Bendahmane M, Koo M, Karrer E, Beachy R N
Department of Cell Biology Division of Plant Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, la Jolla, CA, 92037, USA.
J Mol Biol. 1999 Jul 2;290(1):9-20. doi: 10.1006/jmbi.1999.2860.
The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.
烟草花叶烟草花叶病毒（TMV）外壳蛋白（CP）的生物物理特性使得在TMV表面展示外源肽成为可能。来自狂犬病病毒（RV）糖蛋白的免疫原性表位G5 - 24和来自鼠肝炎病毒（MHV）S糖蛋白的5B19成功展示在TMV表面，并且病毒在烟草品种Xanthi - nn的感染叶片中大量积累。包含一个精氨酸残基加上与CP融合的5B19表位的肽RB19（TMV - RB19），导致在烟草品种Xanthi - nn的接种叶片上诱导坏死性局部病斑以及感染的BY2原生质体的细胞死亡。RNA斑点杂交分析证实，在感染的Xanthi - nn叶片组织中诱导了酸性和碱性病程相关PR2基因的表达。携带来自RV核蛋白的表位31D的TMV在接种的烟草叶片中未积累。对杂交CP的分析预测，野生型CP的等电点（pI）：电荷值为5.31： - 2，CP - RB19为5.64： - 1，CP - 31D为9.14： + 2。当在CP - RB19和CP - 31D中插入酸性氨基酸以使它们的pI：电荷接近野生型CP时，产生的病毒TMV - RB19E和TMV - 4D：31D感染烟草品种Xanthi - nn植株和BY2原生质体而不引起细胞死亡。这些数据表明表位的pI及其对杂交CP pI：电荷值的影响对于成功展示表位的重要性，以及TMV表面对带正电荷表位缺乏耐受性。
