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In this process, the first phosphodiester bond is made between the 3′-OH of the initiating NTP and the second NTP (Fig. 6.7). In these cases, initiation takes place at the exact 3′ end of the template, except during replication of the genomes of some (−) strand RNA viruses, such as bunyaviruses and arenaviruses (Fig. 6.7). Initiation begins at an internal C, and after extension of a few nucleotides, the daughter strand is shifted in the 3′ direction so that the 5′-terminal G residue is not base paired with the template strand. Because the daughter strand slips, this mechanism is called “prime and realign.”

De novo initiation

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.