Principles of Virology. Jane Flint
of Plating Measurement of Virus Particles
Viral Reproduction: the Burst Concept
The One-Step Growth Cycle One-Step Growth Analysis: a Valuable Tool for Studying Animal Viruses
Global Analysis DNA Microarrays Mass Spectrometry Protein-Protein Interactions
LINKS FOR CHAPTER 2
Video: Interview with Dr. Thomas Hope http://bit.ly/Virology_Hope
Cloning HeLa cells with Philip I. Marcus http://bit.ly/Virology_Twiv197
Ode to a plaque http://bit.ly/Virology_Twiv68
Movie 2.1: Plaque formation by vesicular stomatitis virus http://bit.ly/Virology_VZVGFP
Think globally, act locally http://bit.ly/Virology_Twim90
You know my methods, Watson.
SIR ARTHUR CONAN DOYLE
Introduction
Viruses are unique: often made up of nothing more than a nucleic acid molecule wrapped in protein, they parasitize the cellular machinery to produce thousands of progeny. This simplicity is misleading: viruses can infect all known life forms, and they comprise a variety of structures and genomes. Despite such variety, viruses are amenable to study because all viral propagation can be described in the context of three fundamental properties, as noted in Chapter 1: viral genomes are packaged inside particles that mediate their transmission from cell to cell; the viral genome contains the information for initiating and completing an infectious cycle; viruses establish themselves in a host population to ensure virus survival.
How viruses enter individual cells, their genomes are replicated, and new infectious particles are assembled are some of the topics of research in virology. These studies are usually carried out with cell cultures because they are a much simpler and more homogeneous experimental system than animals. Cells can be infected in such a way as to ensure that a single reproduction cycle occurs synchronously in every infected cell, called one-step growth. A full understanding of viral infectious cycles also requires knowledge of cell biology. Consequently, to reproduce the diversity of cells and architectures that are typical of tissues and organs, three-dimensional culture systems have been developed. In this chapter we begin with a brief overview of the infectious cycle, followed by a discussion of methods for cultivating and assaying viruses and detecting viral proteins and genomes and a consideration of viral reproduction and one-step growth analysis.
The Infectious Cycle
The production of new infectious particles can take place only within a cell (Fig. 2.1). Virologists divide viral infectious cycles into discrete steps to facilitate their study, although in virus-infected cells no such artificial boundaries occur. The infectious cycle comprises attachment and entry of the particle, production of viral mRNA and its translation by host ribosomes, genome replication, and assembly and release of progeny particles containing the genome. New virus particles produced during the infectious cycle may then infect other cells. The term virus reproduction is another name for the sum total of all events that occur during the infectious cycle.
Some events are common to virus replication in animals and in cells in culture, but there are also many important differences. While virus particles readily attach to cells in culture, in nature they must encounter a host, no mean feat for nanoparticles without any means of locomotion. After encountering a host, the virus particle must pass through physical host defenses, such as dead skin, mucous layers, and the extracellular matrix. Such barriers and other host defenses, such as antibodies and immune cells, which exist to combat virus infections, are not found in cell cultures. Virus infection of cells in culture has been a valuable tool for understanding viral infectious cycles, but the dissimilarities with infection of a living animal must always be considered.
The Cell
Viral reproduction requires many different functions of the host cell. Examples include the machinery for translation of viral mRNAs, sources of energy, and enzymes for genome replication. The cellular transport apparatus brings viral genomes to the correct cellular compartment and ensures that viral subunits reach locations where they may be assembled into virus particles. Subsequent chapters include a discussion of cellular functions that are important for individual steps in the viral infectious cycle.
PRINCIPLES The infectious cycle
Many distinct functions of the host cell are required to complete a viral infectious cycle.
The synthesis of new virus particles (i.e., a productive infection) requires target cells that are both susceptible (i.e., allow virus entry) and permissive (i.e., support virus reproduction).
Viral nucleic acids must be shielded from harsh environmental conditions in extracellular particles but be readily accessible for replication once inside the cell.
Viruses may be studied by propagation in cells within a laboratory animal or in cells in culture.
The plaque assay is the major way to determine the concentration of infectious virus particles in a sample.
Methods for quantifying and characterizing virus particles evolve rapidly, based on developments in detection, ease, cost, safety, utility in the field, and amenability to large-scale implementation.
Relationships among viruses can be deduced from phylogenetic trees generated from protein or nucleic acid sequences.
Viral reproduction is distinct from cellular or bacterial replication: rather than doubling with each cycle, each single cell cycle of viral reproduction is typically characterized by the release of many (often thousands) of progeny virions.
The multiplicity of infection (MOI) is the number of infectious units added per cell; the probability that any one target cell will become infected based on the MOI can be calculated from the Poisson distribution.
Global analysis of viral, cell, and host responses to virus infection