Dissecting Virus Infectious Cycles by Cryo-Electron Microscopy.

Abstract

In response to events such as receptor binding and endocytic triggers, viruses undergo large-scale, dynamic conformational changes necessary for cell entry and genome delivery. In later stages of the infectious cycle, replication machinery must read and synthesize nucleic acid strands to generate new copies of genetic material, and structural proteins must assemble and package the appropriate contents in order to produce new infectious particles. Structural elucidation of these events is key to understanding them and their inhibition by antiviral agents such as neutralizing antibodies and drugs. Electron microscopy is a versatile technique that offers the ability to resolve three-dimensional structures of individual viral proteins and whole virions in multiple functional states, even in cells at different stages of infection (Fig 1). Here we focus on the use of transmission electron microscopy of frozen-hydrated specimens, i.e., cryo-electron microscopy (cryo-EM). A major advantage of cryo-EM over other structural approaches is that samples of a broad range of sizes can be imaged under near-physiological conditions with native hydration intact. This approach does not require the specimen to be fixed, stained, or coaxed into a crystalline lattice. This versatility has enabled cryo-EM to expand the envelope of structural virology and opened new avenues for understanding the molecular and cellular processes of virus infection and pathogenesis. Fig 1 Cryo-electron microscopy (cryo-EM) enables one to gain insight into viruses over many levels of structural hierarchy, ranging from determination of near-atomic resolution structures of subunits and symmetrical virus assemblies to ultrastructural analysis ... In this micro-review, we examine general approaches and recent applications of cryo-EM to virology. The reader is referred to a number of excellent reviews for more in-depth discussion of current methodology (e.g., [1,2]). Electron microscopy and structural virology have a long, shared history dating to the first images of bacteriophages gathered using some of the earliest electron microscopes [3]. As the technique of cryogenically preserving unfixed samples was being developed, again, viruses were among the first test subjects that helped to demonstrate the utility of cryo-EM for characterizing structure of large biological assemblies [4–6].