Principles of Virology. Jane Flint
that uridylylation of VPg might be accomplished in trans by another 3Dpol molecule. A third binding site on the 3Dpol of enterovirus 71 is at the base of the palm and also would require uridylylation by another polymerase molecule.
When VPg uridylylation begins at the 3′-poly(A) tail of the (+) strand template, the polymerase continues nucleotidyl transfer reactions and copies the entire genome. However, when uridylylation of VPg takes place on the cre, the protein must dissociate and transfer to the 3′ end of the RNA. How this process is accomplished is not known (Fig. 6.10).
Protein priming by the birnavirus RdRP VP1 is unusual because the primer is the polymerase, not a separate protein. Even in the absence of a template, VP1 has self-guanylylation activity that is dependent on divalent metal ions. The guanylylation site is a serine located approximately 23 Å from the catalytic site of the polymerase. The long distance between these sites suggests that guanylylation may be carried out at a second active site. The finding that some altered polymerases that are inactive in RNA synthesis retain self-guanylylation activity supports this hypothesis. After two G residues are added to VP1, it binds to a conserved CC sequence at the terminus of the viral RNA template to initiate RNA synthesis. The 5′ ends of mRNAs and genomic double-stranded RNAs produced by this reaction are therefore linked to a VP1 molecule.
Priming by capped RNA fragments. Influenza virus mRNA synthesis is blocked by treatment of cells with the fungal toxin α-amanitin at concentrations that inhibit cellular DNA-dependent RNA polymerase II. This surprising finding demonstrated that the viral RNA polymerase is dependent on host cell RNA polymerase II. Inhibition by α-amanitin is explained by a requirement for newly synthesized cellular transcripts made by this enzyme to provide primers for influenza viral mRNA synthesis (Fig. 6.11). Presumably, these cellular transcripts must be made continuously because they are exported rapidly from the nucleus once processed. Such transcripts are cleaved in the nucleus by an influenza virus-encoded, cap-dependent endonuclease that is part of the RdRP (Fig. 6.12). The resulting 10- to 13-nucleotide capped fragments serve as primers for the initiation of viral mRNA synthesis.
Figure 6.10 Poliovirus (−) strand RNA synthesis. The precursor of VPg, 3AB, contains a hydrophobic domain and is a membrane-bound donor of VPg. A ribonucleoprotein complex is formed when poly(rC)-binding protein 2 (PCBP2) and 3CDpro bind the cloverleaf structure located within the first 108 nucleotides of (+) strand RNA. The ribonucleoprotein complex interacts with poly(A)-binding protein 1 (PAbp1), which is bound to the 3′ poly(A) sequence, bringing the ends of the genome into close proximity. Protease 3CDpro cleaves membrane-bound 3AB, releasing VPg and 3A. VPg-pUpU is synthesized by 3Dpol using the 3′ poly(A) sequence as a template, and comprises the primer for RNA synthesis. Modified from Paul AV. 2002. p 227–246, in Semler BL, Wimmer E (ed), Molecular Biology of Picornaviruses (ASM Press, Washington, DC).
Figure 6.11 Influenza virus RNA synthesis. (A) Viral (−) strand genomes are templates for the production of either subgenomic mRNAs or full-length (+) strand RNAs. The switch from viral mRNA synthesis to genomic RNA replication is regulated by both the number of nucleocapsid (NP) protein molecules and the acquisition by the viral RdRP of the ability to catalyze initiation without a primer. Binding of the NP protein to elongating (+) strands enables the polymerase to read to the 5′ end of genomic RNA. (B) Capped RNA-primed initiation of influenza virus mRNA synthesis. Capped RNA fragments cleaved from the 5′ ends of cellular nuclear RNAs serve as primers for viral mRNA synthesis. The 10 to 13 nucleotides in these primers do not need to hydrogen bond to the common sequence found at the 3′ ends of the influenza virus genomic RNA segments. The first nucleotide added to the primer is a G residue templated by the penultimate C residue of the genomic RNA segment; this is followed by elongation of the mRNA chains. The terminal U residue of the genomic RNA segment does not direct the incorporation of an A residue. The 5′ ends of the viral mRNAs therefore comprise 10 to 13 nucleotides plus a cap structure snatched from host nuclear pre-mRNAs. Adapted from Plotch SJ et al. 1981. Cell 23:847–858, with permission.
Bunyaviral mRNA synthesis is also primed with capped fragments of cellular RNAs. In contrast to that of influenza virus, bunyaviral mRNA synthesis is not inhibited by α-amanitin because it occurs in the cytoplasm, where capped cellular pre-mRNAs are abundant.
The influenza virus RdRP is a heterotrimer composed of PA, PB1, and PB2 proteins (Fig. 6.12). The PB1 protein is the RNA polymerase, the PB2 subunit binds capped host mRNAs, and the PA protein harbors endonuclease activity. The influenza RdRP binds to the C-terminal domain of RNA polymerase II, an interaction that activates the viral enzyme and allows the capture of capped RNA primers from nascent host mRNAs. In contrast, acquisition of caps by bunyavirus is accomplished by a single protein, the RdRP (L). The N-terminal domains of influenza PA and bunyavirus L have endonuclease activities that participate in such cap snatching. The structures of endonuclease domains from these viruses reveal the presence of a common nuclease fold.
Capping
Most viral mRNAs carry a 5′-terminal cap structure (exceptions include picornaviruses and the flavivirus hepatitis C virus), but the modification is made in different ways. Three mechanisms can be distinguished: acquisition of preformed 5′ cap structures from cellular pre-mRNAs or mRNAs as described above, de novo synthesis by cellular enzymes, or synthesis by viral enzymes. Details of the latter processes can be found in Chapter 8.
Elongation
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 3Dpol 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