[33] Using confocal microscopy to study virus binding and entry into cells

The chapter presents a discussion on the study of virus binding and entry into cells by using confocal microscopy. For the study new approaches to study vaccinia virus (VV), binding and entry based on confocal microscopy are developed. These techniques do not require virus purification or labeling and generate data that reveal the absolute numbers of virus particles that have bound to or have entered into individual cells. The chapter describes these techniques and then illustrates with some of the results obtained. These methods should be applicable to any virus larger than 50 nm. The chapter discusses the way these techniques have generated data that cannot be obtained with classical binding or entry assays. Vaccina virus is the prototype of the poxvirus family. These are DNA viruses that replicate in the cell cytoplasm and have genomes between 150 and 300 kbp. These techniques are presented with a study of the binding and entry of VV. The methods have been particularly useful for studying VV because this virus produces two different forms of infectious virion that are antigenically and biologically distinct and are produced in widely differing amounts. Moreover, the extracellular enveloped virus (EEV) form of VV cannot be purified from contaminating IMV without disrupting the integrity of the outer envelope.