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 NSs^TYRV was just as strong as those by NSs^TSWV and NSs^GRSV, even though NSs^TYRV was expressed in lower amounts. Using the system established, a set of selected NSs^TSWV 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 NSs^TSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.     

Complete Genome Sequence of Mulberry Vein Banding Associated Virus,a New Tospovirus Infecting Mulberry

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.     

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N‐terminal domain for avirulence and RNA silencing suppression

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)‐based resistance. The observation that NSs from two natural resistance‐breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance‐inducing wild‐type strains (NSs^RI), amino acid reversions in NSs from resistance‐breaking strains (NSs^RB), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw‐mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N‐terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs^RI and NSs^RB. Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs^RI showed that Avr functionality could not simply be transferred between species. Although deletion of the C‐terminal domain rendered NSs completely dysfunctional, only a few single‐amino‐acid mutations in the C‐terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference.     

Protease activity, self interaction, and small interfering RNA binding of the silencing suppressor p1b from cucumber vein yellowing ipomovirus

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.     

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.     

A critical domain of Sweet potato chlorotic fleck virus nucleotide‐binding protein (NaBp) for RNA silencing suppression,nuclear localization and viral pathogenesis

RNA silencing is an important mechanism of antiviral defence in plants. To counteract this resistance mechanism, many viruses have evolved RNA silencing suppressors. In this study, we analysed five proteins encoded by Sweet potato chlorotic fleck virus (SPCFV) for their abilities to suppress RNA silencing using a green fluorescent protein (GFP)‐based transient expression assay in Nicotiana benthamiana line 16c plants. Our results showed that a putative nucleotide‐binding protein (NaBp), but not other proteins encoded by the virus, could efficiently suppress local and systemic RNA silencing induced by either sense or double‐stranded RNA (dsRNA) molecules. Deletion mutation analysis of NaBp demonstrated that the basic motif (an arginine‐rich region) was critical for its RNA silencing suppression activity. Using confocal laser scanning microscopy imaging of transfected protoplasts expressing NaBp fused to GFP, we showed that NaBp accumulated predominantly in the nucleus. Mutational analysis of NaBp demonstrated that the basic motif represented part of the nuclear localization signal. In addition, we demonstrated that the basic motif in NaBp was a pathogenicity determinant in the Potato virus X (PVX) heterogeneous system. Overall, our results demonstrate that the basic motif of SPCFV NaBp plays a critical role in RNA silencing suppression, nuclear localization and viral pathogenesis.     

Complementation between Two Tospoviruses Facilitates the Systemic Movement of a Plant Virus Silencing Suppressor in an Otherwise Restrictive Host

Background

New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature.

Methodology/Principal Findings

Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs.

Conclusion/Significance

Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.     

Evidence of HIV exposure and transient seroreactivity in archived HIV-negative severe hemophiliac sera

Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV) 19K protein is a cysteine-rich protein (CRP) and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX) viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP) expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.     

Mycoreovirus genome rearrangements associated with RNA silencing deficiency

Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus.     

NSs Encoded by Groundnut Bud Necrosis Virus Is a Bifunctional Enzyme

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism.     

The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes

RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.     

布尼亚病毒NSs蛋白与RNA沉默机制

布尼亚病毒科含5个属, 350多种病毒, 寄主范围包括植物、动物和人类, 主要通过节肢动物传播（仅有汉坦病毒属通过啮齿动物传播）, 其中大部分病毒危害严重, 影响农业生产, 威胁人类和动物健康, 造成重大经济损失。NSs蛋白是布尼亚病毒S RNA编码的非结构蛋白质, 可以抑制RNA沉默, 在侵染介体和寄主的过程中起时发挥重要作用。在植物寄主中, NSs蛋白通过与病毒dsRNA结合、抑制dsRNA次生扩增、长距离传播等机制抑制RNA沉默; 在动物、人类寄主以及传播介体中, 多种证据表明, NSs蛋白也能抑制RNA沉默。RNA沉默是植物和节肢动物中主要的抗病毒机制之一, NSs蛋白抑制RNA沉默对于病毒成功侵染寄主或传播介体非常重要。本文就近年来NSs蛋白在抑制RNA沉默方面的研究做一综述。     

The C-terminal residues of the 2b protein of Cucumber mosaic virus are important for efficient expression in Escherichia coli and DNA-binding

The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b’s DNA-binding ability, which may partly explain the toxicity of the protein.     

Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus

Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21–114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3.     

Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA

Viral resistance can be effectively induced in transgenic plants through their silencing machinery. Thus, we designed nine short hairpin RNAs (shRNA) constructs to target nuclear inclusion protein b (NIb), helper component proteinase (HC-Pro), cylindrical inclusion protein (CI) and viral protein genome linked (VPg) genes of Potato virus Y (PVY^N) and Tobacco etch virus (TEV-SD1). The shRNAs were completely complementary to the genes of PVY^N, and contained 1–3 nt mismatches to the genes of TEV-SD1. To study the specificity of gene silencing in shRNA-mediated viral resistance, the constructs were introduced into tobacco plants. The results of viral resistance assay revealed that these nine kinds of transgenic tobacco plants can effectively induce viral resistance against both PVY^N and TEV-SD1, and the shRNA construct targeting the NIb gene showed higher silencing efficiency. Northern blot and short interfering RNA (siRNA) analyses demonstrated that the viral resistance can be attributed to the degradation of the target RNA through the RNA silencing system. Correlation analysis of siRNA sequence characteristics with its activity suggested that the secondary structure stability of the antisense strand did not influence siRNA activity; 1 to 3 nt 5’ end of the sense strand caused a significant effect on siRNA activity where the first base such as U was favourable for silencing; the base mismatch between the siRNA and the target gene may be more tolerated in the 5’ end.