The generation and release of mature virions from human cytomegalovirus (HCMV) infected cells is a multistep process, involving a profound reorganization of cellular structures and various stages of virus particle morphogenesis in different cellular compartments. Although the general steps of HCMV morphogenesis are known, it has become clear that the detailed molecular mechanisms are complex and dependent on various viral factors and cellular pathways. The lack of a full understanding of HCMV virion morphogenesis emphasizes the need of imaging techniques to visualize the different stages of virion assembly, such as electron microscopy. Here, we describe various electron microscopy techniques and the methodology of high-pressure freezing and freeze substitution for sample preparation to visualize HCMV morphogenesis. These methods are used in our laboratory in combination with a thorough quantification to characterize phenotypic alterations and to identify the function of viral and cellular proteins for the various morphogenesis stages.
Keywords: Cytomegalovirus; Electron microscopy; HCMV; High-pressure freezing; Morphogenesis; Secondary envelopment; Tomography.