Polycistronic RNA Research Articles

Higher-Order RNA Structural Requirements and Small-Molecule Induction of Tombusvirus Subgenomic mRNA Transcription

Subgenomic (sg) mRNAs are small viral messages that are synthesized by polycistronic positive-strand RNA viruses to allow for the translation of certain viral proteins. Tombusviruses synthesize two such sg mRNAs via a premature termination mechanism. This transcriptional process involves the viral RNA-dependent RNA polymerase terminating minus-strand RNA synthesis prematurely at internal RNA signals during copying of the viral genome. The 3'-truncated minus-strand RNAs generated by the termination events then serve as templates for sg mRNA transcription. A higher-order RNA structure in the viral genome, located just upstream from the termination site, is a critical component of the RNA-based polymerase attenuation signal. Here, we have analyzed the role of this RNA structure in mediating efficient sg mRNA2 transcription. Our results include the following: (i) we define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) we show that the distribution of stability within an attenuation structure affects its function; (iii) we establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) we prove that the higher-order RNA structure forms and functions in the plus strand; (v) we provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; (vi) we demonstrate that sg mRNA transcription can be controlled artificially through small-molecule activation using RNA aptamer technology. These findings provide important new insights into the premature termination mechanism and present a novel approach to regulate the transcriptional process.

The mitochondrial ND1 T3308C mutation in a Chinese family with the secondary hypertension

Mutations in mitochondrial DNA have been associated with hypertension. We report here the clinical, genetic, and molecular characterization of one four-generation Han Chinese family with hypertension. Two matrilineal relatives in this family exhibited the variable degree of a secondary hypertension (renal hypertension) at the age-at-onset of 42 and 56years old, respectively. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the known hypertension-associated ND1 T3308C mutation and 42 other variants, belonging to the Asian haplogroup D4h. The T3308C mutation resulted in the replacement of the first amino acid, translation-initiating methionine with a threonine in ND1. Furthermore, the ND3 T3308C mutation also locates in two nucleotides adjacent to the 3′ end of mitochondrial tRNALeu(UUR). Thus, this T3308C mutation caused an alteration on the processing of the H-strand polycistronic RNA precursors or the destabilization of ND1 mRNA. The occurrence of the T3308C mutation in these genetically unrelated pedigrees affected by diseases but absence of 242 Chinese controls as well as the mitochondrial dysfunctions detected in cells carrying this mutation indicate that this mutation is involved in the pathogenesis of hypertension. However, the mild biochemical defects, the lower penetrance of hypertension in this Chinese family and the presence of some control populations suggested the involvement of other modifier factors in the pathogenesis of hypertension associated with this ND1 T3308C mutation.

RNA Polymerase Transcription Machinery in Trypanosomes

Transcription is a fundamental biological process employed by all living organisms to decode their genetic information. The information stored in genomic DNA is copied into RNA molecules by polymerization of ribonucleotide building blocks, which ultimately gives rise to different classes of transcripts. mRNAs encode polypeptides, rRNAs drive the macromolecular protein-synthesis machinery, and tRNAs act as adaptor molecules to assemble amino acids into proteins. Synthesis of specific transcripts is influenced by environmental and internal cell signals, which in turn are pivotal for the control of cellular regulatory networks. Trypanosomes are unicellular parasitic protozoa, members of the order Kinetoplastidae, which diverged early during evolution. They cause a wide range of debilitating diseases in humans and domestic animals. Trypanosoma brucei, known as the African trypanosome, is transmitted by tsetse flies in subSaharan Africa (15). Infection fulminates into African sleeping sickness in humans and nagana in animals (3). T. brucei is a digenetic parasite that cycles as a procyclic form in the digestive tract of the tsetse vector and as an extracellular bloodstream form in its mammalian host. During its complex life cycle, the parasite passes through five successive morphologically distinct forms (39). Parasites change from the procyclic form, which is characterized by a procyclic-specific surface coat, through two morphologically distinct forms in the fly and then they emerge as long, slender bloodstream forms, covered with a variant surface glycoprotein coat. Once inside the mammalian host, the long slender bloodstream form actively divides and establishes parasitemia. In the late phases of infection, the morphology of the parasite changes to nondividing short stumpy forms, which are ready to be taken up by the insect during a blood meal. The bloodstream form, with a rudimentary mitochondrion, is perfectly adapted to utilize the abundant supply of glucose from the mammalian blood and generate sufficient energy by glycolysis. The insect form, on the other hand, has a functional mitochondrion and generates most of its energy by respiration. These necessary metabolic adaptations depend upon a precise orchestration of numerous metabolic and cell biological activities. Studies of trypanosomes have uncovered several unusual biological phenomena (6). Notable among them are trans splicing and RNA editing (reviewed in references 7, 35, 46, 48, and 59). Protein-coding genes in trypanosomes are transcribed as long polycistronic precursor RNAs. Individual mature mRNAs are formed by trans splicing of a 39-nucleotide spliced leader (SL) RNA at the 5 end and subsequent 3 end maturation. RNA editing, used to produce mitochondrial mRNA, requires extensive alterations of primary transcripts by guide RNAs. Although guide RNA-dependent RNA editing is uniquely observed in trypanosomes, trans splicing has subsequently been observed in several other lower eukaryotes, including nematodes, trematodes, euglenoids, and chordates. Therefore, studies of trypanosomes are expected to uncover cryptic mechanistic components of eukaryotic biology and reveal exotic cellular processes.

Sugar Receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

A ubiquitin‐based vector for the co‐ordinated synthesis of multiple proteins in plants

The genetic engineering of complex traits into crop plants will ultimately require strategies to co-express more than one protein at the same time. Here, we report the development of a ubiquitin (Ub)-based expression method that can generate two proteins from a single transcript. It contains coding regions for the proteins of interest, separated in-frame by the coding region for the C-terminal end of Ub followed by a full-length Ub. On expression in tobacco, this polycistronic messenger RNA (mRNA) is translated to produce a chimeric protein that is rapidly processed by endogenous deubiquitinating proteases to release the two proteins plus a Ub moiety in intact forms. The C-terminal protein domain is released without additional amino acids, whereas the N-terminal protein domain retains the short C-terminal end of Ub. The analysis of vectors with progressively shorter C-terminal ends indicates that only the last six C-terminal amino acids of the proximal Ub domain are needed for efficient processing in plants. By comparing the levels of luciferase and beta-glucuronidase simultaneously expressed by this method in multiple independent tobacco transformants, we synthesized consistently similar ratios of the two proteins over a wide range of protein amounts. Ub-based polyprotein vectors should facilitate the genetic engineering of crops by providing a simple method for the co-ordinated and stoichiometric synthesis of two or more proteins.

Differential Effects of Arginine Methylation on RBP16 mRNA Binding, Guide RNA (gRNA) Binding, and gRNA-containing Ribonucleoprotein Complex (gRNP) Formation

Mitochondrial gene expression in Trypanosoma brucei involves the coordination of multiple events including polycistronic transcript cleavage, polyadenylation, RNA stability, and RNA editing. Arg methylation of RNA binding proteins has the potential to influence many of these processes via regulation of protein-protein and protein-RNA interactions. Here we demonstrate that Arg methylation differentially regulates the RNA binding capacity and macromolecular interactions of the mitochondrial gene regulatory protein, RBP16. We show that, in T. brucei mitochondria, RBP16 forms two major stable complexes: a 5 S multiprotein complex and an 11 S complex consisting of the 5 S complex associated with guide RNA (gRNA). Expression of a non-methylatable RBP16 mutant protein demonstrates that Arg methylation of RBP16 is required to maintain the protein-protein interactions necessary for assembly and/or stability of both complexes. Down-regulation of the major trypanosome type 1 protein arginine methyltransferase, TbPRMT1, disrupts formation of both the 5 and 11 S complexes, indicating that TbPRMT1-catalyzed methylation of RBP16 Arg-78 and Arg-85 is critical for complex formation. We also show that Arg methylation decreases the capacity of RBP16 to associate with gRNA. This is not a general effect on RBP16 RNA binding, however, since methylation conversely increases the association of the protein with mRNA. Thus, TbPRMT1-catalyzed Arg methylation has distinct effects on RBP16 gRNA and mRNA association and gRNA-containing ribonucleoprotein complex (gRNP) formation.

RETRACTED: RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins

RNA silencing is an ancient mechanism of gene regulation with important antiviral roles in plants and insects. Although induction of RNA silencing by RNA viruses has been well documented in plants, the interactions between DNA viruses and the host silencing machinery remain poorly understood. We investigate this question with cauliflower mosaic virus (CaMV), a dsDNA virus that expresses its genome through the polycistronic 35S RNA, which carries an unusually extensive secondary structure known as translational leader. We show that CaMV-derived siRNAs accumulate in turnip- and Arabidopsis-infected plants and that the leader is a major, albeit not exclusive, source for those molecules. Biogenesis of leader-derived siRNA requires the coordinated and hierarchical action of the four Arabidopsis Dicer-like (DCL) proteins. Our study also uncovers a "facilitating" role exerted by the microRNA biosynthetic enzyme DCL1 on accumulation of DCL2-, DCL3-, and DCL4-dependent siRNAs derived from the 35S leader. This feature of DCL1 defines a small RNA biosynthetic pathway that might have relevance for endogenous gene regulation. Several leader-derived siRNAs were found to bear near-perfect sequence complementarity to Arabidopsis transcripts, and, using a sensor transgene, we provide direct evidence that at least one of those molecules acts as a bona fide siRNA in infected turnip. Extensive bioinformatics searches identified >100 transcripts potentially targeted by CaMV-derived siRNAs, several of which are effectively down-regulated during infection. The implications of virus-directed silencing of host gene expression are discussed.

Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei

In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3′-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase ( CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3′→5′ exonuclease complex) or the 5′→3′ exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes.

Cocksfoot mottle virus P1 suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum

The Sobemovirus genome consists of positive sense, single-stranded polycistronic RNA. The 5′-terminal ORF, encoding the protein P1, is its most variable region. Sobemoviral P1 has been described as dispensable for replication but indispensable for systemic infection. The P1 of Rice yellow mottle virus-Nigerian isolate (RYMV-N) is the only RNA silencing suppressor reported for sobemoviruses until now. Using an agrobacterium-mediated transient assay, we demonstrate here that P1 of Cocksfoot mottle virus-Norwegian isolate (CfMV-NO) suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum, two non-host plants. CfMV-NO P1 was able to suppress the initiation and maintenance of silencing. The suppression of systemic silencing was weaker with CfMV-NO P1 than in the case of RYMV-N P1. In the case of suppression at the local level, the reduction in the amount of 25-nucleotide small interfering RNAs (siRNAs) was less pronounced for CfMV-NO P1 than it was when RYMV-N P1 was used. At the same time, we show that CfMV-NO P1 did not bind siRNAs.

Polycistronic RNA polymerase II expression vectors for RNA interference based on miR-155

Polycistronic RNA polymerase II expression vectors for RNA interference based on miR-155

Nidovirales: Evolving the largest RNA virus genome

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32 kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16 kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3′-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Polymorphic and differential expression of the Trypanosoma cruzi alleles containing universal minicircle binding protein

DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite ( TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853–858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62 bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus.

Natural competence of mammalian mitochondria allows the molecular investigation of mitochondrial gene expression

Respiration, a fundamental process in mammalian cells, requires two genomes, those of the nucleus and the mitochondrion (mtDNA). Mutations of mtDNA are being increasingly recognized in disease and may play an important role in the ageing process. Accepting the vital role of mtDNA gene products, our limited knowledge concerning the details of mitochondrial gene expression is surprising. This is, in part, due to our inability to transfect mitochondria and to manipulate their genome. There have been claims of successful DNA import into isolated organelles, but most reports lacked evidence of expression and no method has furthered our understanding of gene expression. Here, we report that mammalian mitochondria possess a natural competence for DNA import. Using five functional assays, we show imported DNA can act as templates for DNA synthesis or promoter-driven transcription, with the resultant polycistronic RNA being processed (5' and 3') and excised mt-tRNA matured. Exploiting this natural competence will allow us to explore mitochondrial gene expression in organello and provides the potential for mitochondrial transfection in vivo.

Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

Differential polyadenylation of ribosomal RNA during post-transcriptional processing inLeishmania

The protozoan parasite Leishmania belongs to the most ancient eukaryotic lineages and this is reflected in several distinctive biological features, such as eukaryotic polycistronic transcription and RNA trans-splicing. The disclosure of this organism's unusual characteristics leads to a better understanding of the origin and nature of fundamental biological processes in eukaryotes. Here we report another unusual phenomenon as we demonstrate that precursor ribosomal RNA can be extensively polyadenylated during post-transcriptional processingt. Furthermore, we demonstrate that the degree of precursor rRNA polyadenylation is variable in different strains and in the different life-stages of a strain.

The Open Reading Frame VI Product ofCauliflower mosaic virusIs a Nucleocytoplasmic Protein: Its N Terminus Mediates Its Nuclear Export and Formation of Electron-Dense Viroplasms

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

The ATPase subunit 6 gene of Leptomonas seymouri (Trypanosomatidae) is transcribed and edited as a polycistronic mRNA

The maturation of mRNA was studied for the Leptomonas seymouri mitochondrial A6 gene coding for ATPase subunit 6. RT-PCR showed that the cytochrome b and A6 genes are transcribed to produce a polycistronic RNA. The RT-PCR products, containing the cDNAs of these genes, were cloned and sequenced. Comparison of the sequence for 12 clones revealed four types of transcripts representing partly or completely edited products of A6. Two different variants of completely edited A6 mRNAs were found.

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana

The P6 protein of Cauliflower mosaic virus (CaMV) transactivates translation of the CaMV 35S polycistronic pregenomic RNA and its spliced versions, and thus allows synthesis of a complete set of viral proteins. Previous studies have shown that P6 interacts with plant L18 and L24 ribosomal proteins and initiation factor eIF3, and it has been proposed that these interactions are involved in the reinitiation of translation of polycistronic viral RNAs. This study characterizes a novel cellular partner of P6, the ribosomal protein L13 from Arabidopsis thaliana. Far-Western assays performed with several P6 deletion mutants have shown that L13 interacts with the miniTAV of P6, which represents the minimal domain for transactivation, suggesting that the P6-L13 interaction might also be involved in this process. L13 and L18 were found to bind to the same region within the miniTAV. Competition assays between L18 and L13 for binding to miniTAV suggest that interactions between P6 and these ribosomal proteins involve separate P6 molecules, and/or occur at different stages of translation or in the context of another function also mediated by P6.

Chloroplast RNA processing and stability

Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their relative longevity. Some of these processes, e.g., psbA splicing and stability of a number of chloroplast mRNAs, are regulated in response to light-dark cycles or nutrient availability. This review highlights recent advances in our understanding of these processes in the model organism Chlamydomonas reinhardtii, focusing on results since the extensive reviews published in 1998 [Herrin DL et al. 1998 (pp. 183-195), Nickelsen Y 1998 (pp. 151-163), Stern DB and Drager RG 1998 (pp. 164-182), in Rochaix JD et al. (eds) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands]. We also allude to studies with other organisms, and to the potential impact of the Chlamydomonas genome project where appropriate.

Control of translation reinitiation on the cauliflower mosaic virus (CaMV) polycistronic RNA.

Translation of the polycistronic 35S RNA of CaMV (cauliflower mosaic virus) occurs via a reinitiation mechanism, which requires TAV (transactivator/viroplasmin). To allow translation reinitiation of the major open reading frames on the polycistronic RNA, TAV interacts with the host translational machinery via eIF3 (eukaryotic initiation factor 3) and the 60S ribosome. Accumulation of TAV and eIF3 in the polysomal fraction isolated from CaMV-infected cells suggested that TAV prevents loss of eIF3 from the translating ribosomes during the first initiation event. The TAV-eIF3-80S complex could be detected in vitro by sucrose-gradient-sedimentation analysis. The question is whether TAV interacts directly with the 48S preinitiation complex or enters polysomes after the first initiation event. eIF4B, a component of the 48S initiation complex, can preclude formation of the TAV-eIF3 complex via competition with TAV for eIF3 binding; the eIF4B- and TAV-binding sites on eIF3g overlap. eIF4B out-competes TAV for binding to eIF3 and to the eIF3-40S complex. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated transactivation of polycistronic translation. Our results thus indicate that eIF4B precludes TAV-eIF3-40S complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only the second and subsequent initiation events. We propose a model in which TAV enters the host translational machinery at the eIF4B-removal step to stabilize eIF3 within polysomes.