Cryo-electron microscopy of cauliflower mosaic virus

Abstract

Cauliflower mosaic virus (CaMV), the type member of the Caulimovirus family, is one of the most extensively studied plant viruses since it was one of the first plant viruses shown to encapsidate a genome of double stranded DNA. This virus has served as a model system for studying plant gene organization, expression and replication and is potentially useful as a gene vector for the genetic manipulation of higher plants. Despite being well characterized in terms of its molecular biology, little is known about the structure, organization and assembly of mature CaMV virions.CaMV virions have a molecular mass of ˜22.8x106 daltons and a sedimentation coefficient of 208S.2 In negatively-stained samples, the diameter of the virus was determined to be 50.3±1.4 nm. The capsid consists of multiple copies of a single, phosphorylated polypeptide (37-42 kDa) which is presumed to be post-translationally modified from the 58kDa primary product of gene IV. The sizes of the virion and mass of the capsid protein indicate that there may be 420 copies of the polypeptide, arranged with T=7 icosahedral lattice symmetry, although no direct evidence exists to support this model. Projected images of CaMV particles, examined by conventional electron microscopy procedures, are difficult to interpret since the details are relatively smooth and featureless. The organization of the circular doubled-stranded DNA molecule (˜4.9xl06 daltons) is unknown: neutron diffraction experiments indicate that it is mainly confined to the region between radii 15.0-21.5 nm. The current study is aimed at obtaining a reliable three-dimensional structural determination of CaMV by examining purified virus samples using recently developed cryo-electron microscopy techniques and image analysis procedures.