Principles of Virology. Jane Flint

1, Fig. 1.13). The reasons for this difference are unclear, but one possibility is that the formation of the eukaryotic nucleus erected a barrier for DNA virus reproduction. On the other hand, the eukaryotic cytoplasm with its extensive membranous system might have been a hospitable location for RNA virus replication.

Viral genomes display a greater diversity of genome composition, structure, and reproduction than any organism. Understanding the function of such diversity is an intriguing goal. As viral genomes are survivors of constant selective pressure, all configurations must provide benefits. One possibility is that different genome configurations allow unique mechanisms for control of gene expression. These mechanisms include synthesis of a polyprotein from (+) strand RNA genomes or production of subgenomic mRNAs from (–) strand RNA genomes (see Chapter 6). There is some evidence that segmented RNA genomes might have arisen from monopartite genomes, perhaps to allow regulation of the production of individual proteins (Box 3.5). Segmentation probably did not emerge to increase genome size, as the largest RNA genomes are monopartite.

Genetic Analysis of Viruses

The application of genetic methods to study the structure and function of animal viral genes and proteins began with development of the plaque assay by Renato Dulbecco in 1952. This assay permitted the preparation of clonal stocks of virus, the measurement of virus titers, and a convenient system for studying viruses with conditional lethal mutations. Although a limited repertoire of classical genetic methods was available, the mutants that were isolated (Box 3.6) were invaluable in elucidating many aspects of infectious cycles and cell transformation. Contemporary methods of genetic analysis based on recombinant DNA technology confer an essentially unlimited scope for genetic manipulation; in principle, any viral gene of interest can be mutated, and the precise nature of the mutation can be predetermined by the investigator. Much of the large body of information about viruses and their reproduction that we now possess can be attributed to the power of these methods.

BOX 3.5

      EXPERIMENTS

       Origin of segmented RNA virus genomes

      Segmented genomes are plentiful in the RNA virus world. They are found in virus particles from different families and can be double stranded (Reoviridae) or single stranded, with (+) (Closteroviridae) or (–) (Orthomyxoviridae) polarity. Some experimental findings suggest that monopartite viral genomes emerged first and then later fragmented to form segmented genomes.

      Insight into how such segmented genomes may have been formed comes from studies with the picornavirus foot-and-mouth disease virus. The genome of this virus is a single molecule of (+) strand RNA. Serial passage of the virus in baby hamster kidney cells led to the emergence of genomes with two different large deletions (417 and 999 nucleotides) in the coding region. Neither mutant genome is infectious, but when they are introduced together into cells, an infectious virus population is produced. This population comprises a mixture of each of the two mutant genomes packaged separately into virus particles. Infection is successful because of complementation: when a host cell is infected with both particles, each genome provides the proteins missing in the other.

      Further study of the deleted viral genomes revealed the presence of point mutations in other regions of the genome. These mutations had accumulated before the deletions appeared and increased the fitness of the deleted genome compared with the wild-type genome.

      These results show how monopartite viral RNAs may be divided, possibly 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