Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp-p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, C Delta 43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp. (C) 2014 The Authors. Published by Elsevier B.V.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-a-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N Delta 65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N Delta 65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process. (C) 2015 Elsevier Inc. All rights reserved.

Enhanced amplified spontaneous emission using layer-by-layer assembled cowpea mosaic virus

Layer-by-layer assembly technique was used to construct ultrathin film of cowpea mosaic virus (CPMV) by electrostatic interactions, and the film was employed as a precursor on which an OF8T2 film was deposited by spin coating. Amplified spontaneous emission (ASE) was observed and improved for the OF8T2 film. Compared with OF8T2 film on quartz, the introduction of CPMV nanoparticles reduced the threshold and loss, and remarkably increased the net gain. The threshold, loss, and gain reached 0.05 mJ/ pulse, 6.9 cm(-1), and 82 cm(-1), respectively. CPMV nanoparticles may enormously scatter light, resulting in a positive feedback, thus the ASE is easily obtained and improved.

First report of the occurence of East African cassava mosaic virus-Uganda (EACMV-UG) in Angola

No abstract is available for this article.

Structure-Based Mutational Analysis of eIF4E in Relation to sbm1 Resistance to Pea Seed-Borne Mosaic Virus in Pea

Background: Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E(S)), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E(R)) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Caracterização de variantes de Grapevine Fanleaf Virus (GFLV), Arabis Mosaic Virus (ARMV) e respectivos RNAS satélites presentes em castas portuguesas de Vitis Vinifera

Dissertação de mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Biochemical and histological investigations of viral localisation in the hypersensitive reaction of Phaselous vulgaris L. var Pinto to tobacco mosaic virus infection

STOBBS, Lorne,W ABSTRACT Biochemical and Histological Investigations of viral localisation in the hypersensitive reaction of Phaseolus vulgaris L. var Pinto to tobacco mosaic virus infection. The infection of Phaseolus vulgaris L. var Pinto with tobacco mosaic virus (TMV) results in the production of distinct necrotic lesions confining the virus to restricted areas of the leaf surface. Biochemical and histological changes in the leaf tissue as a result of infection have been described. Trace accumulations of fluorescent metabolites, detected prior to lesion expression represent metabolites produced, by the cell in response to virus infection. These substances, are considered to undergo oxidation and in diffusing into adjacent cells, react with cellular constituents causing the death of these cells. Such cellular necrosis in advance of infection effectively limits virus spread. Chromatographic studies on extracts from TMV infected Pinto bean leaf tissue suggests that a number of extra-fluorescent metabolites produced on lesion'expression represent end products of phenolic oxidation r,eactionsoccurring earlier in these cells. Inhibition of phenolic oxidation by ascorbate infiltration or elevated temperature treatment resulted in the absence of extra-fluorescent metabolites and the continued movement of virus in the absence of necrosis. Further studies with i ascorbate infiltration indicated that irreversible necrotic events were determined as early as 12 tci 18 hrs after viral inoculation. Histochemical tests indicated that callose formation was initiated at this time, and occurred in response to necrotisation. Inhibition of necrosis by either ascorbate infiltration or elevated temp8rature treatment resulted in the absence of callose deposition. Scanning electron'micrographs of infected tissue revealed severe epidermal and palisade cell damage. Histochemical tests indicated extensive callose formation in cells bordering the lesion, and suggested the role of callose iTh the blockage of intercellular connections limiting virus movement. The significance of these cellular changes is discussed. ii

Economic impact of Turnip mosaic virus, Cauliflower mosaic virus and Beet mosaic virus in three Kenyan vegetables

Screenhouse experiments conducted in Kenya showed that inoculation of cabbage seedlings with Turnip mosaic virus (TuMV), either alone, or in combination with Cauliflower mosaic virus (CaMV), reduced the number and weight of marketable harvested heads. When viruses were inoculated simultaneously, 25% of cabbage heads were non-marketable, representing 20-fold loss compared with control. By contrast, inoculation with CaMV alone had insignificant effects on cabbage yield. This suggests that TuMV is the more detrimental of these pathogens, and its management should be a priority. Early exposure to TuMV produced cabbages that were 50% lighter than non-infected plants, but later infection was less damaging suggesting that controlling virus infection at the seedling stage is more important. TuMV was far less damaging to kale than it was to cabbage; although high proportions of TuMV-inoculated kale plants showed symptoms (> 90%), the marketability and quality of leaves were not significantly reduced, and no clear relationship existed between timing of infection and subsequent crop losses. Early inoculation of Swiss chard with Beet mosaic virus (BtMV) significantly impaired leaf quality (similar to 50% reduction in marketable leaf production), but the impact of disease was greatest in plants that had been inoculated at maturity, where average leaf losses were two and a half times those recorded in virus-free plants. Disease-management of BtMV in Swiss chard is important, therefore, not only at the seedling stage, but particularly when plants are transplanted from nursery to field.

A naturally occurring recombinant isolate of Lettuce mosaic virus

LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1(1) and mo1(2). An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Specific detection of Lettuce mosaic virus isolates belonging to the Most type

Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mol gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination. (C) 2004 Elsevier B.V. All rights reserved.

Analysis of the serological variability of Lettuce mosaic virus using monoclonal antibodies and surface plasmon resonance technology

A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/ binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LIVIV diagnostic and field epidemiology studies.

Molecular mapping of the viral determinants of systemic wilting induced by a Lettuce mosaic virus (LMV) isolate in some lettuce cultivars

The isolate AF199 of Lettuce mosaic virus (LMV, genus Potyvirus) causes local lesions followed by systemic wilting and plant death in the lettuce cultivars Ithaca and Vanguard 75. Analysis of the phenotype of virus chimeras revealed that a region within the PI protein coding region (nucleotides 112-386 in the viral genome) and/or another one within the CI protein coding region (nucleoticles 5496-5855) are sufficient together to cause the lethal wilting in Ithaca, but not in Vanguard 75. This indicates that the determinants of this particular symptom are different in these two lettuce cultivars. The wilting phenotype was not directly correlated with differences in the deduced amino acid sequence of these two regions. Furthermore, transient expression of the LMV-AF 199 proteins, separately or in combination, did not induce local necrosis or any other visible reaction in the plants. Together, these results Suggest that the systemic wilting reaction might be Clue to RNA rather than protein sequences. (c) 2004 Elsevier B.V. All rights reserved.