The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs - PubMed (original) (raw)

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

Hans Hemmes et al. RNA. 2007 Jul.

Abstract

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.

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Figures

FIGURE 1.

Suppression of RNA silencing by NS3 in cultured Drosophila cells. Cells were transfected with pAc-eGFP and empty pMK33 (A) and treated 1 h after transfection with dsRNA specific for eGFP. Cells in panels B and C were transfected with the same transfection mixture containing pAc-eGFP and pMK33-NS3. NS3 expression was induced with CuSO4, resulting in an increase of the eGFP signal (C) compared to the noninduced cells (B). Expression of NS3 was confirmed by Western blot analysis using bacterial expressed HIS-tagged NS3 as positive control (D).

FIGURE 2.

GFP silencing suppression of MBP–NS3 in _Agrobacterium_-infiltrated Nicotiana benthamiana leaves visualized 5 d post-infiltration. From left to right: noninfiltrated wild-type and GFP expression constructs coinfiltrated with an empty binary vector, the MBP construct, and MBP–NS3 binary vector, respectively.

FIGURE 3.

Affinity of MBP–NS3 for different RNA duplexes. A dilution series of MBP–NS3 (0.01–3770 nM) was incubated with 100 pM each of 32P-labeled 21 nt siRNA duplex (A), 19 nt blunt ended RNA duplex (B), or 26 nt siRNA duplex (C) for 20 min, then loaded onto a 5% native gel. The Kd was determined of MBP–NS3 for the different small RNA molecules by plotting the bound RNA fraction as a function of the MBP–NS3 concentration (D). In panels A, B, and C the first lane contains only siRNAs.

FIGURE 4.

Gel filtration of the MBP–NS3–siRNA complex. MBP–NS3 was incubated with 32P-labeled siRNAs and size separated on a Superdex-200 column. Fractions were collected and tested for the presence of 32P-labeled siRNAs (top panel). As control 32P-labeled siRNAs were size separated in the absence of MBP–NS3 (lower panel). The elution position of protein molecular weight markers is indicated by arrows below the picture: 669 kDa, thyroglobulin (9.1 mL); 441 kDa, ferritin (10.5 mL); 158 kDa, aldolase (12.1 mL); 66 kDa, bovine serum albumin (14.3 mL); and 29 kDa, carbonic anhydrase (16.3 mL).

FIGURE 5.

Inhibition of RISC assembly by NS3 in vitro. (A) In direct competition experiments, RISC assembly was monitored by adding 32P-labeled siRNAs and MBP–NS3 (0.4–755.0 nM) to Drosophila embryo extract. (B) Indirect competition assay where RISC assembly was initiated by adding 32P-labeled siRNAs to embryo extract. MBP–NS3 (0.4–755.0 nM) was added to preincubated reactions after 30 min. (A,B) Lane 1 contains only free siRNAs, lane 2 32P-labeled siRNAs and embryo extract, and lane 3 32P-labeled siRNAs and 23.6 nM MBP–NS3. In lanes 4–15 the competition effect of MBP–NS3 on RISC assembly is shown. (C) For direct and indirect competition experiments the formation of the RISC complex as a function of MBP–NS3 concentration is plotted relative to the RISC formation in the absence of MBP–NS3 (lane 2).

FIGURE 6.

NS3 inhibits siRNA-mediated target cleavage in the Drosophila embryo extract in vitro RNA silencing system. (A) In direct competition assays, RISC-mediated target RNA (0.5 nM) cleavage was induced by siRNAs (5 nM) and MBP–NS3 (0.4–755.0 nM) simultaneously added to Drosophila embryo extracts. (B) In indirect competition, RISC was preassembled by adding siRNAs (5 nM) to embryo extract for 30 min and target RNA (0.5 nM) and MBP–NS3 (0.4–755.0 nM) subsequently added. (C) For direct and indirect competition experiments the percentage of cleaved target is plotted as a function of the MBP–NS3 concentration relative to the percentage of cleaved target in the absence of MBP–NS3. (A,B) Lanes 1 include siRNAs and lack MBP–NS3; lanes 2 lack inducer siRNA and MBP–NS3. We note that Drosophila embryo extract was used at the same concentration as we used for RISC assembly experiments (1 μg/μL in the test tube).

FIGURE 7.

Affinity of MBP–NS3 for different miRNA duplexes. A dilution series of MBP–NS3 (0.01–3770 nM) was incubated with 100 pM each of 32P-labeled Ath-miR171a (A), Ath-miR171b (B), or Ath-miR171c (C) for 20 min, then loaded onto a 5% native gel. The Kd was determined of MBP–NS3 for the different small RNA molecules by plotting the bound fraction as a function of the MBP–NS3 concentration (D). In panels A, B, and C the first lane contains only miRNAs.

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The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs - PubMed (original) (raw)