Immuno-electron microscopy at sub-nanometer resolution

Abstract

We have been studying the structure and assembly properties of the capsid of herpes simplex virus, type 1 (HSV-1), whose icosahedral shell (T=16) is ∽ 125 nm in diameter, has a mass of ∽ 200 MDa, and is composed of at least six protein species, ranging from 12 kDa to 149 kDa per monomer. To this end, the structures of empty and full purified capsids have been visualized in three-dimensional reconstructions from cryo-electron micrographs, at a resolution of ∽ 3.5 nm. With the intent of identifying specific structural features of the capsid in terms of particular proteins, or particular segments of these proteins, we have begun to explore the potentialities of cryo-reconstructions of capsids decorated with monoclonal antibodies.B-capsids were purified from BHK cells infected with the MP strain of HSV-1. Some material was analyzed directly by cryo-electron microscopy and image reconstruction as described. Other samples were incubated with approximately equimolar amounts of a monoclonal antibody (8F5) raised against purified VP5 (the major capsid protein, 149 kDa). The resulting precipitate was then washed with buffer to flush out unbound antibodies, and then analyzed in the same way, i.e. by observation in a Philips EM400T equipped with a Gatan 626 cryo-holder. The specificity of Mab.8F5 for VP5 was confirmed by an ELISA with purified VP5.