title: Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.
  
  
  
  It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Tian
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Nan
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Tian
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xu
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, China.
authors.forename: Baicheng
authors.initials: B
authors.lastname: Huang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, China.
authors.forename: Enmin
authors.initials: E
authors.lastname: Zhou
authors.affiliation: NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Hiscox
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, China chenhy@nwsuaf.edu.cn.
authors.forename: Hongying
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D009363:Neoplasm Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases