A plant virus satellite RNA directly accelerates wing formation in its insect vector for spread.

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector.

Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division.

Chromosome segregation requires centromeres on every sister chromatid to correctly form and attach the microtubule spindle during cell division. Even though centromeres are essential for genome stability, the underlying centromeric DNA is highly variable in sequence and evolves quickly. Epigenetic mechanisms are therefore thought to regulate centromeres. Here, we show that the 359-bp repeat satellite III (SAT III), which spans megabases on the X chromosome of Drosophila melanogaster, produces a long noncoding RNA that localizes to centromeric regions of all major chromosomes. Depletion of SAT III RNA causes mitotic defects, not only of the sex chromosome but also in trans of all autosomes. We furthermore find that SAT III RNA binds to the kinetochore component CENP-C, and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. In conclusion, our data reveal that SAT III RNA is an integral part of centromere identity, adding RNA to the complex epigenetic mark at centromeres in flies.

BRCA1 haploinsufficiency for replication stress suppression in primary cells.

BRCA1-a breast and ovarian cancer suppressor gene-promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1(+/+) and BRCA1(mut/+) mammary epithelial cells and fibroblasts. Here we report that all BRCA1(mut/+) cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1(mut/+) cells with wt BRCA1. In addition, we observed 'conditional' haploinsufficiency for HR-DSBR in BRCA1(mut/+) cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue.

Viruses and prions of Saccharomyces cerevisiae.

Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for blocking the propagation of the viruses and prions, and proteins involved in the expression of viral components. Here, we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast.

Cross-protection: a century of mystery.

Cross-protection is a phenomenon in which infection of a plant with a mild virus or viroid strain protects it from disease resulting from a subsequent encounter with a severe strain of the same virus or viroid. In this chapter, we review the history of cross-protection with regard to the development of ideas concerning its likely mechanisms, including RNA silencing and exclusion, and its influence on the early development of genetically engineered virus resistance. We also examine examples of the practical use of cross-protection in averting crop losses due to viruses, as well as the use of satellite RNAs to ameliorate the impact of virus-induced diseases. We also discuss the potential of cross-protection to contribute in future to the maintenance of crop health in the face of emerging virus diseases and related threats to agricultural production.

Downregulation of Bamboo mosaic virus replication requires the 5' apical hairpin stem loop structure and sequence of satellite RNA.

Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.

Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication.

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.

Plant virus satellite and defective interfering RNAs: new paradigms for a new century.

Although many subviral RNAs reduce or intensify disease symptoms caused by the helper virus, only recently have clues concerning the mechanism of disease modulation been revealed. New models for DI RNA-mediated reduction in helper virus levels and symptom attenuation include DI RNA enhancement of posttranscriptional gene silencing (PTGS), which is an antiviral defense mechanism in plants. Symptom enhancement by the satRNA of Cucumber mosaic virus is caused by minus-strand induction of the programmed cell death pathway. In contrast, symptom enhancement by satC of Turnip crinkle virus is due to satC interference with virion formation, leading to increased levels of free coat protein, which is the viral suppressor of PTGS. Mutualism between satRNA and helper virus can be seen for the satRNA of Groundnut rosette virus, which contributes to the virus by allowing virion assembly. These novel findings are leading to re-evaluation of the relationships between subviral RNAs, helper viruses, and hosts.

In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with turnip crinkle virus.

RNA replication enhancers are cis-acting elements that can stimulate replication or transcription of RNA viruses. Turnip crinkle virus (TCV) and satC, a parasitic RNA associated with TCV infections, contain stem-loop structures that are RNA replication enhancers (P. Nagy, J. Pogany, and A. E. Simon, EMBO J. 1999, 18, 5653-5665). We have found that replacement of 28 nt of the satC enhancer, termed the motif1-hairpin, with 28 randomized bases reduced satC accumulation 8- to 13-fold in Arabidopsis thaliana protoplasts. Deletion of single-stranded flanking sequences at either side of the hairpin also affected RNA accumulation with combined alterations at both sides of the hairpin showing the most detrimental effect in protoplasts. In vitro analysis with a partially purified TCV RdRp preparation demonstrated that the motif1-hairpin in its minus-sense orientation was able to stimulate RNA synthesis from the satC hairpin promoter (located at the 3' end of plus strands) by almost twofold. This level of RNA synthesis stimulation is approximately fivefold lower than that observed with a linear promoter, suggesting that a highly stable hairpin promoter is less responsive to the presence of the motif1-hairpin enhancer than a linear promoter. The motif1-hairpin in its plus-sense orientation was only 60% as active in enhancing transcription from the hairpin promoter. Since the motif1-hairpin is a hotspot for RNA recombination during plus-strand synthesis and since satC promoters located on the minus-strand are all short linear sequences, these findings support the hypothesis that the motif1-hairpin is primarily involved in enhancing plus-strand synthesis.

Mutualism, parasitism and competition in the evolution of coviruses.

Coviruses are viruses with the property that their genetic information is divided up among two or more different viral particles. I model the evolution of coviruses using information on both viral virulence and the interactions between viruses and molecules that parasitize them: satellite viruses, satellite RNAs and defective interfering viruses. The model ultimately, and inevitably contains within it single-species dynamics as well as mutualistic, parasitic, cooperative and competitive relationships. The model shows that coexistence between coviruses and the self-sufficient viruses that spawned them is unlikely, in the sense that the quantitative conditions for coexistence are not easy to satisfy I also describe an abrupt transition from mutualistic two-species to single-species dynamics, showing a new sense in which questions such as 'Is a lichen one species or two?' can be given a definite answer.

Symptom attenuation by a satellite RNA in vivo is dependent on reduced levels of virus coat protein.

Many plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck carmovirus [Kong et al. (1995) Plant Cell 7, 1625-1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051-2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CPCPm) was determined. Our results reveal that CPCPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C.

Satellite RNA-mediated resistance to turnip crinkle virus in Arabidopsis involves a reduction in virus movement.

Satellite RNAs (sat-RNAs) are parasites of viruses that can mediate resistance to the helper virus. We previously showed that a sat-RNA (sat-RNA C) of turnip crinkle virus (TCV), which normally intensifies symptoms of TCV, is able to attenuate symptoms when TCV contains the coat protein (CP) of cardamine chlorotic fleck virus (TCV-CPCCFV). We have now determined that sat-RNA C also attenuates symptoms of TCV containing an alteration in the initiating AUG of the CP open reading frame (TCV-CPm). TCV-CPm, which is able to move systemically in both the TCV-susceptible ecotype Columbia (Col-0) and the TCV-resistant ecotype Dijon (Di-0), produced a reduced level of CP and no detectable virions in infected plants. Sat-RNA C reduced the accumulation of TCV-CPm by < 25% in protoplasts while reducing the level of TCV-CPm by 90 to 100% in uninoculated leaves of Col-0 and Di-0. Our results suggest that in the presence of a reduced level of a possibly altered CP, sat-RNA C reduces virus long-distance movement in a manner that is independent of the salicylic acid-dependent defense pathway.

The coat protein of turnip crinkle virus is involved in subviral RNA-mediated symptom modulation and accumulation.

Some satellite (sat-) and defective interfering (DI) RNAs associated with plant viruses intensify or ameliorate the symptoms of the virus. We recently demonstrated that the TCV coat protein (CP) is involved in symptom modulation by sat-RNA C. Two additional subviral RNAs have now been tested for effect of the CP on symptom modulation. DI RNA G, which normally intensifies the symptoms of TCV, is able to attenuate symptoms if the TCV CP is replaced with the CP of cardamine chlorotic fleck virus. DI RNA G had no effect on the symptoms of TCV with a single base alteration in the CP open reading frame, unlike sat-RNA C, which was able to ameliorate the symptoms of the mutant TCV. Using a hybrid sat-RNA constructed from sat-RNA C and TCV (which shares a similar 3'-end region with DI RNA G), the 3'-terminal 53 bases of sat-RNA C were found to be involved in symptom attenuation, which was directly correlated with the lack of detectable viral genomic RNA in whole plants. Sat-RNA D had no effect on the symptoms of mutant or wild-type TCV. The accumulation of TCV subviral RNAs in plants and protoplasts was also found to be strongly influenced by the presence or absence of the wild-type TCV CP.

Trans-acting untranslated elements of groundnut rosette virus satellite RNA are involved in symptom production.

Isolates of groundnut rosette umbravirus (GRV) contain a satellite RNA (sat-RNA), about 900 nucleotides (nt) in length, different variants of which are responsible for the symptoms of different forms of rosette disease in groundnuts and, in the particular instance of sat-RNA YB3b, for the production of yellow blotch symptoms in Nicotiana benthamiana. Sat-RNA YB3b does not affect the accumulation of GRV genomic or subgenomic RNAs in infected plants. Replication of sat-RNA YB3b and induction of yellow blotch symptoms do not require the production of any sat-RNA-encoded proteins. Experiments with deletion mutants identified three functional untranslated elements in sat-RNA YB3b. One (designated R) comprises nt 47-281, is essential for sat-RNA replication and appears to be cis-acting. The other two (designated A and B) comprise nt 280-470 and 629-849, respectively, are both involved in yellow blotch symptom production and can act in trans. Element A contains the determinant that is unique to sat-RNA YB3b. The process of symptom induction by sat-RNA YB3b apparently involves a novel type of specific interaction of two untranslated RNA elements, which can complement each other, with a host factor or factors.

Down-regulation of groundnut rosette virus replication by a variant satellite RNA.

Symptom production in groundnut plants infected with groundnut rosette virus (GRV) depends on the presence of satellite RNA (sat-RNA) in the GRV culture, and sat-RNA variants that induce only mild symptoms are known. One such variant drastically diminished the replication of GRV genomic RNA in infected Nicotiana benthamiana plants. This down-regulating ability did not involve either of the two open reading frames in the sat-RNA but was controlled by a region near its 5' end, which is required for sat-RNA replication. When N. benthamiana plants were inoculated with GRV and the mild satellite and challenged by inoculation with a GRV isolate (YB) containing a sat-RNA that induces yellow blotch symptoms, no symptoms appeared and little GRV genomic RNA or sat-RNA was detected in the plants, provided the two inoculations were no more than 2 days apart. A GRV isolate containing a sat-RNA that neither induces symptoms in N. benthamiana nor affects genomic RNA accumulation also provided protection against yellow blotch symptom production if inoculated before or up to 2 days after isolate YB. However, in this case protection ws incomplete and both GRV RNA and sat-RNA accumulated to normal levels. It is suggested that sequences from the mild sat-RNA may provide a novel source of resistance against rosette disease.