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

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

For negative-strand-specific RT-PCR, we used the approach Figure 2 The influence of aa substitutions in the SDD motif on subgenomic transcription and genomic replication. A, RT-PCR strategy for the detection of genomic negative strands and subgenomic mRNA7s. For the RT-PCR, primers SF12 and SR683 were used to amplify regions specific for plasmid, positiveand negative-stranded genomic RNA. Primer SR683 is directed against sequences within the ORF1a region and SF12 is directed against the leader sequences of the 5′UTR; both are used for priming cDNA synthesis (dashed line) on the genomic negative strands. Primers SF12 and SR15284 are used for the PCR specific for positive-stranded sg mRNA7s. Primer SR15284 is directed against the body sequence of sg mRNA7s and is used for priming cDNA synthesis (dashed line) on the positive strand of sg mRNA7s. B, RT-PCR amplification of leader-body junction specific regions of subgenomic mRNA7s. To study the impact of mutations in the SDD motif on PRRSV transcription, RT-PCR analysis of the sg mRNA7s was performed on the total RNAs obtained from mock-transfected BHK-21 cells and cells transfected with pMDD, pSAD, pSED, pSND, pSGD, pSDA, pSDE, pSDN, pSGA, pGND, pGDD, and pAPRRS, using a forward primer located in the leader region and a reverse primer in ORF7. The bands representing the specific sg mRNA7s are shown in the top panel and semiquantitative RT-PCR analysis of β-actin gene of BHK-21 cells to estimate the amounts sg mRNA7s is shown in the middle panel. To study the impact of mutations in the SDD motif on PRRSV replication, the RNAs were isolated from BHK-21 cells transfected with mock-transfected BHK-21cells and cells transfected with pMDD, pSAD, pSED, pSND, pSGD, pSDA, pSDE, pSDN, pGDN, pSGA, pGDD, and pAPRRS, using a forward primer located in the leader region and a reverse primer in the ORF1. RT-PCR analysis of genomic negative strands is shown in the bottom panel.