Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 292

All nucleic acid polymerases insert incorrect nucleotides during chain elongation. DNA-directed DNA polymerases have proofreading capabilities in the form of exonuclease active sites that can correct such mistakes. Most RdRPs do not possess this capability. The result is that error frequencies in RNA replication can be as high as one misincorporation per 10³ to 10⁵ nucleotides polymerized, whereas the frequency of errors in DNA replication is about 10,000-fold lower. Many of the polymerization errors cause lethal amino acid changes, but other mutations that are not lethal are retained in the genomes of infectious virus particles. This phenomenon has led to the realization that RNA virus populations are quasispecies, or mixtures of many different genome sequences. The errors introduced during RNA replication have important consequences for viral pathogenesis and evolution (Volume II, Chapter 10). Because RNA viruses exist as mixtures of genotypically different viruses, viral mutants may be isolated readily. For example, live attenuated poliovirus vaccine strains are viral mutants that were isolated from an unmutagenized stock of wild-type virus.

Fidelity of copying by RdRPs is determined by how the template, primer, and NTP interact at the active site. Nucleotide binding occurs in two steps: first, the NTP is bound in such a way that the ribose cannot interact properly with the Asp of motif A and the Asn of motif B (Fig. 6.6). If the NTP is correctly base paired with the template, then there is a conformational change in the enzyme, which reorients the triphosphate and allows phosphoryl transfer to occur. The closed active-site polymerase structures reveal a network of hydrogen bonds from the 2′ hydroxyl of the base-paired NTP to motif B in the fingers domain and the top of motif A. This network links a base-paired NTP with structural interactions that stabilize the closed active site and promote catalysis. The conformational change after NTP binding is a major fidelity checkpoint for the picornaviral RdRP.

Further insight into fidelity control in RdRPs comes from the analysis of an altered poliovirus 3D^pol with higher fidelity than the wild-type enzyme. This variant was selected for resistance to ribavirin, an antiviral nucleoside analog that causes transition mutations. The single amino acid change, G64S, slows the conformational change that occurs on NTP base pairing, thereby reducing the elongation rate. Although this amino acid is remote from the active site, it participates in hydrogen bonding to motif A, which is important in holding the NTP in an appropriate conformation for catalysis. Subtle changes in the enzyme caused by this substitution make it more dependent on correct NTP base pairing in the active site, thereby increasing replication fidelity. Of great interest is the observation that a similar interaction between fingers and motif A can be observed in RdRPs from a wide variety of viruses. This mechanism of enhancing fidelity may therefore be conserved in all RdRPs.

The RdRP of members of the Nidovirales (Fig. 6.16) allows faithful replication of the large (up to 41-kb) RNA genomes. The RNA synthesis machinery includes proteins not found in other RNA viruses, such as ExoN, a 3′-5′ exonuclease. Inactivation of this enzyme does not impair viral replication but leads to 15- to 20-fold increases in mutation rates. This observation suggests that ExoN confers a proofreading function upon the viral RNA polymerase, similar to the activity associated with DNA synthesis (Chapter 9). Viruses lacking the ExoN gene display attenuated virulence in mice, and are being considered as vaccine candidates.