- Volume 90, Issue 12, 2009

After serially undiluted passage of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), persistently infected Se301 cells were established. A cell strain, in which no polyhedra or viral particles were observed, was cloned and designated P8-Se301-C1. The P8-Se301-C1 cells are morphologically similar to but grow slower than Se301 cells and they can homologously interfere with SeMNPV. PCR analysis showed that SeMNPV ie-0 and polyhedrin genes were present but DNA polymerase and orf67 genes were absent in P8-Se301-C1, suggesting that the cells harbour incomplete SeMNPV genomes. Dot-blot analysis demonstrated that 0.32±0.16 ng SeMNPV DNA was present in 1.25×105 P8-Se301-C1 cells. A quantitative real-time PCR assay showed that there were 13.2±4.3 copies of the SeMNPV polyhedrin gene in each cell. Nested RT-PCR demonstrated the presence of SeMNPV polyhedrin transcripts in P8-Se301-C1 cells. The fact that P8-Se301-C1 cells carry low levels of partial viral genome but do not produce viral progeny suggests a latent-like viral infection in the cells.

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Δ2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Δ2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Δ2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Δ2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Δ2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Δ2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Δ2b, whereas accumulation of Δ2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

Three ‘triple gene block’ proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-to-cell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection.

Potato virus Y (PVY) is one of the most economically important plant pathogens. The PVY genome has a high degree of genetic variability and is also subject to recombination. New recombinants have been reported in many countries since the 1980s, but the origin of these recombinant strains and the physical and evolutionary mechanisms driving their emergence are not clear at the moment. The replicase-mediated template-switching model is considered the most likely mechanism for forming new RNA virus recombinants. Two factors, RNA secondary structure (especially stem–loop structures) and AU-rich regions, have been reported to affect recombination in this model. In this study, we investigated the influence of these two factors on PVY recombination from two perspectives: their distribution along the whole genome and differences between regions flanking the recombination junctions (RJs). Based on their distributions, only a few identified RJs in PVY genomes were located in lower negative FORS-D, i.e. having greater secondary-structure potential and higher AU-content regions, but most RJs had more negative FORS-D values upstream and/or higher AU content downstream. Our whole-genome analyses showed that RNA secondary structures and/or AU-rich regions at some sites may have affected PVY recombination, but in general they were not the main forces driving PVY recombination.

Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is required for encapsidation and virus systemic movement. To better understand the roles of HCRSV CP in virus infection and its interactions with host proteins, a cDNA library of kenaf (Hibiscus cannabinus L.) was constructed and screened by using a yeast two-hybrid system (YTHS) to identify CP-interacting proteins. One protein identified was sulfite oxidase (SO) and the interaction was confirmed in vitro and in vivo. The interaction was found to be associated with peroxisomes by immunofluorescent labelling of peroxisomes by an anti-SKL signal peptide antibody. Our YTHS results showed that only the P and S domains of CP interacted with SO from kenaf. This is probably due to the exposure of these two domains on the outer surface of the capsid. Peroxisomes were observed to aggregate in HCRSV-infected cells, and biochemical assays of total protein from kenaf leaf extracts showed that SO activity and SO-dependent H2O2-generating activity in the HCRSV-infected leaves increased compared with that in mock-inoculated kenaf plants.

For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV), full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins) proved to be essential for virus replication in planta and symptom production on C. quinoa, whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants, the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to the CP–RT.

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.

Maize streak virus (MSV), which causes maize streak disease (MSD), is one of the most serious biotic threats to African food security. Here, we use whole MSV genomes sampled over 30 years to estimate the dates of key evolutionary events in the 500 year association of MSV and maize. The substitution rates implied by our analyses agree closely with those estimated previously in controlled MSV evolution experiments, and we use them to infer the date when the maize-adapted strain, MSV-A, was generated by recombination between two grass-adapted MSV strains. Our results indicate that this recombination event occurred in the mid-1800s, ∼20 years before the first credible reports of MSD in South Africa and centuries after the introduction of maize to the continent in the early 1500s. This suggests a causal link between MSV recombination and the emergence of MSV-A as a serious pathogen of maize.