Abstract
The re-emergence of poxviral zoonotic infections and the threat of bioterrorism emphasise the demand for effective antipoxvirus therapies. Here, we show that carbenoxolone, a pharmacological inhibitor of gap junction function and a compound widely used in cell culture, is capable of hindering the replication of Vaccinia virus, the prototypical poxvirus, in a gap junction-independent manner in a human keratinocyte cell line. Viral protein synthesis occurs in the presence of carbenoxolone but infectious virion formation is minimal, indicating that carbenoxolone blocks viral morphogenesis. Initial viability tests suggested that carbenoxolone was not toxic to cells. However, electron microscopic analysis of carbenoxolone treated cells revealed that it alters the cellular endomembrane system. This widespread ultrastructural damage prevents Vaccinia virus virion assembly. These results strengthen the need for thorough characterisation of the effects of antiviral compounds on the cellular ultrastructure.
Highlights
Vaccinia virus (VACV) is the prototypical member of the Poxviridae, a family of large DNA viruses that replicate entirely in the cytoplasm of infected cells
As poxviruses are well-known to replicate and cause pathology in keratinocytes, we opted to investigate the effect of CBX in VACV replication in a human keratinocyte cell line
HaCaT cells, a spontaneously immortalised human keratinocyte line[12], were pre-treated for 1 h with different concentrations of CBX or carrier prior to infection with VACV-A5L-EGFP, a VACV construct in which the core A5 protein has been tagged with EGFP13, allowing estimation of virus growth on the basis of fluorescence levels[14]
Summary
HaCaT cells, a spontaneously immortalised human keratinocyte line[12], were pre-treated for 1 h with different concentrations of CBX or carrier prior to infection with VACV-A5L-EGFP, a VACV construct in which the core A5 protein has been tagged with EGFP13, allowing estimation of virus growth on the basis of fluorescence levels[14]. Subsequent steps in cells pre-treated with CBX were carried out in the presence of the compound. VACV infection was carried out at 0.1 pfu/cell (MOI = 0.1) and fluorescence levels were measured at 0, 24 and 48 h p.i. We observed that CBX inhibited VACV replication in a dose-dependent manner (Fig. 1A). ® treated with CBX were compared to the carrier control at 48 h p.i. using Cell-Titer Blue (Promega) which measures toxicity through the ability of viable cells to metabolise resazurin into fluorescent resorufin. No evidence of toxicity was identified up to 30 μM
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