Translation of the RNAs of brome mosaic virus: The monocistronic nature of RNA1 and RNA2

Each of the two largest brome mosaic virus RNAs, RNA1 and RNA2, directs the synthesis of a large protein in cell-free extracts derived from wheat embryo. The size of each protein represents the translation of almost the entire length of the corresponding RNA. It was shown previously that brome mosaic virus RNA4 directs the synthesis of the coat protein and that brome mosaic virus RNA3, although it also contains the coat protein cistron, is translated mostly into a single product unrelated to the coat protein (Shih & Kaesberg, 1973). Thus, the brome mosaic virus genome encodes a total of four proteins.     

Mutually-induced Conformational Switching of RNA and Coat Protein Underpins Efficient Assembly of a Viral Capsid

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T = 3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T = 3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.     

Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.     

Interaction of spermidine with viral RNA and its influence on protein synthesis

Addition of spermidine to a cell-free protein synthesizing system from wheat germ programmed with total brome mosaic virus (BMV) RNA resulted in a several-fold stimulation of amino acid incorporation. Increasing the spermidine concentration in the system led to inhibition of the overall protein synthesis, but the production of longer polypeptides was inhibited much more than that of the coat protein (shorter product). Analysis of the products synthesized under direction of BMV RNA 3 (longer product) and RNA 4 (coat protein) revealed that optimal translation of RNA 3 occurred at a much lower concentration of spermidine than that of RNA 4. Binding experiments with radioactive spermidine and BMV RNAs showed that the saturation of spermidine binding is achieved at a lower concentration of spermidine for RNA 3 than for RNA 4, which may suggest that the structure of RNA 4 is more compact than that of RNA 3. Taking into account the binding obtained at a spermidine concentration corresponding to optimal conditions of protein synthesis, it may be concluded that the optimum translation of these two mRNAs occurs when there is a similar level of RNA charge neutralisation, which implies a similar level of RNA structure stabilisation.     

卫星RNA对黄瓜花叶病毒基因组RNA体外合成的影响

卫星ＲＮＡ对黄瓜花叶病毒基因组ＲＮＡ体外合成的影响杨海花,康良仪,赵大健,田波（中国科学院微生物研究所,北京１０００８０）关键词卫星ＲＮＡ,黄瓜花叶病毒,依赖ＲＮＡ的ＲＮＡ聚合酶,体外合成利用卫星ＲＮＡ生防制剂控制田间的番茄、青椒、烟草等由黄瓜花叶病...     

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.     

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.     

A novel procedure for the localization of viral RNAs in protoplasts and whole plants

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus.

RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.     

RNA sequences and structures required for the recruitment and activity of the dengue virus polymerase

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3' end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3'-UTR, we found that the RNA-RNA interaction mediated by 5'-3'-hybridization was able to release the silencing effect of the 3'-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity.     

Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.     

Energetics of RNA binding by the West Nile virus RNA triphosphatase

The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.     

Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.     

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging,viral movement and interference with antiviral plant defence

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S‐domain builds a robust protein shell around the viral genome, whereas the C‐terminal protruding domain, or P‐domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N‐terminal domain, or R‐domain, and the arm region, which connects the R‐domain and S‐domain, are involved in different key steps of the viral cycle, such as cell‐to‐cell movement and the suppression of RNA silencing and pathogenesis through their RNA‐binding capabilities. Deletion mutants revealed that the CP RNA‐binding ability was abolished only after complete, but not partial, deletion of the R‐domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA‐binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R‐domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full‐length CPs were required to encapsidate full‐length genomes. We also found that the R‐domain carboxyl portion and the arm region were essential for efficient cell‐to‐cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R‐domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.     

Analysis of the pancreatic-ribonuclease-digestion products of alfalfa-mosaic-virus ribonucleic acid. Sequence homologies between the different RNAs.

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24-S, 20-S and 17-s RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12-S RNA also normally present in the virus. This 12-S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12-S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12-S RNA occurs in 17-S RNA. Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20-S and 24-S RNA) shows some oligonucleotides containing seven, eight and nine nucleotides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20-S and 24-S RNA with those of 12-S or 17-S RNA revealed no homologous oligonucleotides, thus the origin of 12-S RNA appears to be 17-S RNA.     

High Plains wheat mosaic virus: An enigmatic disease of wheat and corn causing the High Plains disease

Brief historyIn 1993, severe mosaic and necrosis symptoms were observed on corn (maize) and wheat from several Great Plains states of the USA. Based on the geographical location of infections, the disease was named High Plains disease and the causal agent was tentatively named High Plains virus. Subsequently, researchers renamed this virus as maize red stripe virus and wheat mosaic virus to represent the host and symptom phenotype of the virus. After sequencing the genome of the pathogen, the causal agent of High Plains disease was officially named as High Plains wheat mosaic virus. Hence, High Plains virus, maize red stripe virus, wheat mosaic virus, and High Plains wheat mosaic virus (HPWMoV) are synonyms for the causal agent of High Plains disease.TaxonomyHigh Plains wheat mosaic virus is one of the 21 definitive species in the genus Emaravirus in the family Fimoviridae.VirionThe genomic RNAs are encapsidated in thread‐like nucleocapsids in double‐membrane 80–200 nm spherical or ovoid virions.Genome characterizationThe HPWMoV genome consists of eight single‐stranded negative‐sense RNA segments encoding a single open reading frame (ORF) in each genomic RNA segment. RNA 1 is 6,981‐nucleotide (nt) long, coding for a 2,272 amino acid protein of RNA‐dependent RNA polymerase. RNA 2 is 2,211‐nt long and codes for a 667 amino acid glycoprotein precursor. RNA 3 has two variants of 1,439‐ and 1,441‐nt length that code for 286 and 289 amino acid nucleocapsid proteins, respectively. RNA 4 is 1,682‐nt long, coding for a 364 amino acid protein. RNA 5 and RNA 6 are 1,715‐ and 1,752‐nt long, respectively, and code for 478 and 492 amino acid proteins, respectively. RNA 7 and RNA 8 are 1,434‐ and 1,339‐nt long, code for 305 and 176 amino acid proteins, respectively.Biological propertiesHPWMoV can infect wheat, corn (maize), barley, rye brome, oat, rye, green foxtail, yellow foxtail, and foxtail barley. HPWMoV is transmitted by the wheat curl mite and through corn seed.Disease managementGenetic resistance against HPWMoV in wheat is not available, but most commercial corn hybrids are resistant while sweet corn varieties remain susceptible. Even though corn hybrids are resistant to virus, it still serves as a green bridge host that enables mites to carry the virus from corn to new crop wheat in the autumn. The main management strategy for High Plains disease in wheat relies on the management of green bridge hosts. Cultural practices such as avoiding early planting can be used to avoid mite buildup and virus infections.     

A Point Mutation in the Coat Protein Gene Affects Long-Distance Transport of the Tobacco Mosaic Virus

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic spread of the mutant virus was delayed by 5–6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.