Gabrièle Drugeon

Codon context effect in virus translational readthrough A study in vitro of the determinants of TMV and Mo-MuLV amber suppression

To assess the role of codon context on the efficiency of eukaryotic suppression of termination codons, we have compared, in a rabbit cell‐free translation system, the readthrough efficiency related to two synthetic transcripts differing by the codon context around an amber codon. The codon contexts are derived from tobacco mosaic virus (TMV) and Moloney murine leukemia virus (Mo‐MuLV) RNAs. The Mo‐MuLV‐like codon context does not promote suppression. Substituting TMV‐derived triplets in the Mo‐MuLV‐like codon context shows that the two codons downstrem from the TMV UAG signal are important determinants of suppression, as recently demonstrated in vivo.

Suppression of eukaryotic translation termination by selected RNAs.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

Gene expression during the differentiation of myogenic cells of the L6 line.

The problem of the mechanistic relationship among the different phenotypic expressions in an established myogenic line was approached by blocking cell fusion at different developmental stages, by addition of cytochalasin B. The addition of the drug to cultures at the time when the first two myotubes appeared on the dish, blocked fusion, but did not affect DNA synthesis, expression of myosin, phosphorylase, phosphocreatine kinase, phosphorylase kinase or glycogen synthetase, nor the organization of the elements of the hexagonal lattice. It is concluded that cell fusion is not a prerequisite for the expression of the differentiated phenotype.

Effect of mutations within the Cys-rich region of potyvirus helper component-proteinase on self-interaction

The first approximately 60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region. In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro. Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment.

Plant viruses and new perspectives in cross-protection

Cross-protection in plants is the phenomenon whereby a plant preinoculated with a mild virus strain becomes resistant to subsequent inoculation by a related severe strain. It has been used on a large scale in cases where no resistant plants are available. Although several hypotheses have been proposed to explain the molecular mechanism underlying cross-protection, no single hypothesis can account for all the data obtained. Recently, a phenomenon akin to cross-protection has been achieved in transformed plants harboring the cDNA of a part of a viral RNA genome. These results obtained by genetic engineering raise new hopes for obtaining plants resistant to virus infection.

Comparison of the strategies of expression of five tymovirus RNAs by in vitro translation studies

Total nucleotide sequencing of the RNA genome of various tymoviruses has demonstrated that the overall genome organization of these viruses is identical. Furthermore, the strategies of expression of the turnip yellow mosaic virus (TYMV) genome have been established by in vitro translation studies; these include the synthesis of a subgenomic RNA, the utilization of overlapping open reading frames (ORFs) and maturation of a polyprotein. In the experiments described here, the strategies of expression of other tymovirus (eggplant mosaic virus, ononis yellow mosaic virus, belladonna mottle virus and physalis mottle virus) genomes have been compared to those used by the TYMV genome, in particular to determine whether these tymoviruses also resort to the expression of overlapping ORFs and maturation of a polyprotein.

The strategies of plant virus gene expression: models of economy

Given the small size of their genome, the genetic information of viruses is extremely compact, and non-coding regions are very limited as compared to those of prokaryotic and eukaryotic cell systems. Viruses utilize cell components at all levels of the replication cycle for their own benefit, not the least being the translation machinery. They have also evolved a number of highly sophisticated strategies to produce and regulate the production of the proteins required for their propagation. In addition, these proteins are often multifunctional, encoding several essential virus-specific proteins. At the level of transcription, these strategies include splicing, the production of subgenomic RNAs from virus templates and cap-snatching. At the level of translation, regulation exists at all steps: initiation, elongation and termination. Furthermore, viruses frequently resort to co- and/or post-translational cleavage of a polyprotein precursor to yield the mature proteins.

Strategies of Expression of Turnip Yellow Mosaic Virus in Vivo: Developmental Approach for the Study of the Autocatalytic Cleavage of the 206k Polyprotein

Turnip yellow mosaic virus (TYMV) is the type-member of the tymovirus group which belongs to the Sindbis-like virus supergroup of (+)RNA viruses. TYMV infects members of the Cruciferae family. The genomic RNA directs the synthesis of two nonstructural overlapping proteins. It has recently been demonstrated that the larger one of 206K undergoes autocleavage yielding at least two products of 150K and 78K respectively, in vitro (1). The Smaller one of 69K is probably the movement protein (2).