Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana.

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus-induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants' performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative-stranded TSWV and the positive-stranded potato virus X.

An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in Nicotiana benthamiana.

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development.

A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein.

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined.IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme.

Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes.

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of about 200-400 µg g-1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Potatovirus X and Tobacco mosaic virus-based vectors compatible with the Gateway cloning system.

Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato virus X (PVX) and Tobacco mosaic virus (TMV)-based vectors to allow easy cloning of foreign sequences by the Gateway cloning system. Target genes were cloned efficiently by recombination and successfully expressed in Nicotiana benthamiana following inoculation by Agrobacterium (agroinfection). Using green fluorescent protein (GFP) as marker, high-level expression with both PVX-GW and TMV-GW vectors was confirmed. A Gateway inserted phytoene desaturase gene (pds) fragment in PVX-GW and TMV-GW vectors (PVX-GW-PDS and TMC-GW-PDS), induced gene silencing of the endogenous pds gene in N. benthamiana as evidenced by chlorotic leaves. The PVX-GW vector was adapted further by cloning the GFP gene upstream of the Gateway sequences, allowing the easy production of GFP fusions after recombination of a target gene. Subcellular localization of resulting GFP fusion was validated by recombining and expressing the coat protein gene from Tomato chlorotic mottle virus, revealing its nuclear localization. A PVX-GW transient expression assay of a nucleocapsid protein gene fragment of Tomato spotted wilt virus and of a single chain antibody against this protein was shown to confer effective resistance to TSWV infection.

Assessing the expression of chicken anemia virus proteins in plants.

Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces apoptosis. In this study, CAV proteins were expressed in plants as an alternative for recombinant protein production in animal cells. Additionally, the effect of VP3 expression was tested to evaluate possible involvement in programmed cell death in plants. The CAV genes were cloned in binary vectors with the Green fluorescent protein (GFP) as N terminal fusion, and into a Potato virus X (PVX) and Tobacco Mosaic Virus (TMV)-based vectors. Nicotiana benthamiana plants were inoculated with Agrobacterium tumefaciens containing the binary vector constructs or the PVX and TMV constructs. Upon transient expression GFP:VP1 and GFP:VP2 were observed throughout the nucleoplasm, whereas VP3 formed compact aggregates within the nucleus, indicating functional nuclear localization signals in all three proteins. An intense fluorescence was observed for VP2 and VP3 fusions, whereas GFP:VP1 fluorescence remained faint and was only detected in a limited number of cells. Co-expression of GFP:VP1 and VP2 had a marked alteration on the distribution of GFP:VP1, forming large VP1 aggregates throughout the nucleus, indicating an interaction of the two CAV proteins. No visible alteration on GFP pattern was detected upon co-expression of GFP:VP1 and VP3, or with GFP:VP2 and VP3. Plants infected with PVX or TMV-based vectors expressing VP3 displayed strong necrosis and wilting, however, a direct association with VP3 expression and programmed cell death in plants, could not be established. Overall, our results show that all CAV proteins can be expressed in plant cells, though expression level of VP1 needs to be further optimized before testing its potential as (edible) subunit vaccine.

The Ebola virus VP35 protein is a suppressor of RNA silencing.

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants.

Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants.

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.

Multiple virus resistance at a high frequency using a single transgene construct.

RNA silencing is a natural antiviral defence in plants, which can be exploited in transgenic plants for preprogramming virus recognition and ensuring enhanced resistance. By arranging viral transgenes as inverted repeats it is thus possible to obtain strong repression of incoming viruses. Due to the high sequence specificity of RNA silencing, this technology has hitherto been limited to the targeting of single viruses. Here it is shown that efficient simultaneous targeting of four different tospoviruses can be achieved by using a single small transgene based on the production of minimal sized chimaeric cassettes. Due to simultaneous RNA silencing, as demonstrated by specific siRNA accumulation, the transgenic expression of these cassettes rendered up to 82 % of the transformed plant lines heritably resistant against all four viruses. Thus RNA silencing can be further improved for high frequency multiple virus resistance by combining small RNA fragments from a series of target viruses.

Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells.

It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in different ways, the NS1 protein of Influenza virus, NSs of Tospovirus Tomato spotted wilt virus and HC-Pro of Zucchini yellow mosaic virus were expressed and investigated to determine if they antagonize induced RNAi. Using the recombinant SFV replicon expressing firefly luciferase, silencing was induced and the suppressor activity of these inhibitors during or after initiation of siRNA synthesis was tested, to determine which step of the RNAi pathway is impaired. It was found that these proteins, identified in mammalian or plant systems, also display activity in tick cells. These data suggest that ticks utilize a mechanism similar to the one found in other eukaryotes.

Phage display-selected single-chain antibodies confer high levels of resistance against Tomato spotted wilt virus.

Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field.

The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants.

RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs (dsRNAs) and provide the sequence specificity for degradation of cognate RNA molecules. In plants, RNA silencing plays a key role in developmental processes and in control of virus replication. It has been shown that many plant viruses encode proteins, denoted RNA silencing suppressors, that interfere with this antiviral response. Although RNA silencing has been shown to occur in vertebrates, no relationship with inhibition of virus replication has been demonstrated to date. Here we show that the NS1 protein of human influenza A virus has an RNA silencing suppression activity in plants, similar to established RNA silencing suppressor proteins of plant viruses. In addition, NS1 was shown to be capable of binding siRNAs. The data presented here fit with a potential role for NS1 in counteracting innate antiviral responses in vertebrates by sequestering siRNAs.

Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions.

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the BUNYAVIRIDAE: Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS(S) gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS(S)-expressing leaf sectors suggests that the tospoviral NS(S) protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.

Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance.

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.