New features on the genomic organization of a novel dicistrovirus identified from the sweet potato whitefly Bemisia tabaci.

The whitefly Bemisia tabaci is an agricultural pest causing large economic losses worldwide. We analysed the genomic sequence of a new viral member of the family Dicistroviridae identified by high-throughput sequencing of total RNA extracted from whiteflies. The virus, tentatively named Bemisia-associated dicistrovirus 2 (BaDV-2), has a genome of 8012 nucleotides with a polyadenylated 3' end. In contrast to typical dicistroviruses, BaDV-2 has a genome containing three open reading frames (ORFs) encoding predicted proteins of 1078 (ORF1a), 481 (ORF1b) and 834 (ORF2) amino acids, which correspond to replicase A (containing helicase and cysteine protease domains), replicase B (a domain of an RNA-dependent RNA polymerase - RdRP) and capsid proteins, respectively. The 3' end of ORF1a contains a potential frameshift signal, suggesting that ORF1a and ORF1b may be expressed as a single polyprotein (replicaseFS), corresponding to other dicistroviruses. The BaDV-2 genomic sequence shares the highest nucleotide identity (61.1 %) with Bemisia-associated dicistrovirus 1 (BaDV-1), another dicistrovirus identified from whiteflies. The full BaDV-2 replicaseFS polyprotein clustered with aparaviruses, whereas the capsid polyprotein clustered with cripaviruses in phylogenetic analyses, as with BaDV-1. The intergenic region (IGR) between ORF1b and ORF2 is predicted to adopt a secondary structure with atypical features that resembles the dicistrovirus IGR IRES structure. Our analyses indicate that BaDV-2 is a novel dicistrovirus and that BaDV-2 together with BaDV-1 may not be appropriately grouped in any of the three currently accepted dicistrovirus genera.

Complete genome sequence and phylogenetic analysis of a novel dicistrovirus associated with the whitefly Bemisia tabaci.

A novel single-stranded RNA virus was detected in a whitefly (Bemisia tabaci) sample subjected to high-throughput sequencing. The 8293 nt-long genome presents a polyadenylated 3' end, and contains two ORFs encoding putative 1596 and 849 aa-long proteins. These putative proteins display significant similarity to replicase and capsid polyproteins, respectively, of discitroviruses. Its complete genome sequence shared the highest nucleotide identity (59.8%) with cricket paralysis virus (family Dicistroviridae, genus Cripavirus). Phylogenetic analyses showed that this new virus putative protein sequences clustered with those from members of Dicistroviridae. However, the replicase and capsid polyprotein sequences clustered with those of members of different genera, respectively to Aparavirus and Cripavirus. RT-PCR using newly collected adult and nymph whitefly samples confirmed the presence of this virus in field populations of B. tabaci. Genome sequence and organization, and polyproteins comparison indicate that this virus is a new species of the family Dicistroviridae. The name Bemisia-associated dicistrovirus 1 is proposed for this virus.

Biochemical analysis of NSs from different tospoviruses.

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.

Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

Analysis of the A-U rich hairpin from the intergenic region of tospovirus S RNA as target and inducer of RNA silencing.

Earlier work indicated that Tomato spotted wilt virus (TSWV) messenger transcripts, and not the (anti)genomic RNAs, are targeted by the RNA silencing machinery. Here, the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) of the TSWV S RNA, and present at the 3' end of viral mRNAs, was analyzed as a target and inducer for RNA silencing. Virus-derived siRNAs (vsiRNAs) purified from virus infected plants were found to derive from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the IGR from where hardly any vsiRNAs were found. Similar profiles were observed with the distantly related Tomato yellow ring tospovirus (TYRV). Dicer cleavage assays using Drosophila melanogaster (Dm) embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3' UTR (GFP-HP) did not show more rapid/strong silencing and profiling of the corresponding siRNAs, generated outside the context of a viral infection, still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP is a weak inducer of RNA silencing and only plays a minor role in the amplification of a strong antiviral RNAi response.

Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus.

As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs(RI) protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N(RI) (/) (RB) protein, or when NSs(RB) was expressed. Although NSs(RI) was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs(RB) had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.