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

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

Usually, RdRp domains are readily identifiable by comparative sequence analysis. Little is known about the biochemical and structural properties of the replicase [24] . Although many studies suggest nsp9 is the RdRp of PRRSV [25, 26] , there is no experimental evidence to demonstrate its catalytic activity. A core palm structure containing the four motifs (A-D) found in all classes of polymerases [1] is present in the C-terminal region of nsp9; therefore, it is possible to identify RdRp catalytic activity by testing whether the conserved SDD motif (at residues 3050 to 3052 of nsp9) itself is essential for virus replication (Figure 1) . This work provides an opportunity to better understand the biology of PRRSV, as well as to identify antiviral targets for the prevention of PRRSV. Cells transfected with an SGA mutant of EAV did not show detectable RNA replication or subgenomic mRNA transcription [3, 27] . Moreover, previous studies have focused on the structure-function analysis of the GDD motif and mainly depended on in vitro biochemical experiments. However, characterization of the SDD sequence motif of PRRSV has not yet been reported. Given the heterogeneity observed with regard to the GDD motif in RNA polymerases of PS RNA viruses, we wanted to ascertain whether flexibility exists with respect to amino acid substitutions at this site of the PRRSV RNA polymerase and the influences of amino acid change on virus replication and transcription. We utilized site-directed mutagenesis to construct several different combinations of mutations in the SDD motif in an infectious clone of PRRSV, and investigated the functional role of the motif.