Principles of Virology, Volume 1. Jane Flint

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 can implicate particular cellular pathways in viral reproduction and can reveal signatures of virus-induced lethality or immune protection.

Figure 2.1 The viral infectious cycle. The infectious cycle of poliovirus is shown as an example, illustrating the steps common to most viruses: attachment and entry, translation, genome replication, particle assembly, and release. See Appendix, Fig. 22, for explanation of abbreviations. Inset: Lipid components of the plasma membrane. The membrane consists of two layers (leaflets) of phospholipid and glycolipid molecules. Their fatty acid tails converge to form the hydrophobic interior of the bilayer; the polar hydrophilic head groups (shown as balls) line both surfaces.

Entering Cells

Viral infection is initiated by a collision between the virus particle and the cell, a process that is governed by chance. A virion may not infect every cell it encounters: it must first come in contact with the tissues that contain cells to which it can bind. Such cells are normally recognized by means of the specific interaction of a virus particle with a cell surface receptor. These cellular molecules do not exist for the benefit of viruses: they all perform functions for the cell. Virus-receptor interactions can be either promiscuous or highly selective, depending on the virus and the distribution of the cell receptor. The presence of such receptors determines whether the cell will be susceptible to the virus. However, whether a cell is permissive for the reproduction of a particular virus depends on other, intracellular components found only in certain cell types. Cells must be both susceptible and permissive if an infection is to be successful. Virus entry into cells is the topic of Chapter 5.

Viral RNA Synthesis

      Although the genomes of viruses come in a number of configurations, they share a common requirement: they must be efficiently copied into mRNAs for the synthesis of viral proteins and progeny genomes for assembly. The synthesis of RNA molecules in cells infected with RNA viruses is a unique process that has no counterpart in the cell (see Chapter 6). With the exception of retroviruses, all RNA viruses encode an RNA-dependent RNA polymerase to catalyze the synthesis of both mRNAs and genomes. For the majority of DNA viruses and retroviruses, synthesis of viral mRNA is accomplished by RNA polymerase II, the enzyme that produces cellular mRNA (see Chapter 7). Much of our current understanding of the mechanisms of cellular transcription comes from study of the transcription of viral templates.

Viral Protein Synthesis

      All viruses are parasites of translation: their mRNAs must be translated by the host’s cytoplasmic protein-synthesizing machinery (see Chapter 11). However, viral infection often results in modification of the host’s translational apparatus so that viral mRNAs are translated selectively. The study of such modifications has revealed a great deal about mechanisms of protein synthesis. Analysis of viral translation has also led to the discovery of new mechanisms, such as internal ribosome binding and leaky scanning, that have been subsequently found to occur in uninfected cells.

Viral Genome Replication

      Replication of viral genomes requires the cell’s synthetic machinery