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

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

Construction of SDD mutants. The full-length mutant plasmids were constructed by site-directed PCR mutagenesis and SOE (gene splicing by overlap extension) PCR. A series of mutations was introduced into the nsp9 SDD motif by making single mutations at amino acid residue 3050, 3051, or 3052. The synthetic deoxyribonucleotides used in the mutagenesis are shown in Table 2 . To create plasmids pGDD, pSED, pSDE, and pSGA, a shuttle plasmid named pTB2, representing nucleotide positions 8840-13334 of the viral genome, was constructed to introduce specific modifications to the SDD motif in the full-length genomic clone. The fragment 8840-13334 nt was cloned into the vector pBluescript II SK (+/) (Stratagene, La Jolla, CA, USA). pTB2 plasmid was used as the template in site-directed PCR mutagenesis. PCR-mediated mutagenesis was carried out with the appropriate primers containing the required nucleo-tide changes. To construct plasmids pMDD, pSAD, pSND, pSDA, pSDN, pSGD, and pGDN, SOE PCR was used with pAPRRS as the template and two pairs of primers. The strategy of construction of them was the same with pSND. For mutant pSND, a pair of primers (SF8129 plus SR9348SND) was used to amplify approximately 1.2 kb from nt 8129 to 9348, and the downstream 4.0 kb fragment from nt 9301 to 13334 was obtained by a second PCR using primers, SF9301SND and SR13334. The 5.2 kb fragment containing the desired D→N mutation was generated by the SOE PCR with 1.2 and 4.0 kb fragments as templates, and SF8129 and SR13334 as the primers.