Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable "core" of housekeeping genes accompanied by a much more flexible "shell" consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the "shell" genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution. Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the "shell" genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages. The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses. Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Post-transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV-encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

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Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.

The potyvirus group [named after its type member, potato virus Y (PVY)] is the largest of the 34 plant virus groups and families currently recognized (Ward & Shukla, 1991). It contains at least 180 definitive and possible members (or 30% of all known plant viruses) which cause significant losses in agricultural, pasture, horticultural and ornamental crops (Ward & Shukla, 1991). These viruses are unique in the diversity of inclusion bodies that are formed during the infection cycle (see Lesemann, 1988). A feature shared by all potyviruses is the induction of characteristic pinwheel or scroll-shaped inclusion bodies in the cytoplasm of the infected cells (Edwardson, 1974). These cylindrical inclusion (CI) bodies are formed by a virus-encoded protein and can be considered as the most important phenotypic criterion for assigning viruses to the poty-virus group (Milne, 1988; Shukla et al., 1989; Ward & Shukla, 1991).

Highlights and prospects of potyvirus molecular biology

The RNA genome of tobacco etch virus (TEV), a plant potyvirus, functions as an mRNA for synthesis of a 346-kilodalton polyprotein that undergoes extensive proteolytic processing. The RNA lacks a normal 5' cap structure at its terminus, which suggests that the mechanism of translational initiation differs from that of a normal cellular mRNA. We have identified a translation-enhancing activity associated with the 144-nucleotide, 5' nontranslated region (NTR) of the TEV genome. When fused to a reporter gene encoding beta-glucuronidase (GUS), the 5' NTR results in an 8- to 21-fold enhancement over a synthetic 5' NTR in a transient-expression assay in protoplasts. A similar effect was observed when the 5' NTR-GUS fusions were expressed in transgenic plants. By using a cell-free translation system, the translation enhancement activity of the TEV 5' NTR was shown to be cap independent, whereas translation of GUS mRNA containing an artificial 5' NTR required the presence of a cap structure. Translation of GUS transcripts containing the TEV 5' NTR was relatively insensitive to the cap analog m7GTP, whereas translation of transcripts containing the artificial 5' NTR was highly sensitive. The 144-nucleotide TEV 5' NTR synthesized in vitro was shown to compete for factors that are required for protein synthesis in the cell-free translation reaction mix. Competition was not observed when a transcript representing the initial 81 nucleotides of the TEV 5' NTR was tested. These results support the hypothesis that the TEV 5' NTR promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to internal sites within the NTR.

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Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region.

SUMMARY The complete nucleotide sequence of the genomic RNA of the potyvirus potato virus Y strain N (PVYn) was obtained from cloned cDNAs. This sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. The positions of the cleavage sites at the N terminus of the capsid and cytoplasmic inclusion proteins have been determined. Other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottling virus and of the tobacco etch virus. Comparison of the PVY polyprotein sequence with that of other potyvirus polyproteins shows similarities in genome organization and a high level of identity along most of the polyprotein, except for the putative proteins flanking the helper component. A search for specific protein motifs has revealed the existence of a potential metal-binding site at the putative N terminus of the helper component in potyviruses. The possible functions of this structure are discussed.

Nucleotide sequence of potato virus Y (N Strain) genomic RNA.

Oxylipins, derived from fatty acid hydroperoxides (FAHs), are thought to play different roles during plant pathogen interactions. During hypersensitive response (HR) some of them serve as signals necessary for defence gene activation whereas others could contribute to pathogen killing or could participate in the execution of plant programmed cell death (PCD) associated with this resistance. In order to address the role of these compounds in the latter process, we have closely observed lipid peroxidation, the first step of this metabolic pathway, under different situations which led either to accelerated or inhibited HR cell death. The oxidative process has been studied in cryptogein-elicited tobacco leaves and during Ralstonia solanacearum-induced HR. It was shown that FAH accumulation was preceded by the co-ordinated rise in 9 lipoxygenase (9 LOX) and galactolipase activities in addition to the transcription of a set of four genes encoding 9 LOX and patatin-like proteins, NtPAT1-3. The latter gene expression was at the origin of a metabolic pathway allowing the release of poly-unsaturated fatty acids from plastid galactolipids and their oxidation into free 9 FAHs. This 9 LOX-dependent lipid peroxidation was found to be sufficient to lead to HR cell death. Finally, during the bacterial-induced HR, lipid peroxidation appeared as a composite of metabolites of enzymatic and non-enzymatic origins and suggested the role of H2O2 as an important source of oxidant that might, in synergy with 9 FAHs, contribute to cell death execution.

The combined action of 9 lipoxygenase and galactolipase is sufficient to bring about programmed cell death during tobacco hypersensitive response

Multicopy plasmid RSF1010 and four of its in vitro-constructed derivatives were mobilized by the self-transmissible RP4 plasmid into Azotobacter vinelandii UW. Modifications of the Escherichia coli transformation procedure of Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114, 1972) allowed transformation of A. vinelandii strains UW and ATCC 12837 with purified RP4 or RSF1010 deoxyribonucleic acid.

Transformation of Azotobacter vinelandii with Plasmids RP4 (IncP-1 Group) and RSF1010 (IncQ Group)

Potato virus Y (PVY), a potyvirus, has an RNA genome containing 9704 nucleotides of which 185 belong to the 5' nontranslated region (NTR) Contrary to most eukaryotic mRNAs that have a cap structure, the potyvirus RNA has a genome-linked protein (VPg) In order to understand the mechanisms of PVY RNA translation initiation, hybrid-arrest translation was used to localize sequences involved in binding of proteins and/or ribosomes The 5' NTR was fused to the beta-glucuronidase (GUS) reporter gene Six antisense oligodeoxynucleotides were used for hybridization, and the efficiency of the in vitro translation of the hybridized mRNA was modified to different levels depending upon the position of the oligodeoxynucleotide used The highest inhibition was obtained with an oligodeoxynucleotide hybridized to the 5' end In addition, translation of GUS mRNA containing the PVY 5' NTR was greatly enhanced when this mRNA was capped These results differ from those obtained with the tobacco etch virus (TEV) and three picornaviruses, but are similar to those obtained with capped mRNA

Maurice Tronchet

Papers

Nucleotide sequence of potato virus Y (N Strain) genomic RNA.

The combined action of 9 lipoxygenase and galactolipase is sufficient to bring about programmed cell death during tobacco hypersensitive response

Transformation of Azotobacter vinelandii with Plasmids RP4 (IncP-1 Group) and RSF1010 (IncQ Group)

Effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus Y RNA.