- Volume 80, Issue 8, 1999

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5′-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem–loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5′-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.

Viroids, small single-stranded circular RNAs endowed with autonomous replication, are unique systems to conduct evolutionary studies of complete RNA genomes. The primary structure of 36 progeny variants of peach latent mosaic viroid (PLMVd), evolved from inoculations of the peach indicator GF-305 with four individual PLMVd cDNAs differing in their pathogenicity, has been determined. Most progeny variants had unique sequences, revealing that the extremely heterogeneous character of PLMVd natural isolates most probably results from the intrinsic ability of this RNA to accumulate changes, rather than from repeated inoculations of the same individual trees under field conditions. The structure of the populations derived from single PLMVd sequences differed according to the observed phenotype. Variant gds6 induced a reproducible symptomatic infection and gave rise to a more uniform progeny that preserves some parental features, whereas variant gds15, which induced a variable phenotype, showed a more complex behaviour, generating two distinct progenies in symptomatic and asymptomatic individual plants. Progenies derived from variants esc10 and ls11, which incited latent infections, followed a similar evolutionary pattern, leading to a population structure consisting of two main groups of variants, one of which was formed by variants closely related to the parental sequence. The evolution rate exhibited by PLMVd, considerably higher than that reported for potato spindle tuber viroid, may contribute to the fluctuating symptomatology of the severe PLMVd natural isolates. However, the polymorphism observed in PLMVd progenies does preserve some structural and functional elements previously proposed for this viroid, supporting the fact that they act as constraints limiting the genetic divergence of PLMVd quasispecies generated de novo.