Advanced Freeze-Replica Methods for Virus Cell Biology.

Freeze-replica methods were developed in the early 1950s and have advanced through various improvements and in combination with other specimen preparation methods up to the present day. In particular, the latest immuno-freeze-etching replica of unroofed specimens is the unique method enabling three-dimensional observation of the cytoplasmic side surface structure of the plasma membrane and the membrane cytoskeleton attached to it under an electron microscope. The experimental procedures are described in detail here, listing the essential materials required for each research experiment, ranging from the classical freeze-fracture replica method to the latest unroofing immuno-freeze-etching replica method. These methods are also considered crucial for morphological approaches in the viral infection process. Indeed, in cells infected with influenza A viruses, the present methods have shown that progeny viral ribonucleoproteins (vRNPs), including the genome, are bundled by actin filaments on the cytoplasmic surface of the host cell plasma membrane.