title: Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity.
abstract: The genome of influenza virus (viral RNA [vRNA]) is associated with the nucleoprotein (NP) and viral RNA-dependent RNA polymerases and forms helical viral ribonucleoprotein (vRNP) complexes. The NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using a Saccharomyces cerevisiae replicon system and a single-gene deletion library of Saccharomyces cerevisiae (T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata, Proc Natl Acad Sci USA, 104:18235-18240, 2007, https://doi.org/10.1073/pnas.0705856104). In infected Prp18 knockdown (KD) cells, the synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from the template after the early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates influenza virus RNA synthesis as an NP chaperone for the processive elongation reaction.
  
  
  OBJECTIVE
  Templates for viral RNA synthesis of negative-stranded RNA viruses are not naked RNA but rather RNA encapsidated by viral nucleocapsid proteins forming vRNP complexes. However, viral basic proteins tend to aggregate under physiological ionic strength without chaperones. We identified the pre-mRNA processing factor Prp18 as a stimulatory factor for influenza virus RNA synthesis. We found that one of the targets of Prp18 is NP. Prp18 facilitates the elongation reaction of viral polymerases by preventing the deleterious annealing of newly synthesized RNA to the template. Prp18 functions as a chaperone for NP to stimulate the formation of NP-RNA complexes. Based on these results, we propose that Prp18 may be required to maintain the structural integrity of vRNP for processive template reading.
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Minakuchi
authors.affiliation: Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sugiyama
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Naito
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.|Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: M
authors.identifier: http://orcid.org/0000-0003-2118-442X
authors.initials: M
authors.lastname: Okuwaki
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.|Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Kawaguchi
authors.affiliation: Faculty of Medicine, University of Tsukuba, Tsukuba, Japan knagata@md.tsukuba.ac.jp.
authors.forename: K
authors.initials: K
authors.lastname: Nagata
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D006801:Humans; D009980:Influenza A virus / Q000502:physiology*; D007251:Influenza, Human / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D009698:Nucleoproteins / Q000378:metabolism*; D011485:Protein Binding; D000072260:RNA Splicing Factors / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis*; D012261:Ribonucleoproteins / Q000378:metabolism; D061805:Transcription Elongation, Genetic; D014158:Transcription, Genetic
chemical_list: D009698:Nucleoproteins; C448981:PRPF18 protein, human; D000072260:RNA Splicing Factors; D012367:RNA, Viral; D012261:Ribonucleoproteins
keywords: host factor; protein chaperones; ribonucleoprotein; viral RNA synthesis