Chimeric Virus as a Source of the Potato Leafroll Virus Antigen
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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.
KeywordsAgroinfiltration Binary vector ELISA Northern blotting analysis Phloem-limited virus Recombinant RNA
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).
This study was funded by the Russian Science Foundation (Grant No. 14-24-00007).
Compliance with Ethical Standards
Conflicts of interest
All authors declare no conflict of interest.
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.
Human Rights and Informed Consent
Research involving Human Participants Informed consent: not applicable for this study.
Informed consent was obtained from all individual participants included in the study.
Statement on the Welfare of Animals
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
- 14.Sambrook, J., Fritsch, E. F., & Maniatis, T. (1987). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
- 19.Chomczynski, P., & Mackey, K. (1995). Modification of the TRIZOL reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechniques, 19, 942–945.Google Scholar
- 20.Drygin, Y. F., Afonina, I. A., Bayer, K., Nikolaeva, O. V., & Atabekov, J. G. (1989). Diagnostics of X and M potato virus infections in crude tuber extracts by non-radioactive DNA-probing. Bioorganicheskaya Khimiya, 15, 947–951. (in Russian).Google Scholar
- 21.Harlow, E., & Lane, D. (1988). Antibodies. Cold Spring Harbor Laboratory, NY: A laboratory manual.Google Scholar
- 23.Jeevalatha, A., Kaundal, P., Shandil, R. K., Sharma, N. N., Chakrabarti, S. K., & Singh, B. P. (2013). Complete genome sequence of potato leafroll virus isolates infecting potato in the different geographical areas of India shows low level genetic diversity. Indian Journal of Virology, 24, 199–204.CrossRefGoogle Scholar
- 24.Huehnlein, A., Schubert, J., Thieme, T., Zahn, V., & Steinbach, P. (2012) Potato leafroll virus isolate SymlessLS10, complete genome GenBank 346189.1.Google Scholar
- 25.Franco-Lara, L. F., McGeachy, K. D., Commandeur, U., Martin, R. R., Mayo, M. A., & Barker, H. (1999). Transformation of tobacco and potato with DNA encoding the full-length genome of potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication. Journal of General Virology, 80, 2813–2822.CrossRefGoogle Scholar
- 26.Dorokhov, Y. L., Skurat, E. V., Frolova, O. Y., Gasanova, T. V., Smirnov, A. A., Zvereva, S. D., et al. (2004). Reciprocal dependence between pectinmethylesterase gene expression and tobamovirus reproduction effectiveness in Nicotiana benthamiana. Doklady Biochemistry and Biophysics, 394, 30–42.CrossRefGoogle Scholar
- 30.Gibbs, A. J. (1977). Descriptions of plant viruses. Canberra, Australia: Research School of Biological Sciences, Australian National University.Google Scholar
- 33.Dodds, J. A., & Hamilton, R. I. (1974). Masking of the RNA genome of tobacco mosaic virus by the protein of barley stripe mosaic virus in doubly infected barley. Virology, 59, 418–427.Google Scholar
- 36.Spitsin, S., Steplewski, K., Fleysh, N., Belanger, H., Mikheeva, T., Shivprasad, S., et al. (1999). Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV. Proceedings of the National Academy of Sciences of the United States of America, 96, 2549–2553.CrossRefGoogle Scholar
- 42.Taliansky, M., & Barker, H. (1999). Movement of luteoviruses in infected plants. In H. G. Smith & H. Barker (Eds.), The Luteoviridae (pp. 69–81). Wallingford: CAB International.Google Scholar
- 44.Loniewska-Lwowska, A., Chełstowska, S., Zagórski-Ostoja, W., & Pałucha, A. E. (2009). Elements regulating potato leafroll virus sgRNA1 translation are located within the coding sequences of the coat protein and read-through domain. Acta Biochimica Polonica, 56, 619–625.Google Scholar