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

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

The RdRp of PRRSV is required for PRRSV replication; therefore, the effect of SDD motif mutations on the catalytic activity of RdRp can be determined by analysis of RNA synthesis and virus production. The wt pAPRRS and the mutant pGDD could produce viruses after transfection into BHK-21 cells and inoculation onto MARC-145 cells. The primers used were complementary to the leader sequence (SF12) and the end of ORF7 (SR15284), and primed cDNA synthesis on all subgenomic positive stranded mRNA7s of PRRSV ( Figure 2 ). The mutant PRRSV full-length cDNA clones pMDD, pSAD, pSED, pSND, pSGD, pSDA, pSDE, pSDN, pGDN, and pSGA carrying mutations specifying the SDD motif in the viral replicase (Table 1) , were transfected into BHK-21 cells and failed to produce sg mRNA7s (Figure 2) . No CPE was detected in MARC-145 cells inoculated with undiluted culture medium of BHK-21 cells transfected with the above-described mutants. Moreover, expression of the N protein was not detected by IFAs in BHK-21 cells transfected with the mutant full-length cDNA clones, except for wt pAPRRS and pGDD ( Figure 3) . However, detection of the nsp2 protein demonstrated that the transfection and plasmid vector system were both functional. The results indicated that all the mutations destroyed the transcription function of RdRp (Figure 3) .