Study of a broad-spectrum monoclonal antibody, single-chain variable fragments and epitope of potyviruses, and the pathogenicity determinants of Turnip mosaic virus infectious clone
Date Issued
2016
Date
2016
Author(s)
Liu, Han-Lin
Abstract
The genus Potyvirus is one of the important plant virus genera, and comprises 158 formal species based on ICTV Virus Taxonomy. Potyviruses can be transmitted by aphids and mechanical inoculation. Some potyviruses have broad host range and cause serious economic losses. In Taiwan, calla lily is reported to be infected by five potyviruses, Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Konjak mosaic virus (KoMV), Turnip mosaic virus (TuMV), and Zantedeschia mild mosaic virus (ZaMMV). To reduce the cost and time of virus indexing, the conserved 121-amino-acids core regions of the capsid protein (CP) of DsMV, KoMV, and ZaMMV were concatenated and expressed. The recombinant protein was used as an antigen to prepare and screen the potyvirus group-specific monoclonal antibody (MAb). The selected C4 MAb could detect nine potyviruses in addition to the five calla lily potyviruses. In the first part of this study, we cloned the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb, and then constructed them as C4 single-chain variable fragments (scFvs). In E. coil expression system, a new PelE secretory signal peptide was used to help the secretion of C4 scFv. The data showed not only long but also short PelE signal peptide could secrete C4 scFv to medium and reduce inclusion body formation. According to western blot and I-ELISA, the soluble C4 scFv showed a binding specificity similar to that of the C4 MAb. In the second part, to identify which epitope is recognized by C4 MAb, the phage display peptide library was used to screen the C4 MAb-reactive peptides. The sequence alignment of C4 MAb-reactive peptides with potyviral CP sequences indicated that a conserved 12-amino acid (WxMMDGxxQxxY/F) sequence may be recognized by C4 MAb, and thus it was named as the C4 epitope. The results of amino acid substitution analysis indicated that tryptophan and tyrosine residues of C4 epitope are crucial for reacting with C4 MAb. Furthermore, sequence alignment of Hippeastrum mosaic virus (HiMV), which could not be detected by C4 MAb, and amino acid substitution analysis also showed the aspartic acid is also involved in binding with C4 MAb. These results of epitope mapping demonstrated the C4 epitope is a common CP epitope of potyviruses. We also tried to develop the C4 epitope as a new epitope tag. The epitope sequences of ZaMMV, KoMV, and DsMV were separately fused to the C-terminus of CP of Odontoglossum ringspot virus (ORSV), and then epitope-tagged ORSV CPs were expressed in a bacterial system and purified. The results of Western blotting and ELISA showed the C4 epitope of KoMV (Ko) had the strongest binding affinity to C4 MAb. To examine the applicability of Ko tag in planta, the transiently expressed Ko-tagged GFP and ORSV CP could be successfully detected by C4 MAb, and the Ko-tagged P19 of Tomato bushy stunt virus (TBSV) still maintained its silencing suppressor function. Furthermore, Ko-tagged EGFP could be successfully detected and subsequently immunoprecipitated by C4 MAb in a mammalian cell system. These data proved that Ko tag has the potential to become a new epitope tag in bacterial, plant, mammalian cell systems. In the third part, TuMV was used as an experimental material to develop a reverse genetic system. We obtained four full-length clone of TuMV by a two-step cloning method. In the infectivity assay, the p35S-TuMV-27 clone which had similar infectivity to p35S-TuMV-1 revealed much better infectivity than p35S-TuMV-5 and p35S-TuMV-6. The sequence comparison of TuMV-5, TuMV-6 and TuMV-27 clones indicated that they have only 1~3 nucleotide difference at the extreme 5'' end of viral genome. After 5’ replacement and 5’ G deletion analyses, these data verified that the lacking of the adenine at position 7 of 5’ UTR could reduce the infectivity. Besides, the infectious TuMV-27 clone was used to analyze which amino acid residues responsible for the lack of infectivity of pTuMV-T100 clone. Based on unique restriction enzyme sites, three fragments (AB, BH, and HX) of TuMV-T100 were used to separately replace the corresponding fragments of TuMV-27. The result of infectivity assay indicated all of these fragments contain the amino acid mutations affected infectivity. TuMV-AB and TuMV-BH could replicate in protoplasts, but could not infect N. benthamiana and C. quinoa plants. These results indicated that they may be defective in cell-to-cell movement. In contrast, TuMV-HX could induce small local lesions on C. quinoa, but its CP accumulation was lower than that of TuMV-27. The point mutation assay confirmed that CP mutation of Y219N decreased the infectivity of TuMV-27. In addition, other tyrosine mutants (TuMV-Y191A, TuMV-Y219A, and TuMV-Y224A) had similar phenotype as TuMV-Y219N. In transient expression assay, the tyrosine-mutated CP was less stable than wild-type CP. Thus, we suggested that the stability of TuMV CP may be affected by tyrosine phosphorylation.
Subjects
monoclonal antibody
single-chain variable fragment
capsid protein
secretory signal peptide
epitope
epitope tag
infectious clone
5’UTR
cell-to-cell movement
phosphorylation
Type
thesis
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