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

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

No transcription of sg mRNA7s was detected when mutants pMDD, pSAD, pSED, pSND, pSDA, pSDE, pSDN, pSGD, pGDN, and pSGA were transfected into BHK-21 cells ( Figure 2) ; therefore, it was important to determine whether these mutants were able to replicate and produce genomic negative strands. In the infected cell, the genomic negative strands of arteriviruses, like those of coronaviruses, are present in double-stranded replicative intermediates (RIs). To detect the influences of replicase mutants on replication, we used a more sensitive RT-PCR approach to analyze the presence of negative strands in cells transfected with the above mutants. As outlined in Figure 2 , specificity for the antigenomic template was achieved by using primer pair SF12 and SR683. For the detection of genomic negative strands, primer SR683 matches sequences within the ORF1a region and SF12 matches the leader region within the 5′UTR. BHK-21 cells were transfected with the above described mutant clones and pAS7. In the latter clone, the predicted nsp9 RNA polymerase domain was removed, a deletion which has been shown to render PRRSV replication incompetent. We used this mutant clone as a negative control for replication detection. RNA was isolated 24 h after transfection and analyzed for the presence of genomic negative strands (Figure 2) . The RT-PCR results for pAS7 were negative, confirming the specificity of our RT-PCR approach. In the cases of pMDD, pSAD, pSED, pSND, pSDA, pSDE, pSDN, pSGD, pGDN, pSGA, pGDD, and pAPRRS, the replicase mutants produced detectable amounts of genomic negative strands (Figure 2 ). The results indicated that none of the mutations abolished the replication function of RdRp.