José-Antonio Daròs

Mechanisms of genetic robustness in RNA viruses.

Two key features of RNA viruses are their compacted genomes and their high mutation rate. Accordingly, deleterious mutations are common and have an enormous impact on viral fitness. In their multicellular hosts, robustness can be achieved by genomic redundancy, including gene duplication, diploidy, alternative metabolic pathways and biochemical buffering mechanisms. However, here we review evidence suggesting that during RNA virus evolution, alternative robustness mechanisms may have been selected. After briefly describing how genetic robustness can be quantified, we discuss mechanisms of intrinsic robustness arising as consequences of RNA‐genome architecture, replication peculiarities and quasi‐species population dynamics. These intrinsic robustness mechanisms operate efficiently at the population level, despite the mutational sensitivity shown by individual genomes. Finally, we discuss the possibility that viruses might exploit cellular buffering mechanisms for their own benefit, producing a sort of extrinsic robustness.

The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens

The “survival of the fittest” is the paradigm of Darwinian evolution in which the best-adapted replicators are favored by natural selection. However, at high mutation rates, the fittest organisms are not necessarily the fastest replicators but rather are those that show the greatest robustness against deleterious mutational effects, even at the cost of a low replication rate. This scenario, dubbed the “survival of the flattest”, has so far only been shown to operate in digital organisms. We show that “survival of the flattest” can also occur in biological entities by analyzing the outcome of competition between two viroid species coinfecting the same plant. Under optimal growth conditions, a viroid species characterized by fast population growth and genetic homogeneity outcompeted a viroid species with slow population growth and a high degree of variation. In contrast, the slow-growth species was able to outcompete the fast species when the mutation rate was increased. These experimental results were supported by an in silico model of competing viroid quasispecies.

A chloroplast protein binds a viroid RNA in vivo and facilitates its hammerhead-mediated self-cleavage

Viroids, small single‐stranded circular RNAs (246–401 nucleotides), do not have mRNA capacity and must recruit host proteins to assist in the steps of their biological cycle. The nature of these cellular factors is poorly understood due to a lack of reliable experimental approaches. Here, to screen for host proteins interacting with viroid RNAs in vivo, we UV‐irradiated avocado leaves infected with avocado sunblotch viroid (ASBVd), the type member of chloroplast viroids containing hammerhead ribozymes. This resulted in the detection of several ASBVd–host protein adducts. Tandem mass spectrometry analysis of the most abundant cross‐linked species identified the protein component as two closely related chloroplast RNA‐binding proteins (PARBP33 and PARBP35) of a family whose members previously have been shown to be involved in stabilization, maturation and editing of chloroplast transcripts. PARBP33 behaves as an RNA chaperone that stimulates in vitro the hammerhead‐mediated self‐cleavage of the multimeric ASBVd transcripts that result from rolling circle replication, indicating that this reaction, despite its RNA‐based mechanism, is facilitated by proteins. The structural and functional parallelism between PARBP33 and PARBP35, and some proteins involved in viral RNA replication, indicates that viroids and RNA viruses recruit similar host proteins for their replication.

Viroids: an Ariadne's thread into the RNA labyrinth

Viroids are structurally, functionally and evolutionarily different from viruses. Despite their small, non‐protein‐encoding, single‐stranded circular RNA genome, viroids can infect higher plants and cause certain diseases. Members of the two viroid families, Pospiviroidae and Avsunviroidae, have evolved to usurp the transcriptional machinery of their host nuclei and chloroplasts, respectively, in which replication proceeds through a rolling‐circle mechanism involving RNA polymerization, cleavage and ligation. Remarkably, viroids subvert certain DNA‐dependent RNA polymerases to transcribe RNA templates, and, in the family Avsunviroidae, post‐transcriptional cleavage is catalysed by hammerhead ribozymes. Viroids are models for studying RNA evolution and for analysing RNA transport in plants, because they can move intracellularly, intercellularly through plasmodesmata and to distal parts of the plant through the vascular system. Viroids elicit RNA‐silencing phenomena, which might mediate some of their biological properties, including pathogenesis. As some viroids behave as catalytic RNAs, they are regarded as remnants of the RNA world.

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase.

Avsunviroidae family: viroids containing hammerhead ribozymes.

Publisher Summary This chapter focuses on the second viroid family, whose members are also referred to as hammerhead viroids, taking into account their most outstanding feature. If the word “small” is the first to come to mind when considering viroids, perhaps the second word is “hammerhead,” because this class of ribozymes, which because of its structural simplicity has an enormous biotechnological potential, is described in avocado sunblotch viroid (ASBVd) as well as in a viroid-like satellite RNA. The most outstanding feature of the Avsunviroidae members is their potential to adopt hammerhead structures in both polarity strands and to self-cleave in vitro accordingly. Viroids differ from viruses not only in their genome size but also in other fundamental aspects, prominent among which is the lack of messenger activity of both viroid RNAs and their complementary strands.

One Is Enough: In Vivo Effective Population Size Is Dose-Dependent for a Plant RNA Virus

Effective population size (Ne) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. Experimental estimates of Ne for different plant viruses have, however, rendered diverging results. The independent action hypothesis (IAH) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. A corollary of IAH is that Ne must be dose dependent. A test of IAH for a plant virus has not been reported yet. Here we perform a test of an IAH infection model using a plant RNA virus, Tobacco etch virus (TEV) variants carrying GFP or mCherry fluorescent markers, in Nicotiana tabacum and Capsicum annuum plants. The number of primary infection foci increased linearly with dose, and was similar to a Poisson distribution. At high doses, primary infection foci containing both genotypes were found at a low frequency (<2%). The probability that a genotype that infected the inoculated leaf would systemically infect that plant was near 1, although in a few rare cases genotypes could be trapped in the inoculated leaf by being physically surrounded by the other genotype. The frequency of mixed-genotype infection could be predicted from the mean number of primary infection foci using the independent-action model. Independent action appears to hold for TEV, and Ne is therefore dose-dependent for this plant RNA virus. The mean number of virions causing systemic infection can be very small, and approaches 1 at low doses. Dose-dependency in TEV suggests that comparison of Ne estimates for different viruses are not very meaningful unless dose effects are taken into consideration.

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro

RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.

Eggplant Latent Viroid, the Candidate Type Species for a New Genus within the Family Avsunviroidae (Hammerhead Viroids)

ABSTRACT Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name Elaviroid.

Stability of Tobacco etch virus infectious clones in plasmid vectors

Tobacco etch virus (TEV) has been traditionally used as a model to research many aspects of the molecular biology of plant RNA virus and, more recently, experimental evolution. However, the only plasmid of this virus species with an infectious clone that has been commonly available to research (pTEV7DA) is rather unstable when propagated in the bacterium Escherichia coli. Here, the TEV infectious clone contained in pTEV7DA is used to construct three new plasmids that allowed infecting the host plants from RNA transcripts synthesized in vitro (pMTEV), directly from plasmid DNA (p35TEV) and by agroinoculation (pGTEV). To increase stability of the three constructed plasmids in E. coli, superfluous vector sequences were removed and the virus expression cassettes were inserted between the plasmid replication origins and antibiotic selection markers in reverse orientation to the latter gene. Although the TEV cDNA in these three new plasmids is not interrupted by any exogenous sequence, they are more stable than the parental pTEV7DA during propagation in E. coli, indicating a major contribution of the plasmid context in virus cDNA stability. Using the different inocula produced from the three new plasmids the TEV infectivity was also compared. The results showed that agroinoculation is the most effective inoculation method and is where symptoms unfold earlier.