Mutational analysis of the SDD sequence motif of a PRRSV RNA-dependent RNA polymerase

Zhou, Yan; Zheng, HaiHong; Gao, Fei; Tian, DeBin; Yuan, ShiShan

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase. RdRps are required for the replication and transcription of all positive-strand (PS) RNA viruses, in concert with host proteins and, sometimes, other viral proteins [1, 2] . The RdRp domain is essential for RNA replication [3, 4] . Several conserved sequence motifs have been identified in the putative RdRps of several animal and plant PS RNA viruses [5] . Currently, eight conserved motifs are known to be present in all classes of polymerases, and four of them reside in the catalytic portion of the "palm" domain [1, 2, 6] . The most conserved palm domain contains the four amino acid sequence motifs found in all classes of polymerases, named A, B, C, and D [7] , plus a fifth motif E, unique to RdRps and reverse transcriptases [1] . This structural conservation implies that palm domains of all RdRps may have evolved from a common, ancient ancestor. Motif C forms a "-strand, turn, -strand" structure that contains the highly conserved Gly-Asp-Asp (GDD) motif, with the aspartic acid residues exposed in the loop region found in RdRps, an RdRp hallmark [5, [8] [9] [10] . This structure is very similar in all classes of polymerases, and positions the two DD residues close to the conserved D of motif A [1] . The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme [7, [10] [11] [12] . The RNA-dependent RNA polymerases (RdRps) of all positive stranded RNA viruses and double stranded RNA viruses, such as reoviruses, contain a GDD motif, a highly conserved sequence located at the active site. However, with one exception, the RdRps of nidoviruses have a replacement of the Gly residue by a Ser residue preceding the two key catalytic Asp residues in the GDD motif [4] . Thus, porcine reproductive and respiratory syndrome virus (PRRSV) has an SDD signature instead of the canonical GDD in the RdRp (nsp9) [13] [14] [15] (Figure 1 , Table 1 ). The role of the serine residue in the RdRps of nidovirus has not been studied; however, the corresponding glycine residue is essential for poliovirus RdRp and for other RdRps carrying a GDD signature [15, 16] . It is said that the phylogeny of RdRps mainly parallels the taxonomy of RNA viruses. The other RNA viruses also have a particular conserved motif in the place of the GDD motif, such as the GDN motif of nonsegment-negative stranded RNA viruses [16, 17] , the SDD motif of segment-negative stranded RNA viruses [16, 18, 19] , and the MDD motif of retroviruses [18] ( Table 1 ). The universal occurrence and exceptional conservation of RdRps has led to RdRps, along with a few other replicative proteins, being used for the identification and classification of RNA viruses. What then, is the phylogenetic relationship of these different motifs among RNA viruses? Based on the present studies of the GDD motif, the requirement for the glycine residue of the GDD motif is somewhat flexible for in vitro RNA synthesis [2] . Studies on polioviruses, HCV [1] , RHDV, tombusviruses [20] , rubella viruses (RV) [1] , CSFV [21] , and caliciviruses [22] showed that the first aspartate of the GDD motif appears to be a strict requirement, as any changes to this position are not tolerated for in vivo viral replication and/or in vitro RNA synthesis. Studies of the second aspartate of the GDD motif showed it was not absolutely conserved in all classes of polymerases, suggesting