered for other uses. Sindbis virus vectors have been developed to screen libraries for the identification of new proteins and to devise sensitive assays to detect viruses more difficult to grow in culture. Both Sindbis virus and SFV vectors are serving as tools for fundamental studies in biology, examples include development in insects and analysis of protein functions in neuronal cells. In this article the replication strategy of alphaviruses and the different ways they can be engineered to serve as expression vectors is described. This provides an introduction to the ways these vectors have been used and illustrates the promise these vectors offer.Keywordsalphavirusesexpression vectorsRNA virusesSemliki Forest virusSindbis virusvaccinesVenezuelan equine encephalitisvirus detection assays
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Semliki Forest virus
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Semliki Forest virus
Sindbis virus
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Abstract Semliki Forest virus (SFV) is a neuropathogenic alphavirus which is of interest both as a model neurotropic alphavirus and as an oncolytic virus with proven potency in preclinical cancer models. The route of central nervous system (CNS) entrance of SFV is poorly understood but has been considered to occur through the blood-brain barrier. Here, we show that SFV primarily enters the CNS through the blood-cerebrospinal fluid barrier, and that VLDLR is crucial in enabling SFV infection of the choroid plexus epithelial cells.
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Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.
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Semliki Forest virus (SFV) and Sindbis virus (SIN) are two, positive-strand RNA viruses of the alphavirus genus. Vectors for both have been developed to express high levels of foreign genes in vitro and in vivo. Basic Protocol 1 describes the preparation of packaged SFV and SIN replicons by co-electroporation of helper and vector RNA into baby hamster kidney (BHK)-21 cells. Basic Protocol 2 describes the activation of packaged SFV replicons with a-chymotrypsin. Basic Protocol 3 provides a method for the infection of hippocampal slices. Basic Protocol 4 is a technique for the infection of primary cultures of dispersed neurons with infectious SFV and SIN replicons. The Alternate Protocol describes a method for the cotransfection of in vitro-transcribed vector and helper RNA into BHK-21 cells. Support Protocol 1 describes determining the titers of infectious SFV and SIN replicon stocks, and Support Protocol 2 for metabolic labeling of infected cells.
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Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10 7 infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.
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Chikungunya virus (CHIKV), an alphavirus transmitted by mosquitoes, has experienced a recent re-emergence in various regions of the world, leading to large-scale outbreaks [...]
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Abstract Semliki Forest virus and Sindbis virus belong to the alphavirus genus of the togaviruses and have recently been shown to efficiently infect neurons in vitro and in vivo, i.e., under conditions where glial cells are present. This unit describes the preparation and application of recombinant, neurotrophic replicons derived from Semliki Forest virus and Sindbis virus for gene transfer into hippocampal neurons.
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