title: Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase.
abstract: The porcine reproductive and respiratory syndrome virus (PRRSV) replicase polyproteins pp1a and pp1ab are proteolytically processed by four proteases encoded in ORF1a. In this study, a large set of PRRSV replicase cleavage products were identified and pp1a cleavage sites were verified by using a combination of bioinformatics, proteomics, immunoprecipitation, and site-directed mutagenesis. For genotype 1 PRRSV (isolate SD01-08), proteomic analysis identified H180/S181, G385/A386, and G1446/A1447 as the cleavage sites separating nsp1α/1β, nsp1β/nsp2, and nsp2/nsp3, respectively. Transient expression of nsp2-8, nsp3-8, nsp4-8, nsp5-8 (using the recombinant vaccinia virus/T7 RNA polymerase system) and immunoprecipitation identified the cleavage end products nsp2, nsp3, nsp4, nsp7α and nsp7β, and various processing intermediates. Our studies also revealed the existence of alternative proteolytic processing pathways for the processing of the nsp3-8 region, depending on the presence or absence of nsp2 as a co-factor. The identity of most cleavage products was further corroborated by site-directed mutagenesis of individual cleavage sites in constructs expressing nsp3-8 or nsp4-8. This study constitutes the first in-depth experimental analysis of PRRSV replicase processing and the data are discussed against the background of the processing scheme previously derived for the arterivirus prototype, the distantly related equine arteritis virus (EAV). Despite several differences between the two viruses, of which the functional significance remains to be studied, our study demonstrates the general conservation of the replicase pp1a processing scheme between EAV and PRRSV, and likely also the other members of the arterivirus family.
authors.affiliation: Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, USA; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Medical Microbiology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Ali
authors.initials: A
authors.lastname: Tas
authors.affiliation: Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, USA.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Medical Microbiology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: e.j.snijder@lumc.nl.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.affiliation: Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, USA; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA. Electronic address: yfang@vet.k-state.edu.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
mesh_terms: D000818:Animals; D002460:Cell Line; D019295:Computational Biology; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D019316:Porcine respiratory and reproductive syndrome virus / Q000201:enzymology* / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase
keywords: Arterivirus; Cleavage site; Nidovirus; Nonstructural protein; Protease; Replication