Purification, crystallization and X-ray analysis of Hibiscus chlorotic ringspot virus

Abstract

Hibiscus chlorotic ringspot virus (HCRSV), a Carmovirus, occurs worldwide and induces chlorotic ringspots on leaves, stunting and flower distortion in Hibiscus species, including kenaf. The HCRSV capsid has T = 3 icosahedral symmetry and contains 180 copies of the coat protein. A virus yield of 48-70 mg per 100 g of infected kenaf leaves was achieved with an improved purification scheme involving sucrose-cushion and sucrose density-gradient centrifugation. The virus was crystallized using PEG 8000 and 2,3-butanediol as co-precipitants. The crystals belonged to the cubic space group P23, with unit-cell parameter a = 392 A, and diffracted X-rays to at least 4.5 A resolution.

Three-dimensional reconstruction of hibiscus chlorotic ringspot virus

“…Significantly, in this study, the two different types of HCSRV particles were only distinguishable by UA staining. The two particles were not distinguishable in an HCRSV sample stained with PTA (Lee et al, 2003). Indeed, the two particles would not have been identified had we gone straight to cryo-EM reconstruction.…”

“…This is surprising, considering the fact that the virus was obtained from plants inoculated with an infectious cDNA clone (Huang et al, 2000) and propagated under growth chamber conditions (Lee et al, 2003). From structural considerations it would appear to be a T ¼ 3 or T ¼ 3-like virus.…”

“…From structural considerations it would appear to be a T ¼ 3 or T ¼ 3-like virus. The possible presence of a mixed population might also explain the difficulty of getting well-diffracting crystals of HCRSV (Lee et al, 2003). We have recently been able to separate smooth and rough particles by agarose gel electrophoresis, which offers a potential for producing better quality crystals and may facilitate the biochemical characterization, including sequencing of the newly identified viral genome.…”

“…HCRSV virus was propagated from an infectious cDNA clone in kenaf plants and purified as previously described (Lee et al, 2003). Cryo-EM samples of HCRSV were prepared by standard procedures (Adrian et al, 1984;Dokland, 2000;Dubochet et al, 1988).…”

“…We have developed a protocol for high yield propagation and purification of HCRSV from kenaf leaves (Lee et al, 2003). In the present study, we subject these samples to EM analysis and show three-dimensional (3D) reconstructions of the HCRSV virion both from negatively stained and frozen hydrated samples.…”