Principles of Virology. Jane Flint
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parent. The origins of the RNA segments can be deduced from their migration patterns during gel electrophoresis (Fig. 3.11) or by nucleic acid hybridization. By analyzing a panel of such reassortants, the segment responsible for the phenotype can be identified.
Functional Analysis
Complementation describes the ability of gene products from two different mutant viruses to interact functionally in the same cell, permitting viral reproduction. It can be distinguished from recombination or reassortment by examining the progeny produced by coinfected cells. True complementation yields only the two parental mutants, while wild-type genomes result from recombination or reassortment. If the mutations being tested are in separate genes, each virus is able to supply a functional gene product, allowing both viruses to be reproduced. If the two viruses carry mutations in the same gene, no reproduction will occur. In this way, the members of collections of mutants obtained by chemical mutagenesis were initially organized into complementation groups defining separate viral functions. In principle, there can be as many complementation groups as genes.
METHODS
Spontaneous and induced mutations
In the early days of experimental virology, mutant viruses could be isolated only by screening stocks for interesting phenotypes, for none of the tools that we now take for granted, such as restriction endonucleases, efficient DNA sequencing methods, and molecular cloning procedures, were developed until the mid to late 1970s. RNA virus stocks usually contain a high proportion of mutants, and it is only a matter of devising the appropriate selection conditions (e.g., high or low temperature or exposure to drugs that inhibit viral reproduction) to select mutants with the desired phenotype from the total population. For example, the live attenuated poliovirus vaccine strains developed by Albert Sabin are mutants that were selected from a virulent virus stock (Volume II, Fig. 7.11).
The low spontaneous mutation rate of DNA viruses necessitated random mutagenesis by exposure to a chemical mutagen. Mutagens such as nitrous acid, hydroxylamine, and alkylating agents chemically modify the nucleic acid in preparations of virus particles, resulting in changes in base-pairing during subsequent genome replication. Base analogs, intercalating agents, or UV light are applied to the infected cell to cause changes in the viral genome during replication. Such agents introduce mutations more or less at random. Some mutations are lethal under all conditions, while others have no effect and are said to be silent.
To facilitate identification of mutants, the population must be screened for a phenotype that can be identified easily in a plaque assay. One such phenotype is temperature-sensitive viability of the virus. Virus mutants with this phenotype reproduce well at low temperatures, but poorly or not at all at high temperatures. The permissive and nonpermissive temperatures are typically 33 and 39°C, respectively, for viruses that replicate in mammalian cells. Other commonly sought phenotypes are changes in plaque size or morphology, drug resistance, antibody resistance, and host range (that is, loss of the ability to reproduce in certain hosts or host cells).
TERMINOLOGY
What is wild type?
Terminology can be confusing. Virologists often use terms such as “strains,” “variants,” and “mutants” to designate a virus that differs in some heritable way from a parental or wild-type virus. In conventional usage, the wild type is defined as the original (often laboratory-adapted) virus from which mutants are selected and which is used as the basis for comparison. A wild-type virus may not be identical to a virus isolated from nature. In fact, the genome of a wild-type virus may include numerous mutations accumulated during propagation in the laboratory. For example, the genome of the first isolate of poliovirus obtained in 1909 undoubtedly is very different from that of the virus we call wild type today. We distinguish carefully between laboratory wild types and new virus isolates from the natural host. The latter are called field isolates or clinical isolates.
The field of viral taxonomy has its own naming conventions which can cause some confusion. Viruses are classified into orders, families, subfamilies, genera, and species. These names are always italicized and start with a capital letter (e.g., Picornaviridae). To ensure clarity, the names of viruses (like poliovirus) should be written differently from the names of species (which are constructs that assist in the cataloging of viruses). A species name is written in italics with the first word beginning with a capital letter (other words should be capitalized if they are proper nouns). For example, the causative agents of poliomyelitis, poliovirus types 1, 2, and 3, are members of the species Enterovirus C. A virus name should never be italicized, even when it includes the name of a host species or genus, and should be written in lowercase: for example, Sida ciliaris golden mosaic virus. A good exercise would be to see how often we have accidentally violated these rules in this textbook.
Figure 3.11 Reassortment of influenza virus RNA segments. (A) Progeny viruses of cells that are coinfected with two influenza virus strains, L and M, include both parents and viruses that derive RNA segments from them. Recombinant R3 has inherited segment 2 from the L strain and the remaining seven segments from the M strain. (B) 32P-labeled influenza virus RNAs were fractionated in a polyacrylamide gel and detected by autoradiography. Migration differences of parental viral RNAs (M and L) permitted identification of the origin of RNA segments in the progeny virus R3. Panel B reprinted from Racaniello VR, Palese P. 1979. J Virol 29:361–373.
Engineering Mutations into Viral Genomes
Infectious DNA Clones
Recombinant DNA techniques have made it possible to introduce any kind of mutation anywhere in the genome of most animal viruses, whether that genome comprises DNA or RNA. The quintessential tool in virology today is the infectious DNA clone, a dsDNA copy of the viral genome that is carried on a bacterial vector such as a plasmid. Infectious DNA clones, or in vitro transcripts derived from them, can be introduced into cultured cells by transfection (Box 3.8) to recover infectious virus. This approach is a modern validation of the Hershey-Chase experiment described in Chapter 1. The availability of site-specific bacterial restriction endonucleases, DNA ligases, and an array of methods for mutagenesis has made it possible to manipulate these infectious clones at will. Infectious DNA clones also provide a stable repository of the viral genome, a particularly important advantage for vaccine strains. As oligonucleotide synthesis has become more efficient and less costly, the assembly of viral DNA genomes up to 212 kbp has become possible (Box 3.9).
DNA viruses. Current genetic methods for the study of most viruses with DNA genomes are based on the infectivity of viral DNA. When deproteinized viral DNA molecules are introduced into permissive cells by transfection, they generally initiate a complete infectious cycle, although the infectivity (number of plaques per microgram of DNA) may be low. For example, the infectivity of deproteinized human adenoviral DNA is between 10 and 100 PFU per μg.