Principles of Virology. Jane Flint
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because the concentration of (+) strand acceptors for template switching is 30 to 70 times higher than that of (−) strand acceptors. A prediction of the replicative mechanism, which has been verified experimentally, is that recombination frequencies should be lower between the genomes of different poliovirus serotypes.
Figure 6.27 RNA recombination. Schematic representation of RNA recombination occurring during template switching by RdRP. Two parental genomes are shown as acceptor and donor. The RNA polymerase (purple oval) has copied the 3′ end of the donor genome and is switching to the acceptor genome. The resulting recombinant molecule is shown.
Alteration of amino acids within the poliovirus 3Dpol thumb domain that directly interact with the RNA duplex led to the identification of Leu420 as critical for replicative recombination. This amino acid is located within an α-helix of the thumb domain in the exit channel for product RNA. It interacts with the ribose group of the third nucleotide of the product RNA strand, away from the active site. The change affects genomic recombination by reducing the initiation rate and the stability of 3Dpol elongation complexes without substantially affecting fidelity. The same amino acid change at 3Dpol Leu420 also dramatically increases sensitivity of viral replication to ribavirin. Consequently, it has been suggested that RNA recombination purges lethal mutations from viral genomes, avoiding ribavirin-induced error catastrophe.
Occasionally recombination occurs between viral and cellular RNAs. An example is a recombination reaction that leads to the appearance of cytopathic bovine viral diarrhea viruses (Box 6.6). The insertion of cellular sequences creates a new protease cleavage site at the N terminus of the NS3 protein, and the recombinant viruses also cause severe gastrointestinal disease in livestock.
If the RdRP skips sequences during template switching, deletions will occur. Such RNAs will replicate if they contain the appropriate signals for the initiation of RNA synthesis. Because of their smaller size, subgenomic RNAs replicate more rapidly than full-length RNA, and ultimately compete for the components of the RNA synthesis machinery. Because of these properties, they are called defective interfering viral genomes. Such RNAs can be packaged into viral particles only in the presence of a helper virus that provides viral proteins. Defective interfering particles accumulate during the replication of most, if not all, RNA viruses. These particles can interfere with the replication of nondefective viruses and are strong inducers of interferon. Consequently, they can influence the outcome of virus infections and the establishment of virus persistence (Volume II, Chapter 3).
RNA Editing
Diversity in RNA viral genomes is also achieved by RNA editing. Viral mRNAs can be edited either by insertion of a nontemplated nucleotide during synthesis or by alteration of the base after synthesis. Examples of RNA editing have been documented in members of the Paramyxoviridae and Filoviridae and in hepatitis delta virus. This process is described in Chapter 10.
DISCUSSION
RNA recombination leading to the production of pathogenic viruses
Pathogenicity of bovine viral diarrhea virus is associated with production of the NS3 protein. Two cytopathic viruses, Osloss and CP1, in which the ubiquitin sequence (UCH) has been inserted at different sites, are shown. In Osloss, UCH has been inserted into the NS2-3 precursor, and NS3 is produced. In CP1, a duplication has also occurred such that an additional copy of NS3 is present after the UCH sequence.
A remarkable property of pestiviruses, members of the Flaviviridae, is that RNA recombination generates viruses that cause disease. Bovine viral diarrhea virus causes a usually fatal gastrointestinal disease. Infection of a fetus with this virus during the first trimester is noncytopathic, but RNA recombination produces a cytopathic virus that causes severe gastrointestinal disease after the animal is born.
Pathogenicity of bovine viral diarrhea virus is associated with the synthesis of a nonstructural protein, NS3, encoded by the recombinant cytopathic virus (see the figure). The NS3 protein cannot be made in cells infected by the noncytopathic parental virus because its precursor, the NS2-3 protein, is not proteolytically processed. In contrast, NS3 is synthesized in cells infected by the cytopathic virus because RNA recombination adds an extra protease cleavage site in the viral polyprotein, precisely at the N terminus of the NS3 protein (see the figure). This cleavage site can be created in several ways. One of the most frequent is insertion of a cellular RNA sequence coding for ubiquitin, which targets cellular proteins for degradation. Insertion of ubiquitin at the N terminus of NS3 permits cleavage of NS2-3 by any member of a wide-spread family of cellular proteases. This recombination event provides a selective advantage, because pathogenic viruses outgrow non-pathogenic ones. Why cytopathogenicity is associated with release of the NS3 protein, which is thought to be part of the machinery for genomic RNA replication, is not known.
Retroviruses acquire cellular genes by recombination, and the resulting viruses can have lethal disease potential (Volume II, Chapter 6).
Perspectives
Structures of RdRPs alone or in combination with RNA templates and products have been solved for a large number of (+), (–), and double-stranded RNA viruses. The information collected has had enormous impact on our understanding of the mechanisms of template and primer binding, NTP selection and binding, catalysis, and chain translocation. More recently, the use of cryo-electron microscopy has led to the resolution of very complicated assemblies of replication complexes at atomic detail. A spectacular example is the resolution of the structure of the L protein of vesicular stomatitis virus: the 3.8-Å-resolution density map could be used to build an atomic model for nearly all of the 2,109-amino-acid protein chain. Despite this abundance of structural information, many unsolved questions remain, including how uridylylation of VPg can be accomplished by a second RNA polymerase molecule; the role of oligomerization in RNA polymerase function; and how independent functional domains work together to ensure that a correct RNA product is produced. Additional structures are needed to detail the conformational movements that take place during the switch between initiation and elongation, and the changes that occur as the polymerase moves from an open to a closed conformation.
RNA viral genetic diversity, and the ability to undergo rapid evolution, is made possible by errors made during nucleic acid synthesis, as well as genome recombination and reassortment. The importance of polymerase errors is underscored by the dramatic decrease in poliovirus fitness caused by a single amino acid change in the polymerase that decreases error rate. A different amino acid change in the RNA polymerase, which increases error frequency, has a similar effect. These observations demonstrate that the mutational diversity of RNA viruses is almost precisely where it must be, determined in large part by the error frequency of the RNA polymerase.
Many host proteins that are required for viral RNA synthesis have been identified, but their precise functions remain obscure. We now have the ability to identify cell proteins that are associated with RNA polymerases and to determine the effect on RNA synthesis when they are removed. Lacking are structural and mechanistic insights into how these proteins participate in RNA synthesis.
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