STUDY OF THE INITIAL STEPS OF POTATO VIRUS X ASSEMBLY

Abstract

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. Eeywords: plant viruses, potato virus X, virus-like particles, RNA, coat protein, virion assembly. The study of various stages of the infection process of plant viruses including the assembly of viral particles is of great practical interest. Identifica- tion of these mechanisms can be the basis for developing new approaches in virus-free crop production. Potato virus O (PVX), a typical Potexvirus (Alphaflex- iviridae), is a common pathogen of many plant species of Solanaceae family, including number of economically significant agricultural plants, particularly potato plants. It should be noted that potato, together with rice, wheat and corn, are the main food stuffs in the world. 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. About 1300 subunits of viral coat protein (CP) are polymerized to form polar helix with a pitch of 3.6 nm, each coil consist of 8-9 CP subunits, with viral RNA being encapsulated into the CP helix (1). PVX is the first filamentous plant virus reconstructed in vitro from RNA and CP (2). No differences between native virions and the reconstructed parti-