Anne-Lise Haenni

An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method.

An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

Gene expression from viral RNA genomes

This review is centered on the major strategies used by plant RNA viruses to produce the proteins required for virus multiplication. The strategies at the level of transcription presented here are synthesis of mRNA or subgenomic RNAs from viral RNA templates, and ‘cap-snatching’. At the level of translation, several strategies have been evolved by viruses at the steps of initiation, elongation and termination. At the initiation step, the classical scanning mode is the most frequent strategy employed by viruses; however in a vast number of cases, leaky scanning of the initiation complex allows expression of more than one protein from the same RNA sequence. During elongation, frameshift allows the formation of two proteins differing in their carboxy terminus. At the termination step, suppression of termination produces a protein with an elongated carboxy terminus. The last strategy that will be described is co-and/or post-translational cleavage of a polyprotein precursor by virally encoded proteinases. Most (+)-stranded RNA viruses utilize a combination of various strategies.

The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation

ABSTRACT The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5′-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5′ cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

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.

Identification and Functional Analysis of the Turnip Yellow Mosaic Tymovirus Subgenomic Promoter

ABSTRACT Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.

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.

Interplay between dengue virus and Toll-like receptors, RIG-I/MDA5 and microRNAs: Implications for pathogenesis.

Abstract A growing body of evidence has demonstrated the role of components of innate immunity, including Toll‐like receptors (TLRs), the retinoic acid‐inducible gene I/melanoma‐differentiation factor 5 (RIG‐I/MDA5) and microRNAs (miRNAs) in the recognition of dengue virus (DENV) or its components by infected cells. TLR3, TLR7/8 and RIG‐I/MDA5 sense genomic RNA or dsRNA, the product of an intermediate step of DENV replication, activating intracellular pathways leading to the production of antiviral effectors, including interferon and pro‐inflammatory cytokines. Recognition by TLR2 and TLR4 also promotes the activation of other intracellular pathways and alters viral replication in an interferon‐independent manner. It was also recently demonstrated that cellular miRNAs, a class of post‐transcriptional regulatory small RNAs, can affect replication. To accomplish this, miRNAs bind either directly to viral RNA, through base‐pair complementarity affecting translation, or indirectly through virus‐mediated changes in host protein expression in the viral life cycle. There is also evidence that certain miRNAs can recognize or be recognized by TLRs and RIG‐I/MDA5, resulting in alteration of the innate immune response. In this review, we summarize our present knowledge of DENV‐host factor interactions, emphasizing the role of TLRs, RIG‐I/MDA5 and miRNAs and their possible connection with pathogenesis. Our discussion is based on recent reports suggesting how these different innate immune components might be activated to induce an antiviral response, and how DENV has developed mechanisms to manipulate or evade these antiviral activities. HighlightsInteraction between dengue virus and pathogen recognition receptors alters the production of pro‐inflammatory cytokines.Cellular miRNA could either promote or decrease DENV replication.Viral components such as proteins and sfRNA block the expression of RIG‐I/MDA5.The interaction of NS3 with hHSP70 perturbs RNA‐induced silencing complex formation, impairing miRNA activity.Intracellular PRRs recognize viral RNAs and induce the expression of type I interferon and interferon‐stimulated genes.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Regulation of Translation of Viral mRNAs

Translation constitutes a crucial step in virus development, since it leads to the production of the structural and nonstructural proteins required by the virus. Because of their relative simplicity, viral genomes, and in particular viral RNA genomes, have served as model systems to investigate the steps involved in protein synthesis and have successfully helped to unravel the various strategies of translation known to occur on mRNA templates.