Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants

Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.     

Estimation of the Size of Genetic Bottlenecks in Cell-to-Cell Movement of Soil-Borne Wheat Mosaic Virus and the Possible Role of the Bottlenecks in Speeding Up Selection of Variations in trans-Acting Genes or Elements

Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil-borne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 ± 0.22 on average and 5.02 ± 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.Plant RNA viruses change their genomes so rapidly that variant viruses with altered biological properties are often found after prolonged growth of infected plants or after serial mechanical inoculations (26, 33). Furthermore, inoculation of less-fit artificial mutants produces revertants or pseudo-revertants even after short infection times (12, 14). The rapid evolution of plant RNA viral genomes is achieved not only by high mutation rates due to error-prone replication by the nonproofreading viral RNA-dependent RNA polymerase (19) but also by rapid selection and strong genetic drift. Generally, narrow genetic bottlenecks facilitate the fixation and extinction of variants in populations (15), and viral populations are no exception to this theory.Plant RNA viruses are known to face many narrow genetic bottlenecks during their life cycles (23). The life cycles of most plant RNA viruses are as follows: After replicating in cells, viruses move from cell to cell through plasmodesmata, which connect the cytoplasms of adjacent cells separated by cell walls in plant tissue. Following the establishment of infection in cells and cell-to-cell movements, the viruses expand their infected regions, spreading to the veins and moving through the vascular system and infecting the plant systemically. Some plant RNA viruses are transmitted through the seeds or via mechanical injuries, but most are transmitted from plant to plant by biological vectors such as insects, nematodes, and fungi. Previous studies have found that genetic bottlenecks occur during the transfer from lower leaves to upper leaves in systemic infections of Wheat streak mosaic virus (WSMV) (11), Tobacco mosaic virus (TMV) (24), and Cucumber mosaic virus (CMV) (18) and during the transfer from one tiller to another tiller of WSMV (11). Vector transmissions were also shown to act as genetic bottlenecks for WSMV (11), CMV (1, 3), and Potato virus Y (PVY) (20). With the exception of PVY, the typical method for detecting genetic bottlenecks has been to observe the spatial separation of closely related strains or artificial synonymous mutants inoculated as mixed populations: the narrower the genetic bottleneck, the more frequently the spatial separation should be observed. Using this idea with mathematical analyses, WSMV was estimated to infect a new tiller starting with four genomes (9), TMV was estimated to infect the upper leaves starting with 10 genomes (24), and CMV was estimated to infect a new plant starting with one to two particles after aphid transmission (3). Studies of PVY using sets of host plant cultivars with or without resistance genes and mixed strains of viruses with or without resistance-breaking abilities also estimated the number of virus particles transmitted by an aphid vector to be 0.5 to 3.2 on average (20).However, genetic bottlenecks in cell-to-cell movement of viruses have not been well characterized, although these occurrences are likely (11) and have been expected to be important for understanding the life cycle and population dynamics of plant RNA viruses. The size of genetic bottlenecks in cell-to-cell movement can be referred to as “multiplicity of infection (MOI) in plant tissue colonization,” and only a recent study showing that the estimated MOI of TMV is between 6 and 1 to 2 (10) indicates the occurrence and the size of genetic bottlenecks in cell-to-cell movement of a plant RNA virus. In this paper, we also show the occurrence of narrow genetic bottlenecks during cell-to-cell movement of a plant RNA virus, Soil-borne wheat mosaic virus (SBWMV, type species of the genus Furovirus), by observing the spatial separation of RNA2 vectors carrying different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Both of the fluorescent proteins were expressed as fusion proteins to the N-terminal nuclear localization signal (NLS) peptide from Simian virus 40 (SV40) large T antigen, which enabled us to observe and count the infected cells accurately using nuclear fluorescence. Numerical data were analyzed to estimate the size of bottlenecks. We also carried out a simulation to show that, due to the narrow genetic bottlenecks, rapid selection occurs even on trans-acting elements in plant RNA virus genomes, overcoming the negative effect of complementation among adaptive and defective genomes in each intracellular population. We discuss the possible roles of the bottlenecks in the life cycle and evolution mechanisms of plant RNA viruses.     

Viral AlkB proteins repair RNA damage by oxidative demethylation

Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.     

Role of RNA helicases in HIV-1 replication

Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly >200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle.     

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

Double-stranded RNA immunoprecipitation allows the identification of Arabidopsis host proteins associated with tobacco rattle virus replication complexes.     

MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8

A forwards genetic screen of a chemically mutated plant population identified mitochondrial RNA editing factor 10 (MEF10) in Arabidopsis thaliana. MEF10 is a trans-factor required specifically for the C to U editing of site nad2-842. The MEF10 protein is characterized by a stretch of pentatricopeptide repeats (PPR) and a C-terminal extension domain ending with the amino acids DYW. Editing is lost in mutant plants but is recovered by transgenic introduction of an intact MEF10 gene. The MEF10 protein interacts with multiple organellar RNA editing factor 8 (MORF8) but not with other mitochondrial MORF proteins in yeast two hybrid assays. These results support the model that specific combinations of MORF and MEF proteins are involved in RNA editing in plant mitochondria.     

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.     

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.     

Genetic Variability of Natural Populations of Cotton Leaf Curl Geminivirus, a Single-Stranded DNA Virus

Reports on the genetic variability and evolution of natural populations of DNA viruses are scarce in comparison with the abundant information on the variability of RNA viruses. Geminiviruses are plant viruses with circular ssDNA genomes that are replicated by the host plant DNA polymerases. Whitefly-transmitted geminiviruses (WTG) are the agents of important diseases of crop plants and best exemplify emerging plant viruses. In this report we have analyzed the genetic diversity of cotton leaf curl geminivirus (CLCuV), a typical emerging WTG. No genetic differentiation was observed between isolates from different host plant species or geographic regions. Thus, the analyzed isolates represented a unique, undifferentiated population. Genetic variability, estimated as nucleotide diversities at synonymous positions in open reading frames (ORFs) for the AC1 (=replication) protein and coat protein (CP = AV1), was very high, exceeding the values reported for different genes in several plant and animal RNA viruses. This was unexpected in a virus that uses the DNA replication machinery of its eukaryotic host. Diversities at nonsynonymous positions, on the other hand, indicated that variability may be constrained in the genome of CLCuV. The ratio of nonsynonymous-to-synonymous substitutions varied for the different ORFs: they were higher for CP than for AC1 and lower still for the AC4 and AV2 ORFs, which overlap AC1 and CP ORFs, respectively. Analysis of nucleotide diversities at synonymous and nonsynonymous positions of the AC4 and AV2 ORFs suggest that their evolution is constrained by AC1 and CP, respectively. Data suggest that AC4 and AV2 are new genes that may have originated by overprinting on the preexistent AC1 and CP genes. Evidence for recombination was found for the AC1 and CP ORFs and for the noncoding intergenic region (IR). Data indicate that the origin of replication is a major recombination point in the IR, but not the only one. Analyses of the IR also suggest that recombinants may be frequent in the population and that recombination may have an important role in the generation of CLCuV variability. Received: 26 February 1999 / Accepted: 31 May 1999     

trans-Caryophyllene is a natural agonistic ligand for peroxisome proliferator-activated receptor-α

Intake of dietary aroma compounds may regulate cellular lipid metabolism. We demonstrated that trans-caryophyllene, a flavor compound in plant foods and teas, activates peroxisome proliferator-activated receptor (PPAR)-α through direct interaction with the ligand-binding domain of PPAR-α. The agonistic activity of trans-caryophyllene was investigated by the luciferase reporter assay, surface plasmon resonance, and time-resolved fluorescence resonance energy transfer assay. Following the stimulation of cells with trans-caryophyllene, intracellular triglyceride concentrations were significantly reduced by 17%, and hepatic fatty acid uptake was significantly increased by 31%. The rate of fatty acid oxidation was also significantly increased. The expressions of PPAR-α and its target genes and proteins in fatty acid uptake and oxidation were significantly up-regulated as well. In HepG2 cells transfected with small interfering RNA of PPAR-α, the effects of trans-caryophyllene on PPAR-α responsive gene expressions, intracellular triglyceride, fatty acid uptake and oxidation were disappeared. These results indicate that the aroma compound, trans-caryophyllene, is PPAR-α agonist thus regulates cellular lipid metabolism in PPAR-α dependent manners.     

Cis-acting RNA elements in human and animal plus-strand RNA viruses

The RNA genomes of plus-strand RNA viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. Those structures that are functionally important are called cis-acting RNA elements because their functions cannot be complemented in trans. They can be involved not only in RNA/RNA interactions but also in binding of viral and cellular proteins during the complex processes of translation, RNA replication and encapsidation. Most viral cis-acting RNA elements are located in the highly structured 5′- and 3′-nontranslated regions of the genomes but sometimes they also extend into the adjacent coding sequences. In addition, some cis-acting RNA elements are embedded within the coding sequences far away from the genomic ends. Although the functional importance of many of these structures has been confirmed by genetic and biochemical analyses, their precise roles are not yet fully understood. In this review we have summarized what is known about cis-acting RNA elements in nine families of human and animal plus-strand RNA viruses with an emphasis on the most thoroughly characterized virus families, the Picornaviridae and Flaviviridae.     

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.     

Rescue of Defective Poliovirus RNA Replication by 3AB-Containing Precursor Polyproteins

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) {wild type}] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.     

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.     

Evidence for Multiplicity Reactivation of Ultraviolet Light Irradiated Ribonucleic Acid Isolated from Tobacco Mosaic Virus

Multiplicity reactivation (MR) seems to take place in leaves of Nicotiana glutinosa inoculated with ultraviolet (UV) light irradiated RNA from tobacco mosaic virus (TMV-RNA). A similar phenomenon was not observed with UV-irradiated TMV particles. Considering MR as resulting from genetic recombination between viral genomes, a recombination mechanism, which has been difficult to prove with plant viruses, is proposed as being operative during multiplication of TMV. From the pattern of MR of TMV-RNA, the location of the gene for the RNA replicase within a TMV-RNA strand is discussed.