CEPCEB Members

Shou-Wei Ding Professor Department of Plant Pathology University of California Riverside, CA 92521 Phone: (951) 827-2341 Fax: (951) 827-4294 Areas of Expertise Virus-host Interactions RNA Silencing (RNAi) RNAi-based Antiviral Immunity in Plants and Animals
Background Plant Viral Suppressors of RNA Silencing A New Role for MicroRNAs in Viral Pathogenesis Adaptive Antiviral Defense by RNA Silencing - A Drosophila Model RNA Silencing Is A New Antiviral Immunity in Mosquitoes Mammalian Viral Suppressors of RNA Silencing Selected Publications (Bibliography page)

Background

I received my BSc and MSc from China and PhD from the Australian National University in 1990. I went to the National University of Singapore as an Assistant Professor in July 1996 after postdoctoral studies in the Agricultural Canada Research Station in Vancouver and the Waite Agricultural Research Institute of the University of Adelaide in Australia. I came to UCR in December 2000.

An initial goal of the research in my lab was to determine the function of an overlapping gene (2b) encoded by cucumber mosaic virus (CMV), discovered during my postdoctoral study in Dr. Bob Symons's lab in Australia. This effort resulted in the identification of the CMV 2b protein in 1998 as one of the first two viral suppressors of gene silencing, in collaboration with Dr. David Baulcombe's group in the UK. Our subsequent research has focused on the mechanism of gene silencing suppression by viral proteins and their interactions with other host defense pathways. A new project recently developed in UCR is to determine if gene silencing plays a role in the animal defense against viruses. I was elected as a full member of the Asia-Pacific International Molecular Biology Network in 2000 and presented an invited 'State-of-the-Art' Lecture on viral suppression of gene silencing at the American Society for Virology 2001 Annual Meeting in Madison.

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Plant Viral Suppressors of RNA Silencing

Post-transcriptional gene silencing (PTGS) represents a novel cellular pathway conserved in a diverse group of organisms. PTGS is also referred to as co-suppression in plants, quelling in fungi, and RNA interference (RNAi) in animals, and collectively, these processes are called RNA silencing since all involve a homology-dependent RNA degradation (Ding, 2000). It is now recognized that double-stranded (ds) RNA serves as the initial trigger of RNA silencing and upon recognition, is cleaved by a type III ribonuclease into small fragments of about 21 nucleotides in length, which subsequently prime degradation of homologous RNA targets. Remarkably, RNA silencing initiated locally in plants produces a mobile silencing signal that is able to instruct specific RNA degradation at a distance.

We have previously proposed that the CMV 2b protein (Cmv2b) acted as a suppressor of host antiviral defense, based on its requirement for virulence and spread of CMV in host plant infections (Ding et al., 1994; 1995; 1996). This hypothesis was confirmed in a silencing reversal assay (Brigneti et al., 1998), in which Cmv2b expressed from either CMV or another viral vector prevented spread of the GFP transgene RNA silencing into the newly emerging tissues, although established silencing in older tissues was not affected (Fig. 1, top right). The fact that plant viruses encode proteins that suppress RNA silencing provided not only the strongest support that RNA silencing functions as a natural antiviral defense in plants, but also yielded valuable tools for the dissection of the RNA silencing pathway (Li & Ding, 2001).

Fig. 1. GFP silencing reversal by Cmv2b. Under UV illumination, only the silenced tissues of the GFP transgenic plants appear red fluorescent duo to chlorophyll autofluorescence.	Fig. 2. Cellular distribution of GFP fused with Cmv2b of either wildtype (top row), 6A (the NLS replaced by 6 alanines; middle), or 6A+NLS

We have reported recently that Cmv2b carries an arginine-rich nuclear localization signal (NLS; Fig. 2) and nuclear targeting of Cmv2b is required for its suppression activity in the silencing reversal assay (Lucy et al., 2000). Several lines of evidence indicate that Cmv2b prevents silencing spread by directly inhibiting the activity of the mobile silencing signal, as Cmv2b blocked the signal from either leaving the site of production, passing through tissues expressing Cmv2b (Fig. 3), or activating RNA degradation once it arrived at target cells (Guo & Ding, 2002). Significantly, inactivation of the silencing signal in Cmv2b-expressing tissues was found to correlate with a significantly reduced DNA methylation of the target transgene (Guo & Ding, 2002). This finding suggests that the silencing signal and the putative cytoplasmic signal that guides DNA methylation in the nucleus, may share a key component that is the target of Cmv2b.

Fig. 3. Suppression of the signal-mediated spread of GFP RNA silencing, activated in a lower leaf infiltrated with 35S-GFP, by Cmv2b (right), but not by the CmvD2b mutant (left), introduced both into an upper leaf by Agrobacterium infiltration (Guo & Ding, 2002).

The 2b protein encoded by tomato aspermy virus, a member of the Cucumovirus genus like CMV, also suppressed transgene RNA silencing in N. benthamiana plants; but unlike Cmv2b, it triggered a strong hypersensitive virus resistance in a related host species, N. tabacum (Li et al., 1999), further demonstrating the complexity in the molecular strategies employed by pathogens and their hosts for defense and counter-defense. Moreover, our recent work showed that Cmv2b also interferes with the salicylic acid (SA)-induced virus resistance in tobacco, indicating an intriguing cross-talk between the RNA silencing and SA-mediated virus resistance pathways (Ji & Ding, 2001).

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A New Role for MicroRNAs in Viral Pathogenesis

Small interfering RNAs (siRNAs) and microRNAs (miRNA) are processed by the ribonuclease Dicer from distinct precursors, double-stranded (ds) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral whereas an emerging role for miRNAs is in the control of development. We have recently described a virulence factor encoded by turnip yellow mosaic virus, p69, that suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of all seven miRNAs examined, as well as a correspondingly enhanced miRNA-guided cleavage of four host mRNAs. Since p69-expressing plants exhibited disease-like symptoms in the absence of viral infection (Fig. 4), our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.

Fig. 4. Tymoviral suppressor p69 conferred virulence in A. thaliana. Expression of p69 in a transgenic plant (left) causes pleiotropic developmental defects that resemble the disease symptoms (right) triggered by virus infection (Chen et al., 2004).

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Adaptive Antiviral Defense by RNA Silencing - A Drosophila Model It is known that RNAi is active in invertebrates since 1998 and in mammals since 2001. Before our publication in May of 2002 (Li et al., 2002), however, it was not clear if the RNAi pathway provides protection against virus infection in the animal kingdom as has been established in plants. We addressed this question by firstly determining if the B2 protein encoded by the animal nodavirus flock house virus (FHV), predicted to have functional similarity to the 2b protein of plant cucumoviruses (Ding et al., 1995), could suppress RNA silencing in plants using an established co-infiltration assay. This led to the identification of FHV B2 as the first animal viral suppressor of RNA silencing (Fig. 5). Further analyses carried out in cultured Drosophila cells have demonstrated that (i) FHV infection resulted in a rapid accumulation of FHV-specific 22-nt siRNAs, (ii) an FHV mutant that does not express B2 failed to accumulate to detectable levels, but (iii) the same mutant accumulated to near-wild type levels in Drosophila cells that was defective for RNAi due to depletion of Argonaute-2 protein (Li et al., 2002), which is an essential component of the RNA-induced silencing complex (RISC). The same is also true in the infection of Drosophila cells by another nodavirus, Nodamura virus (NoV), as found in a recent study (Li et al., 2004). These finding establishes RNA silencing as a natural antiviral defense in invertebrate animals because virus infection (i) triggers RNA silencing that specifically targets viral RNAs for degradation and (ii) requires a virus-encoded function to suppress RNA silencing. We have recently established a virus-induced silencing assay in cultured Drosophila cells to screen for (i) novel components in the RNA silencing antiviral immunity and (ii) suppressors of this new animal antiviral immunity encoded by other invertebrate and vertebrate viruses (see below).

Fig. 5. Cross-kingdom suppression of RNA silencing in plants by an animal viral protein. RNA silencing of a GFP transgene (center) in leaves from Nicotiana benthamiana is suppressed by an animal (left; B2 protein of flock house virus) or a plant (right; 2b protein of tomato aspermy virus) viral suppressor, leading to enhanced GFP expression (lighter green/yellow areas).

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RNA Silencing Is A New Antiviral Immunity in Mosquitoes

Mosquitoes are the most dangerous animals in the world, killing an estimated 2-3 million people annually. We have recently demonstrated that NoV RNA replication in cultured malaria mosquito (Anopheles gambiae) cells also triggers NoV-specific RNA silencing and that the B2 protein of NoV is required to suppress RNA silencing for successful NoV accumulation in the mosquito cells. Our findings indicate that the RNAi pathway in mosquitoes provides protection against viruses. Therefore, we may achieve effective control of mosquitoes and mosquito-borne diseases by disrupting their RNA silencing antiviral immunity.

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·Mammalian Viral Suppressors of RNA Silencing

We have recently carried out a screen to determine if mammalian viruses encoded proteins that are able to suppress the nodavirus-induced RNA silencing antiviral response in Drosophila cells (Li et al., 2004). This led to the identification of the vaccinia virus E3L and NS1 encoded by influenza A, B and C viruses as the first mammalian viral suppressors of the animal RNA silencing antiviral defense (Fig. 6). E3L and NS1 are distinct dsRNA-binding proteins and essential for pathogenesis by inhibiting the mammalian interferon-regulated innate antiviral response. We found that the dsRNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. The influenza viruses contain a segmented negatives-strand RNA genome whereas vaccinia virus (the vaccine against small pox virus) has a large dsDNA genome. Our observations that diverse mammalian viruses carry essential proteins that are suppressors of RNA silencing suggest that RNA silencing is a novel nucleic acid-based antiviral immunity in mammalian cells.

Fig. 6. Suppression of the invertebrate RNAi antiviral response visualized by GFP expression. FHV1P is a mutant of FHV RNA1 in which the coding sequence of B2 is replaced by that of GFP and thus does not express GFP after transfection into Drosophila cells (upper left) unless it is co-transfected with a plasmid that expresses a viral suppressor of RNAi.

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Ding Lab, January 2002. Kathy Harper, Wan Xiang Li, Hongwei Li, Li Feng, Shou Wei Ding, Yoon Gi Choi, Michael Shintaku

Selected Publications (Bibliography page)

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