title: African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions.
abstract: Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCEOur findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Eaton
authors.affiliation: Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Departments of Pediatrics and Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada.
authors.forename: Randal N
authors.initials: RN
authors.lastname: Johnston
authors.affiliation: Eukarÿs SAS, Genopole Entreprises Campus III, Evry, France philippe.jais@eukarys.com shmulevi@ualberta.ca.
authors.forename: Philippe H
authors.initials: PH
authors.lastname: Jais
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada philippe.jais@eukarys.com shmulevi@ualberta.ca.
authors.forename: Maya
authors.initials: M
authors.lastname: Shmulevitz
mesh_terms: D000358:African Swine Fever Virus / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D009713:Nucleotidyltransferases / Q000378:metabolism*; D030102:Orthoreovirus, Mammalian / Q000235:genetics* / Q000502:physiology*; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059386:Reverse Genetics; D014771:Virion / Q000235:genetics / Q000502:physiology*; D019065:Virus Assembly; D014779:Virus Replication; D000098556:mRNA Guanylyltransferases
chemical_list: D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases
keywords: C3P3; NP868R; capping; reovirus; reverse genetics