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

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

In this study, we analyzed some of the properties of the PRRSV RdRp by site-directed mutagenesis of the SDD motif, which is associated with the polymerase activity and is particular to nidoviruses. We took two approaches for systematic mutational analysis of the SDD motif: (i) We introduced either conserved or non-conserved amino acid changes in the SDD motif; and (ii) we introduced mutations that resembled the conserved sequence of other viral RdRps (Table 1) . Based on the sequence alignment, SDD motif was predicted to be a variant of the GDD motif of positive-strand RNA viruses. Our observation that the RdRp of PRRSV is active in replication and transcription when the SDD motif is mutated to GDD suggests that, conversely, the polymerase of other positive-strand RNA viruses might function with an SDD motif. It is reported that the requirement for a glycine residue in GDD motif is strict in the positive-strand RNA viruses [1] . Mutational analysis of the GDD motif of RdRp of RV [2] and TBSV confirmed this view, indeed the mutant ADD was found to revert back [20] . However, our results showed that the mutant amino acid residue S3050G of vGDD did not revert back and was stable until P5. The serine residue of the SDD motif could be substituted by glycine residue. This was the first demonstra-tion of flexibility of the serine residue of SDD motif in PRRSV. In terms of genetic stability, the mutant vGDD could be used as a candidate for genetic marker vaccines. However, it is not obvious why nidoviruses have evolved an SDD sequence in place of the GDD motif. From the multi-step virus growth curve, although vGDD maintained a growth pattern similar to that of vAPRRS, the peak titers of vGDD were lower than those of vAPRRS ( Figure 4) . In terms of how the mutant GDD motif of RdRp affected the replication and transcription of PRRSV, it produced less mRNA7s than vAPRRS, despite producing more sg mRNA7.2 than vAPRRS ( Figure 2) . These results were confirmed in three independent experiments. It has been reported that there is a quantitative balance among sg mRNAs species [30] ; thus, we proposed that a quantitative balance between sg mRNA7.1 and sg mRNA7.2 exists, which was confirmed by our lab (data not shown). RdRp interacts with the promoters of every sg mRNA to produce the sg mRNAs, and a change in the active site of RdRp may influence this interaction, disturbing the quantitative balance between sg mRNA7.1 and sg mRNA7.2.