Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 275

After an RdRP has associated stably with the nucleic acid template, the enzyme then adds nucleotides without dissociating from the template. Most RdRPs are highly processive; that is, they can add thousands of nucleotides before dissociating. The poliovirus RdRP 3D^pol can add 5,000 and 18,000 nucleotides in the absence or presence, respectively, of the accessory protein 3AB. The vesicular stomatitis virus P protein enhances the processivity of the RdRP (L protein), possibly as a result of conformational changes that occur upon binding of P. The increased processivity induced by P protein is enhanced in the presence of N, perhaps because the template must be kept unstructured so as not to impede the progress of L. Full processivity of the influenza virus RNA polymerase also requires the presence of NP.

In general, nucleic acid synthesis begins with the formation of a complex of RdRP, template-primer, and initiating NTP. The NTP α-phosphate undergoes nucleophilic attack by the 3′-OH of the primer strand. The nucleotidyl transfer reaction then takes place, pyrophosphate is released, and the template-primer moves by one base. Many elongation complex structures have been determined that provide insight into the steps that occur during this phase of RNA synthesis. Based on these structures, it has been proposed that the catalytic cycle comprises six structural states: template-primer binding, NTP binding, active-site closure, catalysis, opening of the active site, and translocation and pyrophosphate release.

Figure 6.12 Activation of the influenza virus RNA polymerase by specific virion RNA sequences. (A) Space-filling model of the trimeric influenza virus RdRP showing PB1 (cyan), PA (magenta), and PB2 (green) subunits (PDB file 4WSB). (B) Cartoon model of the trimeric influenza virus RdRP colored as above with PB1 (cyan), PA (magenta), and PB2 (green) subunits. (C) Model for activation of RdRP by virion RNA. The three P proteins form a multisubunit assembly that can neither bind to capped primers nor synthesize mRNAs. Addition of a sequence corresponding to the 5′-terminal 11 nucleotides of the viral RNA, which is highly conserved in all eight genome segments, activates the cap-binding activity of the P proteins. The PB1 protein binds this RNA sequence and activates the cap-binding PB2 subunit, probably by conformational change. Concomitantly with activation of cap binding, the PB1 protein acquires the ability to bind to a conserved sequence at the 3′ ends of genomic RNA segments. This second interaction activates the endonuclease that cleaves host cell RNAs 10 to 13 nucleotides from the cap, producing the primers for viral mRNA synthesis. The RNA polymerase can then carry out initiation and elongation of mRNAs. p, polymerase active site. 5′ and 3′ indicate the binding sites for the 5′ and 3′ ends, respectively, of (−) strand genomic RNA. Gray indicates an inactive site, and red indicates an active site.

In most cases, the RdRP first binds the RNA template-primer such that the templating base is above the active site. In this state, the RdRP conformation is the same as in the unbound form. Nucleotides enter the catalytic site via a large opening on one side of the enzyme. NTP selection is via interactions between the ribose 2′ and 3′ hydroxyl groups and three conserved residues on motifs B and A. These interactions cause a subtle restructuring of the palm domain, closing the active site. Incorrect NTPs can bind, but their ribose hydroxyls will not be properly positioned to cause active-site closure, and hence they will be inefficiently incorporated. After catalysis, the active site is opened by movement of motif A, and the template moves one base to place the next base in the active site.

Closure of the active site by movements of the palm domain appears to be a feature of the RdRPs of all (+) RNA viruses but not (–) strand or double-stranded RNA viruses; their palm domains are already structured in the unbound form. This simple nucleotide selection mechanism greatly influences polymerase fidelity. In T7 RNA polymerase and Taq DNA polymerase, a pre-insertion site is utilized to first bind the incoming NTP to the templating base. Next, the template-NTP base pair undergoes a major movement that places the triphosphate into the active site. These higher-fidelity enzymes therefore select for proper template-NTP pairing at two different binding sites, in contrast to the single site used by (+) strand RNA virus RdRPs.