title: Mutational analysis of the SDD sequence motif of a PRRSV RNA-dependent RNA polymerase.
abstract: The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conserved among the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether the conserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue on RdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9 substitution, of serine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signature sequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viral polymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicated that nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the two aspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequence variant of the GDD motif in PS RNA viruses.
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Haihong
authors.initials: H
authors.lastname: Zheng
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.forename: Debin
authors.initials: D
authors.lastname: Tian
authors.forename: Shishan
authors.initials: S
authors.lastname: Yuan
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*
chemical_list: D012324:RNA-Dependent RNA Polymerase