In vitro identification and in vivo confirmation of inhibitors for SPCSV 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 loss 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 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 a kinetic-based HTS using fluorescence resonance energy transfer technology that isolated 112 compounds. These compounds were validated with dose-response assays including the 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. Significance statement We report here a high-throughput inhibitor identification that targets a severe sweetpotato virus disease caused by co-infection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield loss. 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.

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Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

PLoS ONE ◽

10.1371/journal.pone.0081479 ◽

2013 ◽

Vol 8 (11) ◽

pp. e81479 ◽

Cited By ~ 18

Author(s):

Arthur K. Tugume ◽

Robert Amayo ◽

Isabel Weinheimer ◽

Settumba B. Mukasa ◽

Patrick R. Rubaihayo ◽

...

Keyword(s):

Genetic Variability ◽

Sweet Potato ◽

Wild Species ◽

Rna Silencing ◽

Silencing Suppressor ◽

Suppressor Genes ◽

Rna Silencing Suppressor ◽

Related Wild Species ◽

Virus Isolates

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The P1N-PISPOtrans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

Journal of Virology ◽

10.1128/jvi.02360-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3543-3557 ◽

Cited By ~ 33

Author(s):

Ares Mingot ◽

Adrián Valli ◽

Bernardo Rodamilans ◽

David San León ◽

David C. Baulcombe ◽

...

Keyword(s):

Mass Spectrometry ◽

Sweet Potato ◽

Rna Silencing ◽

Mottle Virus ◽

Gene Products ◽

Silencing Suppressor ◽

Content Type ◽

Rna Silencing Suppressor ◽

Silencing Suppression ◽

Polymerase Slippage

ABSTRACTThe positive-sense RNA genome ofSweet potato feathery mottle virus(SPFMV) (genusPotyvirus, familyPotyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the −1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965–6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infectedIpomoea batatasplants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene inNicotiana benthamianarevealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within thePotyviridaefamily.IMPORTANCEGene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of thePotyviridaefamily.

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Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism

Journal of General Virology ◽

10.1099/vir.0.83471-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 573-582 ◽

Cited By ~ 49

Author(s):

Wilmer J. Cuellar ◽

Fred Tairo ◽

Jan F. Kreuze ◽

Jari P. T. Valkonen

Keyword(s):

Sweet Potato ◽

Rna Silencing ◽

Gene Content ◽

Gene Gain ◽

Virus Species ◽

Silencing Suppressor ◽

Virus Family ◽

Rna Silencing Suppressor ◽

African Strain ◽

The Family

Sweet potato chlorotic stunt virus (genus Crinivirus) belongs to the family Closteroviridae, members of which have a conserved overall genomic organization but are variable in gene content. In the bipartite criniviruses, heterogeneity is pronounced in the 3′-proximal region of RNA1, which in sweet potato chlorotic stuat virus (SPCSV) encodes two novel proteins, RNase3 (RNase III endonuclease) and p22 (RNA silencing suppressor). This study showed that two Ugandan SPCSV isolates contained the p22 gene, in contrast to three isolates of the East African strain from Tanzania and Peru and an isolate of the West African strain from Israel, which were missing a 767 nt fragment of RNA1 that included the p22 gene. Regardless of the presence of p22, all tested SPCSV isolates acted synergistically with potyvirus sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) in co-infected sweetpotato plants (Ipomoea batatas), which greatly enhanced SPFMV titres and caused severe sweetpotato virus disease (SPVD). Therefore, the results indicate that any efforts to engineer pathogen-derived RNA silencing-based resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene. The data from this study demonstrate that isolates of this virus species can vary in the genes encoding RNA silencing suppressor proteins. This study also provides the first example of intraspecific variability in gene content of the family Closteroviridae and may be a new example of the recombination-mediated gene gain that is characteristic of virus evolution in this virus family.

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