Inhibition of Poxvirus Gene Expression and Genome Replication by Bisbenzimide Derivatives

Abstract

Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or antiviral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole), and its derivatives as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double-stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating postreplicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of their class, which appear to directly target the viral genome without affecting cell viability.IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus life cycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and block viral replication.

Label-free digital holo-tomographic microscopy reveals virus-induced cytopathic effects in live cells

“…A progressive and prominent change in RI was observed in the infected cells, visualized in scaled pseudo-color. In contrast, the uninfected cells as well as the VACV-GFP infected cells treated with the deoxy-nucleoside analogue cytarabine (AraC), an inhibitor of VACV late gene expression (16,17), showed less prominent RI changes, although AraC-treated cells inoculated with VACV-GFP exhibited a strong increase in RI in the cell nucleus. We quantified the RI change by deriving the RIG values across the entire cells ( Fig.…”

“…All cell cultures were maintained in a cell bank system and kept in low passages for all experiments. Vaccinia virus strain International Health Department J (VACV-IHD-J) containing early / late (E/L) GFP transgene was kindly provided by J. Mercer (University College London, UK) (17,62). To obtain the purified mature virions (MVs) cytoplasmic lysates were pelleted through a 36% sucrose cushion for 90 min with Optima XPN-100 ultracentrifuge (Beckmann Coulter) SW32Ti rotor at 18,000 rpm.…”

Antivirals in medical biodefense

“…Anti-poxvirus drugs effective in animal models are reviewed in more detail elsewhere [133]. Further candidate anti-poxvirus drugs include kinase inhibitors imatinib (Gleevec/STI-571; [122,123]) and olomoucine [61], terameprocol [119], mitoxandrone [5], the membrane targeting ddBCNA cf2642 [99], bisbenzimide derivatives [151], FC-6407, a OPV D4 processivity factor mimic [111], and a number of natural extracts that have shown interesting antiviral activity against OPV in in vitro infection models [27,153] (Table 1).…”

Concepts in Light Microscopy of Viruses

“…A prototypic cell-permeable small molecular weight dye is Hoechst 33342, which has been used for DNA staining in live cells since the 1970s [ 114 ]. The Hoechst dyes absorb and emit light in the ultraviolet range, and are frequently used in live imaging, for example, to monitor DNA contents during the cell cycle, or viral replication compartments in the cytoplasm [ 62 , 115 , 116 ]. Other dyes that are frequently used in live cell imaging are DRAQ5 to stain DNA in the far-red excitation spectra [ 117 ], the mitochondrial dye chloromethyl-X-rosamine (MitoTracker) [ 118 ] and a series of lysosomotropic dyes for acidic vesicles [ 119 ].…”