title: Pathogenic effects of Rift Valley fever virus NSs gene are alleviated in cultured cells by expressed antiviral short hairpin RNAs.
abstract: BACKGROUND
  Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, may cause severe hepatitis, encephalitis and haemorrhagic fever in humans. There are currently no available licensed vaccines or therapies to treat the viral infection in humans. RNA interference (RNAi)-based viral gene silencing offers a promising approach to inhibiting replication of this highly pathogenic virus. The small (S) segment of the RVFV tripartite genome carries the genetic determinates for pathogenicity during infection. This segment encodes the non-structural S (NSs) and essential nucleocapsid (N) genes.
  
  
  METHODS
  To advance RNAi-based inhibition of RVFV replication, we designed several Pol III short hairpin RNA (shRNA) expression cassettes against the NSs and N genes, including a multimerized plasmid vector that included four shRNA expression cassettes.
  
  
  RESULTS
  Effective target silencing was demonstrated using full- and partial-length target reporter assays, and confirmed by western blot analysis of exogenous N and NSs expression. Small RNA northern blots showed detectable RNAi guide strand formation from single and multimerized shRNA constructs. Using a cell culture model of RVFV replication, shRNAs targeting the N gene decreased intracellular nucleocapsid protein concentration and viral replication. The shRNAs directed against the NSs gene reduced NSs protein concentrations and alleviated NSs-mediated cytotoxicity, which may be caused by host transcription suppression.
  
  
  CONCLUSIONS
  These data are the first demonstration that RNAi activators have a potential therapeutic benefit for countering RVFV infection.
authors.affiliation: Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Johannesburg, Gauteng, South Africa.
authors.forename: Tristan
authors.initials: T
authors.lastname: Scott
authors.forename: Janusz T
authors.initials: JT
authors.lastname: Paweska
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
mesh_terms: D001681:Biological Assay; D003588:Cytopathogenic Effect, Viral / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000276:immunology*; D012296:Rift Valley fever virus / Q000502:physiology*; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering; D049410:Luciferases, Renilla