a pathway to a segmented genome. It is interesting that the point mutations that gave the RNAs a fitness advantage over the standard RNA arose before fragmentation occurred, implying that the changes needed to occur in a specific sequence. The authors of the study conclude: “Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.” While the fragmentation of the foot-and-mouth disease virus genome may represent a step on the path to segmentation, its relevance to what occurs in nature is unclear, because the results were obtained in cells in culture.
A compelling picture of the genesis of a segmented RNA genome comes from the discovery of a new tick-borne virus in China, Jingmen tick virus. The genome of this virus comprises four segments of (+) strand RNA. Two of the RNA segments have no known sequence homologs, while the other two are related to sequences of flaviviruses. The RNA genome of flaviviruses is not segmented: it is a single strand of (+) sense RNA. The proteins encoded by RNA segments 1 and 3 are nonstructural proteins that are clearly related to the flavivirus NS5 and NS3 proteins.
The genome structure of this virus suggests that at some point in the past a flavivirus genome fragmented to produce the RNA segments encoding the NS3and NS5-like proteins. This fragmentation might have initially taken place as shown for foot-and-mouth disease virus in cells in culture, by fixing of deletion mutations that complemented one another. Next, coinfection of this segmented flavivirus with another unidentified virus could have produced the precursor of Jingmen tick virus.
RNA genome of JMTV virus. The viral genome comprises four segments of single-stranded, (+) sense RNA. Proteins encoded by each RNA are indicated. RNA segments 1 and 3 encode flavivirus-like proteins.
The results provide new clues about the origins of segmented RNA viruses.
Moreno E, Ojosnegros S, García-Arriaza J, Escarmís C, Domingo E, Perales C. 2014. Exploration of sequence space as the basis of viral RNA genome segmentation. Proc Natl Acad Sci U S A 111:6678–6683.
Qin XC, Shi M, Tian JH, Lin XD, Gao DY, He JR, Wang JB, Li CX, Kang YJ, Yu B, Zhou DJ, Xu J, Plyusnin A, Holmes EC, Zhang YZ. 2014. A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors. Proc Natl Acad Sci U S A 111:6744–6749.
Classical Genetic Methods
Mapping Mutations
Before the advent of recombinant DNA technology, it was extremely difficult for investigators to determine the locations of mutations in viral genomes. The marker rescue technique (described in “Introducing Mutations into the Viral Genome” below) was a solution to this problem, but before it was developed, other, less satisfactory approaches were exploited.
Recombination mapping can be applied to both DNA and RNA viruses. Recombination results in genetic exchange between genomes within the infected cell. The frequency of recombination between two mutations in a linear genome increases with the physical distance separating them. In practice, cells are coinfected with two mutants, and the frequency of recombination is calculated by dividing the titer of phenotypically wild-type virus (Box 3.7) obtained under restrictive conditions (e.g., high temperature) by the titer measured under permissive conditions (e.g., low temperature). The recombination frequency between pairs of mutants is determined, allowing the mutations to be placed on a contiguous map. Although a location can be assigned for each mutation relative to others, this approach does not result in a physical map of the actual location of the base change in the genome.
In the case of RNA viruses with segmented genomes, the technique of reassortment allows the assignment of mutations to specific genome segments. When cells are coinfected with both mutant and wild-type viruses, the progeny includes reassortants that inherit RNA segments from either 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).
