Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Abstract

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

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Acknowledgments

We deeply appreciate Drs. A. Agranovsky, V. Hallan and E. Gavryushina for their critical comments and helpful discussion. We are thankful to Dr. Yu. A. Varitzev for the wild-type isolate of PLRV and gift of antibodies against PLRV from Agdia (USA).

Funding

This study was funded by the Russian Science Foundation (Grant No. 14-24-00007).

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Correspondence to Yuri F. Drygin.

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All experiments on animals (rabbits) were carried out in accordance with the animal care regulations of the M.V. Lomonosov Moscow State University. The protocol was approved by the Bioethics Committee of the Faculty of Biology, M.V. Lomonosov Moscow State University.

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Skurat, E.V., Butenko, K.O., Kondakova, O.A. et al. Chimeric Virus as a Source of the Potato Leafroll Virus Antigen. Mol Biotechnol 59, 469–481 (2017). https://doi.org/10.1007/s12033-017-0035-6

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Keywords

  • Agroinfiltration
  • Binary vector
  • ELISA
  • Northern blotting analysis
  • Phloem-limited virus
  • Recombinant RNA