Calcium Ions Modulate the Mechanics of Tomato Bushy Stunt Virus

Viral particles are endowed with physicochemical properties whose modulation confers certain metastability to their structures to fulfill each task of the viral cycle. Here, we investigate the effects of swelling and ion depletion on the mechanical stability of individual tomato bushy stunt virus nanoparticles (TBSV-NPs). Our experiments show that calcium ions modulate the mechanics of the capsid: the sequestration of calcium ions from the intracapsid binding sites reduces rigidity and resilience in ∼24% and 40%, respectively. Interestingly, mechanical deformations performed on native TBSV-NPs induce an analogous result. In addition, TBSV-NPs do not show capsomeric vacancies after surpassing the elastic limit. We hypothesize that even though there are breakages among neighboring capsomers, RNA-capsid protein interaction prevents the release of capsid subunits. This work shows the mechanical role of calcium ions in viral shell stability and identifies TBSV-NPs as malleable platforms based on protein cages for cargo transportation at the nanoscale.     

Rapid Delivery of Foreign Genes into Plants by Direct Rub-Inoculation with Intact Plasmid DNA of a Tomato Bushy Stunt Virus Gene Vector

Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified cauliflower mosaic virus 35S promoter and the hepatitis delta virus antigenomic ribozyme with a downstream nopaline synthase gene polyadenylation signal, established infections upon rub-inoculation of plants with intact plasmids. Application of this methodology produced a TBSV DNA-based gene vector which yielded readily detectable levels of localized foreign gene expression in inoculated leaves. This is the first demonstration of an infectious DNA from a member of the Tombusviridae which permits rapid TBSV-mediated foreign-gene expression upon direct rub-inoculation of miniprep DNA onto a variety of plant species.     

Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.     

p33-Independent Activation of a Truncated p92 RNA-Dependent RNA Polymerase of Tomato Bushy Stunt Virus in Yeast Cell-Free Extract

Plus-stranded RNA viruses replicate in membrane-bound structures containing the viral replicase complex (VRC). A key component of the VRC is the virally encoded RNA-dependent RNA polymerase (RdRp), which should be activated and incorporated into the VRC after its translation. To study the activation of the RdRp of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we used N-terminal truncated recombinant RdRp, which supported RNA synthesis in a cell-free yeast extract-based assay. The truncated RdRp required a cis-acting RNA replication element and soluble host factors, while unlike the full-length TBSV RdRp, the truncated RdRp did not need the viral p33 replication cofactor or cellular membranes for RNA synthesis. Interestingly, the truncated RdRp used 3′-terminal extension for initiation and terminated prematurely at an internal cis-acting element. However, the truncated RdRp could perform de novo initiation on a TBSV plus-strand RNA template in the presence of the p33 replication cofactor, cellular membranes, and soluble host proteins. Altogether, the data obtained with the truncated RdRp indicate that this RdRp still requires activation, but with the participation of fewer components than with the full-length RdRp, making it suitable for future studies on dissection of the RdRp activation mechanism.     

A Primary Determinant of Cap-Independent Translation Is Located in the 3′-Proximal Region of the Tomato Bushy Stunt Virus Genome

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus Tombusvirus. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5' cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3' terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845-5851, 1998). In the present report, we provide evidence that a 3'-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5' cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3' proximal element, designated as the 3' cap-independent translational enhancer (3'CITE), that is required for proficient cap-independent translation; (iv) the 3'CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3'CITE activity; and (vi) no significant 3'CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3'CITE from other characterized plant viral 3'-proximal cap-independent translational enhancers. Additionally, because the 3'CITE overlaps with cis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.     

MicroRNA靶向病毒携带的microRNA模拟靶序列对病毒的抑制作用

Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.     

Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.     

Proteolytic Processing of Oligopeptides Containing the Target Sequences by the Recombinant Tobacco Vein Mottling Virus NIa Proteinase

Tobacco vein mottling virus (TVMV) belongs to the potyviridae that consists of about 200 plant viruses. Potyviruses have RNA genomes of approximately 10,000 bases from which a single polyprotein is expressed from each virus upon infection. The NIa proteinase is known to process the polyprotein at seven distinct junctions between proteins. Kinetic constants were determined for the reactions of the recombinant TVMV NIa protease (27 kDa) with synthetic oligopeptides containing the sequences for the cleavage sites. For optimum activity, the substrate needs to have six amino acids (P6–P1) in the amino region and four (P1–P4) in the carboxy region, including four conserved amino acids (V-R-F-Q) in P4P1 positions. Mutation of any of four conserved amino acids to Gly made the substrate inert to the enzyme. Among the substrates, the oligopeptides containing the sequences for junctions, P3–6K1, NIa (VPg-Pro), and NIa-NIb were not processed by the NIa protease. Those junctions have Glu at P3, Glu at P1, and Thr at P2. The implications of high substrate specificity and size dependence in polyprotein processing and viral replication are discussed.