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

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

MARC-145 cells (ATCC, Manassas, VA, USA) and baby hamster kidney (BHK-21) cells were propagated in Eagle's minimum essential medium (EMEM; Gibco-BRL, Gaithers- Figure 1 Schematic representation of PRRSV genome showing the location of the conserved SDD motif within RdRp. Shown is a part of domains in the pp1ab replicase polyproteins of PRRSV. Arrows represent sites in pp1ab that are cleaved by chymotrypsin-like (3CLpro) proteinase. The proteolytic cleavage products are numbered according to full cleavage site map. Within the cleavage products, the location and names of the domains that have been identified as structurally and functionally related are highlighted. These include diverse domains with conserved Cys and His residues (C/H), RNA-dependent RNA polymerase (RdRp), helicase (HEL) and uridylate-specific endoribonuclease (N or NendoU) activities. The number of amino acid D indicates the location of SDD in pp1ab. burg, MD, USA) with 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD, USA), and maintained in EMEM with 2% fetal bovine serum at 37°C with 5% CO 2 . BHK-21 cells and MARC-145 cells were used for incubation of PRRSV. The viruses were rescued from the Type II PRRSV infectious clone pAPRRS (with CMV promoter) [28] and used as parental virus in all experiments.