Purification and characterization of a virus-like particle of Leucostoma persoonii

Abstract

Abundant isometric virus-like particles were visible in hyphae of a hypovirulent isolate of Leucostoma persoonii, NC14.4A, that also contained at least six segments of double-stranded RNA. We purified and characterized the virus-like particles and determined their relationship to the double-stranded RNA segments. Particles were concentrated with polyethylene glycol, precipitated, and clarified by differential centrifugation. Spectrophotometric analysis of sucrose and cesium chloride density gradients revealed two UV-absorbing components. Electron microscopic analysis revealed that the components consisted of 40-nm-diameter isometric particles that had buoyant densities in cesium chloride of 1.313 g/cm³ and 1.339 g/cm³. Sedimentation coefficients were determined from linear-log sucrose gradients to be 107 s and 151 s, respectively. Extraction of protein from each of the two components yielded a major polypeptide, presumably the capsid protein, with a molecular weight of 32 000 daltons. The top component (1.313 g/cm³) contained five segments of double-stranded RNA, approximately 3.0, 2.8, 2.6, 2.3, and 0.7 kb. These were somewhat larger than five corresponding double-stranded RNA segments isolated from hyphae. The bottom component contained the same five segments of double-stranded RNA plus an additional large segment that was somewhat smaller than the largest segment present in hyphal extracts, approximately 7.9 kb. Strain HT, derived from NC14.4A by sequential hyphal tip isolation, retained only two segments of double-stranded RNA (7.9 and 2.3 kb) and lacked virus-like particles. Hybridization analysis indicated that these two segments shared limited sequence homology with each other but shared significant homology with the identical size segments isolated from the virus-like particle and the corresponding hyphal segments. The four other double-stranded RNAs in NC14.4A, and their corresponding segments in the virus-like particle, were each unique. Antiserum specific for the virus-like particle reacted with mycelial extracts of isolate NC14.4A but not with extracts of strain HT. The virus-like particle, designated mycovirus Lp-V, was unique among the six proposed groups of fungal viruses because of its low bouyant density, single small capsid protein, bipartite particle structure, and encapsidation of five or six segments of double-stranded RNA.

Key Words:

• Cytospora
• double-stranded RNA
• hypovirulence
• Leucocytospora
• mycovirus
• reovirus
• Valsa