Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 293

Reassortment is the exchange of entire RNA molecules between genetically related viruses with segmented genomes. In cells coinfected with two different influenza viruses, the eight genome segments of each virus replicate. When new progeny virus particles are assembled, they can package RNA segments from either parental virus. Because reassortment is the simple exchange of RNA segments, it can occur at high frequencies.

In contrast to reassortment, recombination is the exchange of nucleotide sequences among different genomic RNA molecules (Fig. 6.27). Recombination, a feature of many RNA viruses, is an important mechanism for producing new genomes with selective growth advantages. This process has shaped the RNA virus world by rearranging genomes and creating new ones. RNA recombination was first discovered in cells infected with poliovirus and was subsequently observed for other (+) strand RNA viruses. The frequency of recombination can be relatively high: it has been estimated that 10 to 20% of polioviral genomic RNA molecules recombine in a single growth cycle. Recombinant polioviruses are readily isolated from the feces of individuals immunized with the three serotypes of Sabin vaccine. The genome of such viruses, which are recombinants of the vaccine strains with other enteroviruses found in the human intestine, may possess an improved ability to reproduce in the human alimentary tract and have a selective advantage over the parental viruses.

Recombination can occur by two different mechanisms: nonreplicative, the nonhomologous end joining of two different RNA molecules; or replicative, the switching of templates. Nonreplicative recombination is highly inefficient and thought to influence virus evolution minimally. Replicative recombination mainly occurs between nucleotide sequences of two parental genome RNA strands that have a high percentage of nucleotide identity. This mechanism of RNA recombination is coupled with the process of genome RNA replication: it occurs by template switching during (−) strand synthesis. The RNA polymerase first copies the 3′ end of one parental (+) strand, then switches templates and continues synthesis at the corresponding position on a second parental (+) strand. The exact mechanism of template exchange is not known, but it might be triggered by pausing of the polymerase during chain elongation or damage to the template. Template switching in poliovirus-infected cells occurs predominantly during (−) strand synthesis 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 3D^pol 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 3D^pol elongation complexes without substantially affecting fidelity. The same amino acid change at 3D^pol 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).