Identification of a Broad-Spectrum Inhibitor of Viral RNA Synthesis: Validation of a Prototype Virus-Based Approach

Filone, Claire Marie; Hodges, Erin; Honeyman, Brian; Bushkin, G. Guy; Boyd, Kathryn E.; Platt, Andrew; Feng, Na; Strom, Kyle R.; Hensley, Lisa E.; Snyder, John K.; Connor, John H.

doi:10.1016/j.chembiol.2013.02.011

Cited by 21 publications

(9 citation statements)

References 30 publications

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“…However, our data clearly show that under such conditions GFP-expressing viruses provide significantly lower sensitivity than luciferase-expressing viruses, and require much longer assay times. As a consequence, the only study that has employed this approach so far used a high infectious dose (MOI of 1) and readout times of 5 days after infection for EC50 determination, and 3 days after infection for direct visualization of GFP expression (Filone et al, 2013), which corresponds well to our own results (Fig. 3A).…”

Section: Resultssupporting

confidence: 84%

“…In contrast, measuring reporter activity of rgEBOV-luc2 represents an end-point assay, since cells have to be lysed prior to measurement. Another alternative that has only very recently been explored is the use of rgEBOV-GFP for screening purposes in the absence of high-content imaging, just relying on overall GFP expression in a well (Filone et al, 2013). Such an approach offers low equipment costs, comparable to luciferase-based assays, and is even less labor intensive, since no reagents have to be added for measurement.…”

Section: Resultsmentioning

confidence: 99%

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A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals

et al. 2013

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Ebola virus (EBOV) causes a severe hemorrhagic fever with case fatality rates of up to 90%, for which no antiviral therapies are available. Antiviral screening is hampered by the fact that development of cytopathic effect, the easiest means to detect infection with wild-type EBOV, is relatively slow. To overcome this problem we generated a recombinant EBOV carrying a luciferase reporter. Using this virus we show that EBOV entry is rapid, with viral protein expression detectable within 2 hours after infection. Further, luminescence-based assays were developed to allow highly sensitive titer determination within 48 hours. As a proof-of-concept for its utility in antiviral screening we used this virus to assess neutralizing antibodies and siRNAs, with significantly faster screening times than currently available wild-type or recombinant viruses. The availability of this recombinant virus will allow for more rapid and quantitative evaluation of antivirals against EBOV, as well as the study of details of the EBOV life cycle.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals

et al. 2013

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“…DNA encoding the minigenome is cloned into an expression plasmid and co-transfected into cells along with plasmids encoding the viral proteins required for EBOV genome replication and transcription. Importantly, EBOV minigenome systems have also been used extensively for antiviral drug screening (Edwards et al, 2015; Enterlein et al, 2006; Filone et al, 2013; Jasenosky et al, 2010; Olsen et al, 2016; Uebelhoer et al, 2014). Most of the currently existing EBOV minigenomes are under the control of the T7 RNA polymerase (T7) promoter and thus require T7 expression in the transfected cells (Garcia-Dorival et al, 2016; Mühlberger et al, 1999; Uebelhoer et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

et al. 2017

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Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

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“…The exact mechanism of FGI-103, FGI-106 and derivatives is unknown. Although the molecular target is not known, CMLDBU3402 (25) exhibited broad spectrum activity against a number of non-segmented single RNA viruses including EBOV and inhibited RNA transcription in EBOV (Filone et al, 2013). Other compounds, which were shown to have activity against Ebola virus include antioxidants , selective estrogen receptor modulators (Johansen et al, 2013), cell cycle inhibitors and kinase inhibitors (Kolokoltsov et al, 2012).…”

Section: Other Potential Targets and Miscellaneous Compounds With Actmentioning

confidence: 97%

Ebola Virus Potential Drug Targets and Prospects for Small Molecule Drug Discovery

Assefa¹

2014

j pharmaceut sci pharmacol

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Ebola virus is an enveloped non-segmented negative-sense single strand RNA virus. It causes a fetal severe hemorrhagic fever or Ebola virus disease with very low recovery rate. Since its occurrence in the 1970s in northern Zaire and south Sudan, there has been numerous outbreaks, all of which in the tropical regions of Africa. The latest and the largest one and still ongoing was first identified in March 2014. Although the outbreaks were in the tropical Africa, other countries have been affected because of the travel and highly contagious nature of the Ebola virus disease. In addition, there is a potential for the virus to be used as a bioterrorism weapon. Therefore, Ebola virus disease poses a global health threat. Due to the frequent outbreaks, highly contagious and fast replication of the virus, high mortality rate from Ebola virus disease, and the potential of the virus for being used as bioweapon, there is an urgent need for the discovery of Ebola virus disease therapeutic agents. Numerous compounds have been found to inhibit Ebola virus infection in vitro and in animal models, and several viral and host proteins have been identified as potential targets. Among these, the viral RNA-dependent RNA polymerase inhibitors, Viral entry inhibitors, the host S-adenosylhomocysteine hydrolase inhibitors are the most promising. Due the fast replication and spread of the virus, a combination of the RNA polymerase, entry and S-adenosylhomocysteine hydrolase inhibitors would be very effective in controlling the disease and decreasing mortality.

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Identification of a Broad-Spectrum Inhibitor of Viral RNA Synthesis: Validation of a Prototype Virus-Based Approach

Cited by 21 publications

References 30 publications

A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals

A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

Ebola Virus Potential Drug Targets and Prospects for Small Molecule Drug Discovery

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