doi: 10.15389/agrobiology.2014.5.28eng
UDC 632:578.2'21
STUDY OF THE INITIAL STEPS OF POTATO VIRUS X ASSEMBLY
N.A. Nikitin1, E.A. Trifonova1, E.K. Petrova1, O.V. Borisova1,
O.V. Karpova1, I.G. Atabekov1, 2
1M.V. Lomonosov Moscow State University, Biological Faculty, str. 12, 1, Leninskie gory, Moscow, 119234 Russia;
2A.N. Belozerskii Institute of Physicochemical Biology, M.V. Lomonosov Moscow State University, str. 40, 1, Leninskie gory, Moscow, 119991 Russia,
е-mail nikitin@mail.bio.msu.ru
Received September 20, 2014
Potato virus X (Potexvirus) is an important pathogen of number of economically significant agricultural plants of the Solanaceae family. Potato virus X (PVX) virions are flexible filamentous particles 515 nm in length and 13.5 nm in diameter with a helical structure. PVX genome consists of a single-stranded RNA (6435 nucleotides) which is capped and polyadenylated. The study of various stages of the infection process of plant viruses during infection, including the assembly of viral particles is of great practical interest. Identification of these mechanisms can be the basis for developing new approaches in virus-free crop production. Herein, the initial stages of potato virus X (PVX) virion assembly were examined on the example of virus-like particles (VLPs) produced by incubation of PVX RNA with PVX coat protein (CP) in vitro. The formation of identical set of VLP with discrete size under different incubation conditions (ionic strength, pH) was shown. However, the amount of VLP of a different size changes depending on incubation conditions. Most efficient VLP similar to the native virion size assembly has been achieved in a buffer of low ionic strength at pH 8.5. PVX RNA fragments from 800 to 5700 nt in length which were protected by coat protein within VLPs were isolated. Their individual analysis confirmed that all of them represent the 5´-terminal fragments of the genomic PVX RNA of different lengths. Thus, it was revealed that RNAs within VLP are genomic RNA 5´-terminal fragments of different lengths. PVX virions assembly initiation at the RNA 5´-end which is cooperatively extending in the 5´- to 3´-end direction was confirmed. Nucleotide sequence analysis of RNA fragments isolated from VLP of different sizes showed that sites capable to form RNA hairpins were discovered near the RNA 3´-end. They could act as «stop-signals» that prevent CP and RNA interaction and continuous cooperative assembling PVX VLP and virions. Probably, CP could «melt» RNA hairpins more or less efficiently and overcome «stop-signals» during the viral particles formation, depending on the conditions of incubation with the RNA in vitro.
Кeywords: plant viruses, potato virus X, virus-like particles, RNA, coat protein, virion assambly.
REFERENCES
1. Atabekov J.G., Dobrov E.N., Karpova O.V., Rodionova N.P. Potato virus X: structure, disassembly and reconstitution. Mol. PlantPathol., 2007, 8: 667-675. CrossRef
2. Novikov V.K., Kimazev V.Z., Atabekov I.G. DokladyAkademiinauk, 1972, 204: 1259-1262.
3. Kaftanova A.S., Kiselev N.A., Novikov V.K., Atabekov J.G. Structure of products of protein reassembly and reconstruction of potato virus X. Virology, 1975, 65: 283-287.
4. Nikitin N.A., Sushko A.D., Arkhipenko M.V., Rodionova N.P., Karpova O.V., Yaminskii I.V. Kolloidnyi zhurnal, 2011, 73(4): 512-519.
5. Arkhipenko M.V., Petrova E.K., Nikitin N.A., Sushko A.D., Dubro-
vin E.V., Yaminskii I.V., Rodionova N.P., Karpova O.V., Atabekov I.G. Acta naturae, 2011, 3(3/10): 42-48.
6. Kim K.H., Hemenway C. The 5´ nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis. J. Virol., 1996, 70(8): 5533-5540.
7. Kwon S.J., Park M.R., Kim K.W., Plante C.A., Hemenway C.L., Kim K.H. cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X. Virology, 2005, 334(1): 83-97. CrossRef
8. Lough T.J., Lee R.H., Emerson S.J., Forster R.L., Lucas W.J. Functional analysis of the 5´ untranslated region of potexvirus RNA reveals a role in viral replication and cell-to-cell movement. Virology, 2006, 351(2): 455-465.
9. Karpova O.V., Zayakina O.V., Arkhipenko M.V., Sheval E.V., Kiselyova O.I., Poljakov V.Y., Yaminsky I.V., Rodionova N.P., Atabekov J.G. Potato virus X RNA-mediated assembly of single-tailed ternary «coat protein—RNA—movement protein» complexes. J. Gen. Virol., 2006, 87: 2731-2740.
10. Petrova E., Nikitin N., Protopopova A., Arkhipenko M., Yaminskii I., Karpova O., Atabekov J. The role of the 5´-cap structure in viral ribonucleoproteins assembly from Potato Virus X coat protein and RNAs. Biochimie, 2013, 95(12): 2415-2422.
11. Karpova O.V., Arkhipenko M.V., Zayakina O.V., Nikitin N.A., Kisele-
va O.I., Kozlovskii S.V., Rodionova N.P., Atabekov I.G. Molekulyarnaya biologiya, 2006, 40, 4: 703-710.
12. Goodman R.M., Horne R.W., Hobart J.M. Reconstruction of potato virus X in vitro. II. Characterization of the reconstituted product. Virology, 1975, 68: 299-308.
13. Fraenkel-Conrat H., Singer B., Tsugito A. Purification of viral RNA by means of bentanite. Virology,1961, 14: 54-58.
14. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucl. Acids Res., 2003, 31(13): 3406-3415. CrossRef
15. Erickson J.W., Bancroft J.B. The assembly of papaya mosaic virus coat protein with DNA. Progress in Clinical and Biological Research, 1980, 40: 293-300.
