Fluorescence studies on the coat protein of alfalfa mosaic virus

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (Mr 24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinized forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5* 10(8) M-1 sec-1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer. The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

Identification of Alfalfa Mosaic Virus in Greek Alfalfa Crops

Alfalfa mosaic virus (AMV) was found to be the prevalent virus disease in alfalfa crops in Central and Northern Greece. The virus was identified on the basis of the host symptomatology, aphid and seed transmission, particle morphology and serological tests. AMV was largely detected in commercial seeds of local grown alfalfa cvs by ELISA. Infected seed lots appear to be the main source responsible for the widespread distribution of the virus in Greek alfalfa fields.     

Effect of transgenic alfalfa plants with introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial community composition and physiological profile

The aim of this study was to evaluate the effect of transgenic alfalfa (Medicago sativa L.) plants, in comparison to their non-transgenic counterpart, on the density and physiological profiles of aerobic bacteria in the rhizosphere. Plants of transgenic alfalfa expressing the AMVcp-s gene coding for Alfalfa Mosaic Virus coat protein were cultivated in a climatic chamber. Two methods were used to determine the microbial diversity in rhizospheres of transgenic plants. First, the cultivation-dependent plating method, based on the determination of the density of colony-forming bacteria, and second, a biochemical method using the Biolog™ system, based on the utilization of different carbon sources by soil microorganisms. Statistically significant differences in densities of rhizospheric bacteria between transgenic and non-transgenic alfalfa clones were observed in ammonifying bacteria (GTL4/404-1), cellulolytic bacteria (GTL4/404-1, GTL4/402-2, A5-3-3), rhizobial bacteria (GTL4/402-2), denitrifying bacteria (A5-3-3) and Azotobacter spp. (GTL4/402-2). The highest values of substrate utilization by microbial communities and average respiration of C-sources were determined in non-transgenic alfalfa plants of the isogenic line SE/22-GT2. Carbohydrates, carboxylic acids and amino-acids were the most utilized carbon substrates by both Gram-negative and Gram-positive bacteria. Both, the community metabolic diversity and the utilization of C-sources increased in all alfalfa lines with culture time and regardless of transgenic or non-transgenic nature of lines.     

菜豆黄色花叶病毒(BYMV)—新疆苜蓿分离株的鉴定

从新疆苜蓿黄斑花叶病株上分离到病毒分离物M-4,该分离物能引起多种豆科值物系统花叶,并在藜科植物上产生局部褪绿斑,易经汁液摩擦接种和蚜虫传毒,不经菜豆种子传毒。病毒致死温度60—65℃,体外保毒期4—5天,稀释限点10~(-3)—10~(-4)。病毒粒体线状,长约660—740nm,宽15nm;在感病的寄主叶片细胞中,电镜观察到风轮状、带状和环状内含体。免疫电镜法测定,该分离物与菜豆黄色花叶病毒(BYMV)抗血清有血清反应。经SDS-聚丙烯酰胺凝胶电泳和氨基酸自动分析仪分析分别测得该病毒的衣壳蛋白亚基分子量为16,200道尔顿,氨基酸残基数128个。鉴定结果认为,分离物M-4是BYMV的一个株系。     

An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ～900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.     

In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5′ end and lack polyadenylation at the 3′ terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3′ end of their genomic RNAs, a prominent feature of bromo- and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3′ end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. ​(Fig.1)1) turned out to be the replicative form of the 3′ termini (19, 20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16-18). Although other models have been forwarded (9), we have proposed that switching between these two conformations, mediated by CP binding, plays a fundamental role in the life cycle of AMV and ilarviruses by regulating the competing processes of translation and replication of the viral RNAs.Open in a separate windowFIG. 1.The CPB and the TL conformations of the AMV RNA3 3′ terminus. The two conformers of AMV RNA3 3′ 145 nt are shown. (A) CPB conformer. The two major CP binding sites are indicated by brackets. Base pairing between loop D and stem A promotes TL conformation. (B) Secondary structure of the TL conformer.In the present study, the distribution between CPB and TL conformers was further investigated. We addressed how changes in this distribution would affect recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding ezyme) and the viral polymerase in vitro. We also monitored how the binding affinity to CP is affected by this conformational switch in vivo using the yeast three-hybrid (Y3H) system (2, 11, 24). Together, the in vitro and in vivo data clearly demonstrate the existence and function of the conformational switch in the 3′ UTR of AMV RNAs.     

大豆不同花叶病毒抗性品种胼胝质荧光标记初探

选用6个大豆品种与4个不同的大豆花叶病毒株系,分别组成抗病级别不同的组合,通过对接种叶片与上位叶症状观察、苯胺蓝染色辅以荧光显微镜观察和药物学试验,探讨了不同抗病级别组合中胼胝质(即β-l,3-葡聚糖)积累的特点及其在大豆抵抗大豆花叶病毒侵染过程中的作用。试验结果表明,大豆接种病毒后,在抗病级别分别为0～3的各个组合的叶肉细胞中,在侵染早期(接种后6、72 h)不同的组合在不同时间点分别观察到了胼胝质荧光,且胼胝质荧光出现的时间与抗病级别密切相关,即抗病性越强的组合在侵染点处观察到胼胝质的时间越早;而在抗病级别为5的组合中一直未能观察到胼胝质荧光。另外,在抗病级别为0级和1级的各组合中给叶片预注射2-DDG(2-deoxy-D-glucose,一种胼胝质合成抑制剂)再接种病毒,在上位叶能观察到坏死斑的出现并且通过RT-PCR能够检测到大豆花叶病毒外壳蛋白基因。以上结果表明,大豆被大豆花叶病毒侵染后,抗病性越强的品种就会在侵染点处越早地积累胼胝质,胼胝质的沉积与大豆抗病毒侵染密切相关。     

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.     

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.     

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

Samples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593 ) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.