Eucaryotic initiation factor 4B controls eIF3-mediated ribosomal entry of viral reinitiation factor

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.     

Targeting of dicer-2 and RNA by a viral RNA silencing suppressor in Drosophila cells

RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2-Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.     

The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition

We report the structure of the flock house virus B2 protein, a potent suppressor of RNA interference (RNAi) in animals and plants. The B2 protein is a homodimer in solution and contains three alpha-helices per monomer. Chemical shift perturbation shows that an antiparallel arrangement of helices (alpha2/alpha2') forms an elongated binding interface with double-stranded RNA (dsRNA). This implies a novel mode of dsRNA recognition and provides insights into the mechanism of RNAi suppression by B2.     

Editing independent effects of ADARs on the miRNA/siRNA pathways

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR‐B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir‐376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase‐inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA‐binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.     

The 2b protein of cucumoviruses has a role in promoting the cell-to-cell movement of pseudorecombinant viruses

Pseudorecombinant viruses (i.e., those containing a reassorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.     

Molecular dissection of the cauliflower mosaic virus translation transactivator.

The cauliflower mosaic virus (CaMV) transactivator (TAV) is a complex protein that appears to be involved in many aspects of the virus life cycle. One of its roles is to control translation from the polycistronic CaMV 35S RNA. Here we report a molecular dissection of TAV in relation to its ability to enhance dicistronic translation in transient expression experiments. We have identified a protein domain that is responsible and sufficient for that activity. This 'MiniTAV domain' consists of only 140 of the 520 amino acids in the full-length sequence. A further domain located outside the MiniTAV, and therefore dispensable for transactivation, is probably involved in interactions with other molecules. This was identified by its ability to compete with wild-type TAV and some of its deletion mutants. We found, furthermore, that the TAV protein binds RNA. Two regions needed for RNA-binding properties were defined outside the MiniTAV domain and RNA binding seems not to be directly involved in the transactivation mechanism.     

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.     

Biological and Molecular Characterization of Three Isolates of Tomato aspermy virus Infecting Chrysanthemums in India

Severe mosaic, chlorotic ringspots and flower deformation were observed during the winter of November 2006–February 2007 on chrysanthemums ( Chrysanthemum morifolium ) at three locations in India: Lucknow (UP), Dhanbad (MP) and Kolkata (WB). Tomato aspermy virus (TAV) was detected in affected plants by ELISA and by RT-PCR using TAV specific primers. These TAV isolates were mechanically transmitted to test plant species and also by aphids ( Aphis gossypii ) to Lycopersicon esculentum . The complete RNA 3 of each TAV isolate was cloned and sequenced and determined to be 2386 nucleotides (nt) long, and to encode two open reading frames (ORFs): the movement protein (MP) of 741 nt and the coat protein (CP) of 657 nt translating in to 246 and 218 amino acid (aa), respectively. When RNA 3 sequences of the Indian isolates were multiple aligned with seven other strains of TAV occurring worldwide, Indian isolates shared 98–99% identities among themselves and with the KC, V, P, B, I and C strains of TAV. In phylogenetic analysis, the Lucknow and Kolkata isolates of TAV clustered together and showed a close relationship with the KC-TAV strain from South Korea, whereas the Dhanbad isolate formed an independent cluster and showed closeness with the V-TAV strains from Spain and Australia. Recombination events were also observed in the CP region of the Dhanbad isolate, supporting its diverse behaviour. This is the first report of the complete RNA 3 sequence of these three Indian TAV isolates.     

A virus-encoded inhibitor that blocks RNA interference in mammalian cells

Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.     

Constrained peptides mimic a viral suppressor of RNA silencing

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.     

Thermodynamics of RNA-RNA binding

BACKGROUND: Reliable prediction of RNA-RNA binding energies is crucial, e.g. for the understanding on RNAi, microRNA-mRNA binding and antisense interactions. The thermodynamics of such RNA-RNA interactions can be understood as the sum of two energy contributions: (1) the energy necessary to 'open' the binding site and (2) the energy gained from hybridization. METHODS: We present an extension of the standard partition function approach to RNA secondary structures that computes the probabilities Pu[i, j] that a sequence interval [i, j] is unpaired. RESULTS: Comparison with experimental data shows that Pu[i, j] can be applied as a significant determinant of local target site accessibility for RNA interference (RNAi). Furthermore, these quantities can be used to rigorously determine binding free energies of short oligomers to large mRNA targets. The resource consumption is comparable with a single partition function computation for the large target molecule. We can show that RNAi efficiency correlates well with the binding energies of siRNAs to their respective mRNA target. AVAILABILITY: RNAup will be distributed as part of the Vienna RNA Package, www.tbi.univie.ac.at/~ivo/RNA/     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.     

RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies

Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHVΔB2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHVΔB2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHVΔB2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHVΔB2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHVΔB2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.     

Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC. Although several studies have been conducted to evaluate the specific functions of hTRBP and hPACT in RNAi, exact mechanisms and modes of action are still unknown. Here, we present a biochemical study further evaluating the role of hTRBP and hPACT in hAgo2-loading. We found that both proteins enhance hAgo2-mediated RNA cleavage significantly; even a hAgo2 mutant impaired in siRNA binding shows full cleavage activity in the presence of hTRBP or hPACT. Pre-steady state binding studies reveal that the assembly of wildtype-hAgo2 (wt-hAgo2) and siRNAs remains largely unaffected, whereas the binding of mutant hAgo2-PAZ9 to siRNA is restored by adding either hTRBP or hPACT. We conclude that both proteins assist in positioning the siRNA within hAgo2 to ensure optimal binding and cleavage. Overall, our data indicate that hTRBP and hPACT are part of a regulative system of RNAi that is important for efficient target RNA cleavage.     

Inhibition of herpesvirus-6B RNA replication by short interference RNAs

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.     

Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.     

TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp.     

Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.