The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.
The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced HR was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn 2+ ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn 2+ -dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.
Plant viruses cause a variety of diseases in susceptible hosts. The disease symptoms often include leaf malformations and other developmental abnormalities, suggesting that viruses can affect plant development. However, little is known about the mechanisms underlying virus interference with plant morphogenesis. Here, we show that a C-4 type zinc-finger (ZF) protein, p12, encoded by a carlavirus (chrysanthemum virus B) can induce cell proliferation, which results in hyperplasia and severe leaf malformation. We demonstrate that the p12 protein activates expression of a regulator of cell size and proliferation, designated upp-L (upregulated by p12), which encodes a transcription factor of the basic/helix-loop-helix family sufficient to cause hyperplasia. The induction of upp-L requires translocation of the p12 protein into the nucleus and ZF-dependent specific interaction with the conserved regulatory region in the upp-L promoter. Our results establish the role of the p12 protein in modulation of host cell morphogenesis. It is likely that other members of the conserved C-4 type ZF family of viral proteins instigate reprogramming of plant development by mimicking eukaryotic transcriptional activators.
Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5′ third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3′ two-thirds of the genome. The polypeptide translation of this 3′ open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. An inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and in silico assembly of RNA isolated from Solenopsis invicta and four other ant species, followed by targeted Sanger sequencing, we obtained nearly complete genomes for four further viruses in the group. Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent (pairwise amino acid identities of as low as 17 % in the non-structural polyprotein), but possess the same overall polycistronic genome structure, which is distinct from all other characterized picorna-like viruses. Consequently, we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species) and Sopolycivirus (11 species), with members of the latter infecting ants in at least 3 different subfamilies.
The Potato mop-top virus (PMTV) genome encodes replicase, movement, and capsid proteins on three different RNA species that are encapsidated within tubular rod-shaped particles. Previously, we showed that the protein produced on translational readthrough (RT) of the coat protein (CP) gene, CP-RT, is associated with one extremity of the virus particles, and that the two RNAs encoding replicase and movement proteins can move long distance in the absence of the third RNA (RNA-CP) that encodes the capsid proteins, CP and CP-RT. Here, we examined the roles of the CP and CP-RT proteins on RNA movement using infectious clones carrying mutations in the CP and CP-RT coding domains. The results showed that, in infections established with mutant RNA-CP expressing CP together with truncated CP-RT, systemic movement of the mutant RNA-CP was inhibited but not the movement of the other two RNAs. Furthermore, RNA-CP long-distance movement was inhibited in a mutant clone expressing only CP in the absence of the CP-RT polypeptide. CP-RT was not necessary for particle assembly because virions were observed in leaf extracts infected with the CP-RT deletion mutants. RNA-CP moved long distance when protein expression was suppressed completely or when CP expression was suppressed so that only CP-RT or truncated CP-RT was expressed. CP-RT but not CP interacted with the movement protein TGB1 in the yeast two-hybrid system. CP-RT and TGB1 were detected by enzyme-linked immunosorbent assay in virus particles and the long-distance movement of RNA-CP was correlated with expression of CP-RT that interacted with TGB1; mutant RNA-CP expressing truncated CP-RT proteins that did not interact with TGB1 formed virions but did not move to upper noninoculated leaves. The results indicate that PMTV RNA-CP can move long distance in two distinct forms: either as a viral ribonucleoprotein complex or as particles that are most likely associated with CP-RT and TGB1.
Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.
During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5′-3′ mRNA decapping is a late step in NMD-related mRNA degradation in plants.
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