this time. Later, DNA replaced RNA as cellular genomes, perhaps through the action of reverse transcriptases. With the emergence of DNA genomes probably came the evolution of DNA viruses. However, those with RNA genomes were and remain evolutionarily competitive, and hence they continue to survive to this day.
Analysis of sequences of more than 4,000 RNA-dependent RNA polymerases is consistent with the hypothesis that the first RNA viruses to emerge after the evolution of translation were those with (+) strand RNA genomes. The last common ancestor of these viruses encoded only an RNA-dependent RNA polymerase and a single capsid protein. Double-stranded RNA viruses evolved from (+) strand RNA viruses on at least two different occasions, and (–) strand RNA viruses evolved from dsRNA viruses. The emergence of viruses with the latter genome types was likely facilitated by the capture of genes such as those encoding RNA helicases, to allow for the production of larger genomes.
Single-stranded DNA viruses of eukaryotes appear to have evolved from genes contributed from both bacterial plasmids and (+) strand RNA viruses. Different dsDNA viruses originated from bacteriophages at least twice. The larger eukaryotic DNA viruses form a monophyletic group based on analysis of 40 genes that derive from a last common ancestor. These viruses appear to have emerged from smaller DNA viruses by the capture of multiple eukaryotic and bacterial genes, such as those encoding translation system components.
There is no evidence that viruses are monophyletic, i.e., descended from a common ancestor: there is no single gene shared by all viruses. Nevertheless, viruses with different genomes and replication strategies do share a small set of viral hallmark genes that encode icosahedral capsid proteins, nucleic acid polymerases, helicases, integrases, and other enzymes. For example, as discussed above, the RNA-dependent RNA polymerase is the only viral hallmark protein conserved in RNA viruses. Examination of the sequences of viral capsid proteins reveals at least 20 distinct varieties that were derived from unrelated genes in ancestral cells on multiple occasions. The emerging evidence therefore suggests that viral replication enzymes arose from precellular self-replicating genetic elements, while capsid protein genes were captured from unrelated genes in cellular hosts.
The compositions of the eukaryotic and bacterial viromes differ substantially (Chapter 1, Fig. 1.13). In bacteria, most known viruses possess dsDNA genomes; fewer viruses have ssDNA genomes, and there is a very limited number of viruses with RNA genomes. In eukaryotes, most of the virome diversity is accounted for by RNA viruses, but ssDNA and dsDNA viruses are common (Chapter 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.
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
