A Structure-Function Diversity Survey of the RNA-Dependent RNA Polymerases From the Positive-Strand RNA Viruses

Jia, Hengxia; Gong, Peng

In order to assign or identify possible functional regions beyond the RdRP catalytic module, we use the RdRP catalytic motifs and highly conserved residues to help estimate the boundaries of the catalytic module. The RdRP active site is surrounded by the palm, fingers, and thumb domains with seven catalytic motifs (motifs A-G) distributed within the palm (motifs A-E) and fingers (motifs F-G) (Poch et al., 1989; Gorbalenya et al., 2002; Bruenn, 2003; te Velthuis, 2014; Wu et al., 2015) (see an alignment of motif A-C of the 49 representative RdRP sequences in Figure 2 ). RdRPs share motifs A/C/D with DNAdependent polymerases and A-F with the reverse transcriptases (RTs, RNA-dependent DNA polymerases) (Poch et al., 1989; Delarue et al., 1990; Gong and Peersen, 2010) , while motif G is an RdRP hallmark motif that may participate in RNA template binding and post-catalysis RdRP translocation on the template (Gorbalenya et al., 2002; Shu and Gong, 2016) . Although each catalytic motifs may be well conserved at the levels of virus genus and family, highly conserved residues across different virus families can only be identified in motifs A/B/C/F, and only three residues are absolutely conserved (Figure 2 ). Among these three residues, two aspartic acid residues in motifs A and C (corresponding to the PV RdRP residues D233 and D328) participate in the coordination interactions with the two divalent metal ions essential for the phosphoryl transfer reaction, and are also required for other classes of polymerases (Beese and Steitz, 1991; Huang et al., 1998; Li et al., 1998; Yin and Steitz, 2004; Zamyatkin et al., 2008; Gong and Peersen, 2010; Appleby et al., 2015) . The third absolutely conserved residue is a glycine (corresponding to the PV RdRP residue G289) in motif B. This residue is typically adjacent to an serine and this SG dipeptide plays essential roles in recognizing the 2 -hydroxyl group of the nucleotide triphosphate (NTP) substrate, while the corresponding peptide bond flip accompanies a subtle conformational change of the NTP-induced RdRP active site closure identified by crystallography (Gong and Peersen, 2010; Appleby et al., 2015; Shu and Gong, 2016) . It has also been suggested that this glycine residue may be essential for a 3-Å tip movement of the motif B loop (corresponding to the PV RdRP residues 288-292) that could participate in the aforementioned post-catalysis RdRP translocation during each nucleotide addition cycle (NAC) (Sholders and Peersen, 2014) . Under either situation, the backbone flexibility of this glycine residue likely explains its requirement at this position. In some cases, the serine residue is replaced by a threonine or even rarely by other residues, and the threonine substitution can also be found in nucleoside analog drug-resistant virus stains (Figure 2 ; Dutartre et al., 2006; Lam et al., 2012; Flint et al., 2014) , suggesting that the side-chain hydroxyl group is the core conservative part of this residue. Motif F typically a−c "-" indicates the omitted suffix "virus," "viridae," or "virales," respectively. d PDB entries chosen in Figure 3 for seven RdRP-structure available virus families. e Nontype species. "/" indicates that virus order has not been assigned. References listed in the abbreviation column are used to define the N-and C-terminal boundaries of the RdRP proteins. Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | Sequence alignment of motifs A-C of RdRPs from representative positi