Positive-strand Viruses Research Articles

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection.

BackgroundLethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.ResultsThe effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.Conclusions(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

MEK5/ERK5/mef2: A novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.

RNA structure: new messages in translation, replication and disease

The ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs took place between 27 and 29 October 2008, in Baeza, Spain, and was organized by E. Martinez‐Salas, G. Belsham & J. Gomez. ![][1] See Glossary for abbreviations used in this article Since the elucidation of the genetic code approximately 40 years ago, the role of RNA in crucial biological processes has gained increasing attention, and is now one of the fastest developing areas in molecular biology and biochemistry. The advances made in structural studies of transfer RNA (tRNA), ribozymes, riboswitches, the ribosome and viral IRES elements in particular have shown the importance of secondary and tertiary structures in RNA biology, and have significantly changed our understanding of the translational control of gene expression (Fig 1). The World Heritage town of Baeza in Andalucia, Spain, hosted the ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs. About 45 participants met in the beautiful buildings of the Sede Antonio Machado at the Universidad Internacional de Andalucia. Figure 1. Schematic illustration of discreet RNA regulatory elements. The translation of an mRNA is regulated by diverse mechanisms that involve both structural and non‐structural RNA elements, as well as interactions with RNA‐binding proteins. IRESes are found in the 5′ UTR and promote cap‐independent translation. Pseudoknots can be located in the 5′ UTR, the 3′ UTR, or the coding region, and their localization influences their effect on translation; for example, initiation, frameshifting and termination. 3′ UTR structural elements such as CREs, CITEs and SLs often function through long‐range RNA interactions. The miRNA target sites are located in the 3′ UTR. The canonical mRNA features of the cap (m7G) and poly(A) are targets of several regulatory interactions. The … [1]: /embed/graphic-1.gif

Metabolite profiling studies in Saccharomyces cerevisiae: an assisting tool to prioritize host targets for antiviral drug screening

BackgroundThe cellular proteins Pat1p, Lsm1p, and Dhh1p are required for the replication of some positive-strand viruses and therefore are potential targets for new antiviral drugs. To prioritize host targets for antiviral drug screening a comparative metabolome analysis in Saccharomyces cerevisiae reference strain BY4742 Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 and deletion strains pat1Δ, lsm1Δ and dhh1Δ was performed.ResultsGC/MS analysis permitted the quantification of 47 polar metabolites and the identification of 41 of them. Metabolites with significant variation between the strains were identified using partial least squares to latent structures discriminate analysis (PLS-DA). The analysis revealed least differences of pat1Δ to the reference strain as characterized by Euclidian distance of normalized peak areas. The growth rate and specific production rates of ethanol and glycerol were also most similar with this strain.ConclusionFrom these results we hypothesize that the human analog of yeast Pat1p is most likely the best drug target candidate.

Functional analysis of structural motifs in dicistroviruses

The family Dicistroviridae is composed of positive-stranded RNA viruses which have monopartite genomes. These viruses carry genome-linked virus proteins (VPg) and poly (A) tails. The 5′ untranslated region (UTR) is approximately 500 nucleotides and contains an internal ribosome entry site (IRES). These features resemble those of vertebrate picornaviruses, but dicistroviruses have other distinct characteristics. Picornaviruses have a single large open reading frame (ORF) encoding the capsid proteins at the 5′-end and the replicases at the 3′-end. In contrast, dicistroviruses have two nonoverlapping ORFs. The 5′-proximal ORF encodes the replicases and the 3′-proximal ORF encodes the capsid proteins. Usually, positive-stranded viruses which have capsid protein genes in the 3′ part of the genome produce subgenomic RNA for synthesis of the capsid proteins, because abundant quantities of the capsid proteins are required for the viral replication cycle. In dicistroviruses, translation of the capsid proteins is controlled by an additional IRES. This IRES is located in the intergenic region (IGR) between the replicase and capsid coding regions, and mediates the initiation of translation for the capsid proteins. The IGR-IRES has a multiple stem-loop structure containing three pseudoknots. We describe the characteristics of dicistroviruses, including the RNA elements and viral proteins.

New insights into internal ribosome entry site elements relevant for viral gene expression

A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5' UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA-RNA contacts between the 5' and 3' ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.

Recombination and Selection in the Evolution of Picornaviruses and Other Mammalian Positive-Stranded RNA Viruses

Picornaviridae are a large virus family causing widespread, often pathogenic infections in humans and other mammals. Picornaviruses are genetically and antigenically highly diverse, with evidence for complex evolutionary histories in which recombination plays a major part. To investigate the nature of recombination and selection processes underlying the evolution of serotypes within different picornavirus genera, large-scale analysis of recombination frequencies and sites, segregation by serotype within each genus, and sequence selection and composition was performed, and results were compared with those for other nonenveloped positive-stranded viruses (astroviruses and human noroviruses) and with flavivirus and alphavirus control groups. Enteroviruses, aphthoviruses, and teschoviruses showed phylogenetic segregation by serotype only in the structural region; lack of segregation elsewhere was attributable to extensive interserotype recombination. Nonsegregating viruses also showed several characteristic sequence divergence and composition differences between genome regions that were absent from segregating virus control groups, such as much greater amino acid sequence divergence in the structural region, markedly elevated ratios of nonsynonymous-to-synonymous substitutions, and differences in codon usage. These properties were shared with other picornavirus genera, such as the parechoviruses and erboviruses. The nonenveloped astroviruses and noroviruses similarly showed high frequencies of recombination, evidence for positive selection, and differential codon use in the capsid region, implying similar underlying evolutionary mechanisms and pressures driving serotype differentiation. This process was distinct from more-recent sequence evolution generating diversity within picornavirus serotypes, in which neutral or purifying selection was prominent. Overall, this study identifies common themes in the diversification process generating picornavirus serotypes that contribute to understanding of their evolution and pathogenicity.

A comparative survey, by expressed sequence tag analysis, of genes expressed in peach leaves infected with Plum pox virus (PPV) and free from PPV

Plum pox virus (PPV), genus Potyvirus, is a serious threat to the fruit and ornamental horticulture industry in Canada. Genomic resources of its natural hosts, Prunus spp., currently available to researchers studying PPV are very poor. Systematic sequencing of expressed sequence tags (ESTs) can be used as a discovery base for genes associated with PPV infection, replication, and symptom development. Here we describe the construction of two directional, size-selected cDNA libraries derived from PPV-infected and PPV-free peach leaves, and the generation of 3839 ESTs. Based on their partial sequences and putative functions, the ESTs are classified into 611 clusters and 1153 singletons. About 65.36% of the genes (2509 ESTs) show homology to genes from other organisms, and 34.64% of cDNAs (1330 ESTs) have no matches in the GenBank database. Comparison analyses suggest that genes involved in defence, cellular transport, development, protein synthesis, proteins with binding function in the PPV-infected peach leaf tissue are more active than those in PPV-free leaves. Moreover, a number of gene transcripts, including β-1,3- glucanase, cytochrome-P450-like protein, cytochrome P450 monooxygenase, heat-shock protein 70, thioredoxin, alcohol dehydrogenase, catalase, cysteine protease inhibitor, translation factor EF-1α, and pathogenesis-related protein (PR1) are highly prevalent only in the PPV-infected peach leaf. Consistent with results in several recent publications from other laboratories, our finding supports the view that positive-stranded viruses may elicit common responses in susceptible plants. The results of this study help better understand the molecular mechanisms associated with susceptibility to PPV disease in Prunus persica. and provide a studying point for the development of novel strategies to control the virus.

Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA

Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5' end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression.

Bioinformatic mapping of AlkB homology domains in viruses

BackgroundAlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes.ResultsProfile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system.ConclusionsThe AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment.

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses.

RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3' poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5'-UTR and 3'-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses.

Autogenous Translational Inhibition of Core Protein: Implication for Switch from Translation to RNA Replication in Hepatitis C Virus

Positive-stranded viruses use the genomic RNA as a common template for translation and RNA replication which proceed in inverse direction; a certain regulatory mechanism for translation control is probably required to coordinate these two antagonistic processes. Hepatitis C virus (HCV) core protein is a good candidate that might play a role in such a regulation. In this study, we further investigated whether HCV core protein modulates internal ribosome entry site (IRES)-directed translation. The inclusion of the core-coding sequence significantly suppressed translation initiated by HCV IRES in monocistronic and bicistronic reporter systems. The region mainly responsible for this inhibition was mapped to nt 441–473 of the core-coding sequence. This suppression was eliminated by frameshift mutations introduced into this region, suggesting that it is the core protein expressed in cis, rather than the core-coding nucleotide sequence that negatively modulates the efficiency of HCV IRES-dependent translation. Furthermore, the core protein provided in trans also specifically decreased the IRES activity in directing cap-independent translation both in transfected cells and in cell-free translation study. Consistently, a gel mobility shift assay showed a specific interaction between the core protein and HCV IRES-containing RNA transcript. These findings suggest that HCV core protein may down-regulate the cap-independent translation as a regulatory mechanism required for initiation of transcription.

Synthesis of Subgenomic RNAs by Positive-Strand RNA Viruses

f b w t ( t g s p t n p Many RNA viruses encode more than one gene on a single genomic RNA. Yet only the first gene, or open reading frame (ORF), on a normal eukaryotic mRNA is translated. Thus, downstream genes on viral genomes are expressed either via novel translational events or, more commonly, by deployment of subgenomic mRNAs (sgRNAs). Subgenomic RNAs of positive-strand viruses have the same 39 ends as genomic RNA, but have deleions at the 59 ends to bring the 59 end of the RNA in proximity with the start codon of downstream (on genomic RNA) ORFs. Because replication is required for sgRNA synthesis, the RNA-dependent RNA polymerase (RdRp) is always translated first, directly from genomic RNA of positive-strand RNA viruses. sgRNAs express products needed during intermediate and late stages of infection, such as structural or movement proteins. Taxa of (1) sense viruses that produce sgRNAs inlude animal viruses in the order Nidovirales (Coronairidae and Arteriviridae families) and the families Togairidae, Caliciviridae, Nodaviridae, and Astroviridae, plant iruses of the Luteoviridae, Bromoviridae, Tombusviridae, nd Closteroviridae families, and the Tobravirus, Carlairus, Tymovirus, Potexvirus, Hordeivirus, Tobamovirus, obemovirus, and Furovirus genera. Subgenomic RNA ynthesis has been studied more in plant viruses than in nimal viruses. This is probably because a greater perentage of all plant viruses make sgRNAs and also ecause plant viruses, with smaller genomes and highly fficient replication, are often more amenable than anial viruses to studies of RNA replication mechanisms, specially in cell-free extracts. The similarities between

Alphavirus expression vectors

Publisher Summary This chapter describes the mechanics, including how alphaviruses function as expression vectors and some features that make them useful. It also describes some of the ways in which alphaviruses have been used and some potential ways they can be of value. Alphaviruses are positive-strand RNA-enveloped viruses. Three of them, Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, are becoming widely recognized as expression vectors. The most versatile of these vectors are replicons in which the structural protein genes are replaced by a heterologous gene. They can be packaged into virus-like particles by the use of defective helpers that provide the structural proteins. Alphaviruses and their replicons are normally cytopathic in cultured vertebrate cells, but replicons that have a mutation in one of the nonstructural protein genes have a decreased level of viral RNA synthesis and show either reduced or no cytopathogenicity in some cultured cells, in particular baby hamster kidney cells. Apart from these improvements, alphavirus vectors are beginning to demonstrate their value as vaccines and possibly also for expression in neurons.

Influenza C virus persistence depends on exceptional steps in viral RNA synthesis and transport.

The cell line MDCK-pi, which is persistently infected with a variant of influenza C/AnnArbor/1/50 virus (C/AA-pi), was studied as a long-term persistence model by means of a strand-specific in situ hybridization assay. As a typical feature of the persistence, we identified a continuous synthesis of antigenomic positive-strand RNA encoded by segment 7 (NS) during virus production. In contrast, infection with the parental wild-type virus led to a rapid reduction of antigenomic RNA as observed in the late period of replication particularly for RNA segment 7. Furthermore, the replication cycle of the persistent variant did not show the switch from early to late replication events followed by clearance of intracellular virus, but was regulated in terms of productive and nonproductive phases. Nonproductive phases were reversible and characterized by a low level of virus-specific RNA signals. In the productive phase, a difference in cytoplasmic RNA transport was detected for the two viruses: a marked cytoplasmic accumulation of negative- and positive-strand wild-type virus RNAs stood in contrast to a RNA localization in different cellular compartments for the persistent virus. Also, Vero cells infected with the C/AA-pi variant were restricted to a transient, non-persistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends on a distinctly modified and cell type-specific regulation of virus-specific RNA synthesis and transport.

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.

New approaches to the development of virus vaccines for veterinary use.

The marked progress in recombinant deoxyribonucleic acid (DNA) technology during the past decade has led to the development of a variety of safe new vaccine vectors which are capable of efficiently expressing foreign immunogens. These have been based on a variety of virus types--poxviruses, herpesviruses and adenoviruses--and have led to the production of many new potential recombinant vaccines. Of these recombinant vaccines, the rabies vaccine, in which the rabies G protein is expressed in a vaccinia vector, has been widely used in the field to prevent the spread of rabies both in Europe and in the United States of America. A recombinant Newcastle disease virus vaccine, using fowlpox virus as the vector to express immunogenic proteins from the Newcastle disease virus, has been licensed as the first commercial recombinant vectored vaccine. Many other recombinant virus vaccines are still at the stage of laboratory or field testing. The most recent breakthrough in vaccinology has been the success with the use of naked DNA as a means of vaccination. This approach has shown great promise in mouse model systems and has now become the most active field in new vaccine development. Molecular redesigning of conventional ribonucleic acid (RNA) viruses to obtain more stable attenuated vaccines was previously possible only for positive-strand RNA viruses, such as poliovirus. However, recent advances in molecular biological techniques have enabled the rescuing of negative-strand viruses from DNA copies of their genomes. This has made it possible to engineer specific changes in the genomes of Rhabdoviridae and Paramyxoviridae, both of which include several viruses of veterinary importance. The authors describe the current progress in the development of vector vaccines, DNA vaccines and vaccines based on engineered positive- and negative-strand RNA virus genomes, with special emphasis on their application to diseases of veterinary importance.

Switch from translation to RNA replication in a positive-stranded RNA virus.

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5' end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication.

Transfer RNA Mimicry in a New Group of Positive-Strand RNA Plant Viruses, the Furoviruses: Differential Aminoacylation between the RNA Components of One Genome

Recent sequencing of the genomes of several furoviruses—fungus-transmitted rod-shaped positive-strand plant viruses—has suggested the presence of tRNA-like structures (TLSs) at the 3′ ends of the genomic RNAs. We show here that the genomic RNAs of soil-borne wheat mosaic virus (SBWMV), beet soil-borne virus (BSBV), potato mop-top virus (PMTV), peanut clump virus (PCV), and Indian peanut clump virus (IPCV) all possess functional TLSs that are capable of high-efficiency valylation. While the SBWMV, BSBV, and PMTV TLSs are similar to those found in tymoviruses, the PCV and IPCV TLSs harbor an insertion of about 40 nucleotides between the two halves of the TLS. The valylated SBWMV and BSBV RNAs formed tight complexes with wheat germ EF-1α · GTP (Kd= 2 to 11 nM), whereas valylated PMTV, PCV, and IPCV RNAs bound EF-1α · GTP weakly (Kd≥ 50 nM). The TLS of PCV RNA2 differs from PCV RNA1 in lacking the major valine identity nucleotide in the anticodon and consequently is capable of only very inefficient valylation. This is the first case of differential aminoacylation between the RNA components of one genome.