In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

Development of FRET-based high-throughput screening for viral RNase III inhibitors.

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.

Design, Synthesis, and Anti-ToCV Activity of Novel 4(3H)-Quinazolinone Derivatives Bearing Dithioacetal Moiety.

Tomato chlorosis virus (ToCV) has caused great harm to the production of tomato worldwide. To develop efficient anti-ToCV agents, some novel 4(3H)-quinazolinone derivatives containing dithioacetal were designed and synthesized, and their anti-ToCV activities were evaluated by microscale thermophoresis (MST) using ToCV coat protein (ToCV-CP) as a new target. The results showed that some compounds had a strong binding capacity to ToCV-CP. In particular, compounds C5 and C22 have an excellent binding capacity to ToCV-CP, with binding constant values of 0.24 and 0.25 µM, respectively. Additionally, reduced ToCV-CP gene expression levels of 81.05 and 87.59% could be achieved when tomato was treated with compounds C5 and C22, respectively, which were obviously higher than the levels after ningnanmycin (NNM) treatment (43.88%) and lead compound Xiangcaoliusuobingmi (XCLSBM) treatment (63.56%). Therefore, this work indicates that 4(3H)-quinazolinone derivatives containing dithioacetal moiety can be used as novel anti-ToCV agents.

Design, Synthesis, and Anti-ToCV Activity of Novel Pyrimidine Derivatives Bearing a Dithioacetal Moiety that Targets ToCV Coat Protein.

Novel pyrimidine sulfide derivatives containing a dithioacetal and strobilurin moiety were designed and synthesized. Their antiviral activities against tomato chlorosis virus (ToCV) were investigated through the tomato chlorosis virus coat protein (ToCVCP)-oriented screening method. Microscale thermophoresis was used to study the interaction between the compound and the ToCVCP. Compounds B13 and B23 interacted better with ToCVCP than the other compounds and had dissociation constant (Kd) values of 0.09 and 0.06 µM, respectively. These values were lower than those of the control agents, ningnanmycin (0.19 µM) and ribavirin (6.54 µM), which indicated that the compounds had a strong binding effect with ToCVCP. Quantitative real-time polymerase chain reaction was used to evaluate the role of compounds B13 and B23 in the gene regulation of ToCVCP. Both compounds significantly reduced the expression level of the ToCVCP gene in Nicotiana benthamiana with reduction values of 88 and 83%, which were better than those of ningnanmycin (65%) and lead compound C14 (73%). Pyrimidine sulfide containing a dithioacetal and strobilurin moiety is significant in the research and development of novel anti-ToCV agents.

Discovery of Potent and Novel Quinazolinone Sulfide Inhibitors with Anti-ToCV Activity.

A series of novel quinazolinone sulfide derivatives containing a dithioacetal moiety were designed and synthesized using Tomato chlorosis virus coat protein (ToCVCP) as a potential drug target, and the inhibitory effect of ToCV was systematically evaluated in vitro and in vivo. The experimental results showed that most of the compounds presented a strong affinity. Notably, the binding abilities of compounds D8 and D16 to ToCVCP both reached a micromolar level, which were 0.19 and 0.83 µM, respectively. The relative expression level of ToCVCP gene was detected using real-time quantitative polymerase chain reaction in Nicotiana benthamiana. Compounds D8 and D16 significantly reduced the relative expression level of ToCVCP gene by 93.34 and 83.47%, respectively, which were better than those of conventional antiviral agents. This study lays a good foundation for the structural design and modification of quinazolinone sulfide derivatives as anti-ToCV drugs.

First Anti-ToCV Activity Evaluation of Glucopyranoside Derivatives Containing a Dithioacetal Moiety through a Novel ToCVCP-Oriented Screening Method.

Tomato chlorosis virus (ToCV) is a newly reported plant virus that has rapidly spread to all parts of the world, resulting in a serious decline in tomato quality and yield due to the lack of effective control agents. In this study, the ToCV coat protein (ToCVCP) was expressed and purified in Escherichia coli, and a series of novel glucopyranoside derivatives containing a dithioacetal moiety was designed and synthesized. The binding affinity of these compounds to ToCVCP was determined using microscale thermophoresis. Results revealed that compounds 6b and 8a interacted with ToCVCP with Kd values of 0.12 and 0.21 µM, respectively. Quantitative reverse transcription polymerase chain reaction was used to evaluate the anti-ToCV activity of 6b and 8a in vivo, and both significantly reduced the expression level of ToCVCP gene in tomato compared with commercial antivirus agents. This study provides an efficient and convenient screening method for anti-ToCV agents and reliable support for the development of novel agrochemicals for ToCV.

Induction of antiviral responses by acibenzolar-s-methyl against cucurbit chlorotic yellows virus in Melon.

Cucurbit chlorotic yellows virus (CCYV) (family Closteroviridae, genus Crinivirus) is an emerging virus which causes severe diseases on melon (Cucumis melo) plants. CCYV-infected melon plants display yellowing, mottling, chlorosis, or chlorotic spots on leaves. To develop a new control strategy, the potential for 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester (ASM) to suppress CCYV infection was evaluated. ASM treatment on melon plants greatly increased the expression levels of pathogenesis-related 1a gene, a marker gene for systemic acquired resistance. ASM treatment on melon plants before inoculation of CCYV suppressed systemic symptoms and decreased CCYV accumulation. ASM treatment on melon even after inoculation of CCYV reduced disease severity and accumulation levels of CCYV. The results show the potential for ASM treatment on attenuation of the CCYV disease symptoms.

Disease control and its selection for damaging plant virus strains in vegetatively propagated staple food crops; a theoretical assessment.

Viral diseases are a key constraint in the production of staple food crops in lesser developed countries. New and improved disease control methods are developed and implemented without consideration of the selective pressure they impose on the virus. In this paper, we analyse the evolution of within-plant virus titre as a response to the implementation of a range of disease control methods. We show that the development of new and improved disease control methods for viral diseases of vegetatively propagated staple food crops ought to take the evolutionary responses of the virus into consideration. Not doing so leads to a risk of failure, which can result in considerable economic losses and increased poverty. Specifically in vitro propagation, diagnostics and breeding methods carry a risk of failure due to the selection for virus strains that build up a high within-plant virus titre. For vegetatively propagated crops, sanitation by roguing has a low risk of failure owing to its combination of selecting for low virus titre strains as well as increasing healthy crop density.