Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

Abstract

Potato virus X (PVX) and some other potexviruses can be reconstitutedin vitrofrom viral coat protein (CP) and RNA. PVX CP is capable of forming viral ribonucleoprotein complexes (vRNP) not only with homologous, but also with foreign RNAs. This paper presents the structure and properties of vRNP assembledin vitroupon incubation of PVX CP and RNAs of various plant and animal viruses belonging to different taxonomic groups. We have shown that the morphology and translational properties of vRNPs containing foreign (heterologous) RNA are identical to those of homological vRNP (PVX RNA - PVX CP). Our data suggest that the assembly of the "mixed" vRNPin vitrocould be started at the 5'-proximal region of the RNA, producing a helical structure of vRNPs with foreign nucleic acids. The formation of heterologous vRNPin vitrowith PVX CP appears not to require a specific 5' end RNA nucleotide sequence, and the PVX CP seems to be able to pack foreign genetic material of various sizes and compositions into artificial virus-like particles.

Keywords: RNA; plant viruses; translation activation; viral ribonucleoproteins.

Fig. 1

TEM images of the vRNPs assembled in vitro from homologous or foreign RNA and PVX CP. (A) PVX RNA; 
(B) NMV RNA; (C) PAMV RNA; (D) total BMV RNA; (E) Mengo virus RNA; (F) TMV RNA; (G) AltMV RNA. 
The RNA : CP ratio (w/w) = 1 : 10. The samples were stained with 2% uranyl acetate. Scale bars represent 100 nm.

Fig. 2

AFM images of the vRNPs assembled in vitro from homologous or foreign RNA and PVX CP. (A) PVX RNA on mica; (B) TMV RNA on mica; (C) NMV RNA on graphite; (D) total BMV RNA on mica; (E) Mengo virus RNA on mica; (F) PAMV RNA on mica. The RNA : CP ratio (w/w) = 1 : 10. The samples were air-dried. Cantilever oscillation frequency 300–350 kHz. Arrowheads point to protein-free RNA tails. Scale bars represent 1 μm.

Fig. 3

Histograms showing height and length distribution of vRNP on the basis of the AFM data. PVX CP was incubated with RNA at RNA : PVX CP (w/w) ratio = 1 : 10. (A) PVX RNA; 
(B) total BMV RNA; 
(C) Mengo virus RNA.

Fig. 4

In vitro translational activation of RNA within vRNP. vRNP are formed upon incubation of PVX CP with homologous and foreign RNAs at weight ratio = 10 : 1, except for lane 4 at sections B, D, E, where the CP : RNA ratio = 30 : 1. Electrophoretic analysis (in 8–20% denaturating polyacrylamide gel) of ³⁵ S labeled translation products produced in wheat germ extract. (A) PVX RNA; (B) total BMV RNA; (C) PAMV RNA; (D) NMV RNA; (E) TMV RNA; (F) Mengo virus RNA. Lane 1 is RNA; Lane 2 – RNA + PVX CP; Lane 3 – (RNA + PVX CP) + MP1.