Principles of Virology. Jane Flint

Figure 6.7 Mechanisms of initiation of RNA synthesis. De novo initiation may occur at the 3′ end of the viral RNA or from an internal base. When a primer is required, it may be a capped or protein-linked oligonucleotide.

Structural comparisons of viral RdRPs that catalyze de novo initiation reveal larger thumb subdomains with structural elements that fill most of the active-site cavity and provide a platform for initiating nucleotides. For example, the structure of the RdRP of hepatitis C virus indicates that a dinucleotide is synthesized by the polymerase using a β-loop insertion in the thumb domain as a “protein platform” in the active site (Fig. 6.8). After the product reaches a certain length, the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA to exit as the enzyme moves along the template strand.

      A protein platform also appears to participate in de novo priming by the reovirus RdRP, a cube-like structure with a catalytic site in the center that is accessible by four tunnels. One tunnel allows template entry, one serves for the exit of newly synthesized double-stranded RNA, a third permits exit of mRNA, and a fourth is for substrate entry. A priming loop that is not observed in this region of other RNA polymerases is present in the palm domain. The loop supports the initiating NTP, then retracts into the palm and fits into the minor groove of the double-stranded RNA product. This movement assists in the transition between initiation and elongation, and also allows the newly synthesized RNA to exit the polymerase.

      Protein platforms also appear to participate in the de novo priming of RNA synthesis by flaviviruses other than hepatitis C virus (dengue and West Nile viruses), influenza virus genome RNA synthesis, all known (–) strand RNA viruses, and bacteriophage Φ6.

       Primer-Dependent Initiation

      Protein priming. A protein-linked oligonucleotide serves as a primer for RNA synthesis by RdRPs of members of the Picornaviridae and Caliciviridae. Protein priming also occurs during DNA replication of adenoviruses, certain DNA-containing bacteriophages (Chapter 10), and hepatitis B virus (Chapter 7). A terminal protein provides a hydroxyl group (in a tyrosine or serine residue) to which the first (priming) oligonucleotide can be linked by viral polymerases, via a phosphodiester bond. The primer is then elongated.

Polioviral genomic RNA, as well as newly synthesized (+) and (−) strand RNAs, are covalently linked at their 5′ ends to the 22-amino-acid protein VPg (Fig. 6.9A), initially suggesting that VPg might function as a primer for RNA synthesis. This hypothesis was supported by the discovery of a uridylylated form of the protein, VPg-pUpU, in infected cells. VPg can be uridylylated in vitro by 3D^pol and can then prime the synthesis of VPg-linked poly(U) from a poly(A) template. The template for uridylylation of VPg is either the 3′ poly(A) on (+) strand RNA [during synthesis of (−) strand RNA] (Fig. 6.10) or an RNA hairpin, the cis-acting replication element (cre), located in the coding region [during synthesis of (+) strand RNA] (Fig. 6.9B and C).

Figure 6.8 Mechanism of de novo initiation. (A) Ribbon diagram of RdRP of hepatitis C virus. Fingers, palm, and thumb domain are colored blue, green, and magenta, respectively. The C-terminal loop that blocks the active site is shown in brown. Active-site residues are yellow (PDB file 4WTM). (B) Swinging-gate model of initiation. With the RNA template (green) in the active site of the enzyme, a short β-loop (red) provides a platform on which the first complementary nucleotide (light green) is added to the template (left). The second nucleotide is then added, producing a dinucleotide primer for RNA synthesis (middle). At this point, nothing further can happen because the priming platform blocks the exit of the RNA product from the enzyme. The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (right) to exit as the enzyme moves along the template strand.

      Structures of the RdRPs of different picornaviruses and caliciviruses indicate that the active site is more accessible than in polymerases with a de novo mechanism of initiation. The small thumb domains of these polymerases leave a wide central cavity that can accommodate the template and the protein primer.