title: West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals.
abstract: BACKGROUND
  West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals.
  
  
  RESULTS
  We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed -1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals.
  
  
  CONCLUSIONS
  N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.
authors.affiliation: Histology and Molecular Biology Section, Army Medical and Veterinary Research Center Via Santo Stefano Rotondo, 4 00184 Rome, Italy. giovanni.faggioni@gmail.com
authors.forename: Giovanni
authors.initials: G
authors.lastname: Faggioni
authors.forename: Alice
authors.initials: A
authors.lastname: Pomponi
authors.forename: Riccardo
authors.initials: R
authors.lastname: De Santis
authors.forename: Laura
authors.initials: L
authors.lastname: Masuelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Ciammaruconi
authors.forename: Federica
authors.initials: F
authors.lastname: Monaco
authors.forename: Annapia
authors.initials: A
authors.lastname: Di Gennaro
authors.forename: Laura
authors.initials: L
authors.lastname: Marzocchella
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sambri
authors.forename: Rossella
authors.initials: R
authors.lastname: Lelli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Rezza
authors.forename: Roberto
authors.initials: R
authors.lastname: Bei
authors.forename: Florigio
authors.initials: F
authors.lastname: Lista
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D002522:Chlorocebus aethiops; D019295:Computational Biology; D016679:Genome, Viral / Q000235:genetics; D006734:Horse Diseases / Q000276:immunology* / Q000821:virology; D006736:Horses; D006801:Humans; D016366:Open Reading Frames; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology; D014901:West Nile Fever / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D014902:West Nile virus / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D014764:Viral Proteins; C542426:WARF4 protein, West Nile virus