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.

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.

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.

Deletion mapping of the potyviral helper component-proteinase reveals two regions involved in RNA binding.

The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA. To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins.

Infection of barley protoplasts with rice hoja blanca tenuivirus. Brief report.

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.

Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro.

Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames.

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.

Interference with Physalis mottle tymovirus replication and coat protein synthesis by transcripts corresponding to the 3'-terminal region of the genomic RNA--role of the pseudoknot structure.

The role of the 3' noncoding (NC) region of Physalis mottle tymovirus genomic RNA in the multiplication of the virus was examined using an in vivo protoplast assay system. Coat protein (CP) synthesis was specifically inhibited by sense 3' NC region transcripts. To establish the role of the pseudoknot structure present in the NC region in virus multiplication, four site-specific mutants, two of which disrupted the pseudoknot structure while the other two restored the structure, were constructed. Interestingly, none of the four sense mutant transcripts inhibited CP synthesis, suggesting that the specific sequence representing the 3' terminal pseudoknot structure may play an important role in virus multiplication. However, the wild-type antisense 3' NC transcript as well as the four antisense mutant transcripts inhibited CP synthesis, suggesting that the inhibitions by antisense transcripts could be due to the formation of RNA-RNA hybrids at the 3' end of the genomic RNA.

Viruses: exquisite models for cell strategies.

Because of the small size of their genome, viral genes have been forerunners in helping us understand gene expression. It is also because of their small size that viruses have elaborated the amazing variety of strategies that enables them to produce all the proteins they require for their multiplication. As a consequence, many of the strategies of expression known to occur in cell systems were first demonstrated in viruses. The aim of this review is to highlight the contribution of viruses to our knowledge of cell processes.

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Characterization of the in vitro activity of the RNA-dependent RNA polymerase associated with the ribonucleoproteins of rice hoja blanca tenuivirus.

An RNA-dependent RNA polymerase (RdRp) activity associated with the ribonucleoproteins of rice hoja blanca tenuivirus (RHBV) was detected and analyzed. Conditions for in vitro RNA synthesis and for coupled RNA synthesis-translation of RHBV were established. In both cases, synthesis of the viral and viral complementary genomic and subgenomic RNA3 and RNA4 were observed, demonstrating that both transcription and replication occurred. Though coupling of RNA synthesis to translation allowed efficient translation of the newly synthesized subgenomic RNAs, studies of the effect of various inhibitors of protein synthesis revealed that RNA synthesis was independent of translation. Primer extension experiments demonstrated that in the presence of capped exogenous RNAs, a stretch of 10 to 16 nonviral nucleotides was added to the 5' end of a population of newly synthesized viral complementary RNA4. It appears that in addition to RdRp activity, RHBV-associated protein(s) also possessed cap-snatching capacity.

Comparison of Colombian and Costa Rican strains of rice hoja blanca tenuivirus.

The sequences of RNA-3 and RNA-4 of rice hoja blanca tenuivirus isolates from Colombia and from Costa Rica were determined and analyzed. These isolates were 98.9% and 98.6% identical in the coding and non-coding regions of RNA-3, and 96.9 and 91.5% identical in the coding and non-coding regions of RNA-4, and are therefore strains of the same virus. There is about three times as much variation between isolates (based on consensus sequences) as there is within isolates (based on sequences of individual clones). There is also considerably more variation for RNA-4 (both between and within isolates) than there is for RNA-3, even though between tenivirus species RNA-3 has diverged more than RNA-4, implying that the evolution of the tenuivirus RNAs is not necessarily dependent on the amount of variation found for these RNAs.

An enigma: the role of viral RNA aminoacylation.

The first demonstration on the aminoacylation capacity of the RNA genome of a plant virus appeared more than 25 years ago. Shortly thereafter, aminoacylation of the RNA genome of a number of other plant viruses was observed. This led to considerable work on the tRNA-like region of these viral RNAs, and to the first demonstration of the presence of pseudoknots in their folding pattern. In spite of the vast amount of efforts put into trying to understand the reason for the aminoacylation capacity of certain viral RNA genomes, as yet no clear general conclusion emerges. It rather looks as though the reason for aminoacylation may be different for different viruses, and that aminoacylation may operate at different levels in the virus life cycle. Given that certain RNA viruses possess structures which resemble that of tRNAs at their 5'- or 3'-termini, it is most likely that convergent evolution may have dominated the appearance of such structures in the virus world.

ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus.

The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T. A portion of this protein (amino acids [aa] 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity. In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro. A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity. The protein also possessed nonspecific RNA binding capacity. Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding. The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.