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

Virology Journal

Florigio Lista⁶  Roberto Bei³  Giovanni Rezza¹  Rossella Lelli⁵  Vittorio Sambri²  Laura Marzocchella³  Annapia Di Gennaro⁵  Federica Monaco⁵  Andrea Ciammaruconi⁶  Laura Masuelli⁴  Riccardo De Santis⁶  Alice Pomponi⁶  Giovanni Faggioni⁶ 

[1] Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy;Regional Reference Centre for Microbiological Emergencies (CRREM), Microbiology Unit, Azienda Ospedaliero-Universitaria di Bologna, Policlinico S. Orsola–Malpighi, Bologna, Italy;Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Rome, Italy;Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy;Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Teramo, Italy;Histology and Molecular Biology Section, Army Medical and Veterinary Research Center Via Santo Stefano Rotondo, 4 00184, Rome, Italy

关键词: Alternative open reading frame;    N-NS4B/WARF4;    WARF4;    WNV;    West Nile Virus;   

Others  :  1153082
DOI  :  10.1186/1743-422X-9-283

 received in 2011-09-01, accepted in 2012-11-12,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Faggioni et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

【 参考文献 】

• [1]Monath TP: Flaviviruses. In Virology. Edited by Fields BN, Knipe DM. New York: Raven Press; 1990:763-814.
• [2]Smithburn KC, Hughes TP, Burke AW, Paul JH: A Neurotropic Virus Isolated from the Blood of a Native of Uganda. Am J Trop Med 1940, 4:471-492.
• [3]Calistri P, Giovannini A, Hubalek Z, Ionescu A, Monaco F, Savini G, Lelli R: Epidemiology of west nile in europe and in the mediterranean basin. Open Virol J 2010, 4:29-37.
• [4]Hall RA, Broom AK, Smith DW, Mackenzie JS: The ecology and epidemiology of Kunjin virus. Curr Top Microbiol Immunol 2002, 267:253-269.
• [5]Murgue B, Zeller H, Deubel V: The ecology and epidemiology of West Nile virus in Africa, Europe and Asia. Curr Top Microbiol Immunol 2002, 267:195-221.
• [6]Tsai TF, Popovici F, Cernescu C, Campbell GL, Nedelcu NI: West Nile encephalitis epidemic in southeastern Romania. Lancet 1998, 352:767-771.
• [7]Angelini P, Tamba M, Finarelli AC, Bellini R, Albieri A, Bonilauri P, Cavrini F, Dottori M, Gaibani P, Martini E, Mattivi A, Pierro AM, Rugna G, Sambri V, Squintani G, Macini P: West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009. Euro Surveill 2010, 15:pii: 19547.
• [8]Papa A, Danis K, Baka A, Bakas A, Dougas G, Lytras T, Theocharopoulos G, Chrysagis D, Vassiliadou E, Kamaria F, Liona A, Mellou K, Saroglou G, Panagiotopoulos T: Ongoing outbreak of West Nile virus infections in humans in Greece, July-August 2010. Euro Surveill 2010, 15:pii: 19644.
• [9]Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S, Wakem EM, French RA, Garmendia AE, Van Kruiningen HJ: Isolation of West Nile virus from mosquitoes, crows, and a Cooper’s hawk in Connecticut. Science 1999, 286:2331-2333.
• [10]Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ: Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 1999, 286:2333-2337.
• [11]Gould LH, Fikrig E: West Nile virus: a growing concern? J Clin Invest 2004, 113:1102-1107.
• [12]Kramer LD, Li J, Shi PY: West Nile virus. Lancet Neurol 2007, 6:171-181.
• [13]Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ: Structure of West Nile virus. Science 2003, 302:248.
• [14]Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Summers MD, M.A.M: Virus taxonomy, classification and nomenclature of viruses. Arch Virol 1995, 10(Suppl.)):1-586.
• [15]Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, Bowen M, McKinney N, Morrill WE, Crabtree MB, Kramer LD, Roehrig JT: Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 2002, 298:96-105.
• [16]Beasley DW, Li L, Suderman MT, Barrett AD: Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 2002, 296:17-23.
• [17]Platonov AE, Shipulin GA, Shipulina OY, Tyutyunnik EN, Frolochkina TI, Lanciotti RS, Yazyshina S, Platonova OV, Obukhov IL, Zhukov AN, Vengerov YY, Pokrovskii VI: Outbreak of West Nile virus infection, Volgograd Region, Russia, 1999. Emerg Infect Dis 2001, 7:128-132.
• [18]Coia G, Parker MD, Speight G, Byrne ME, Westaway EG: Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J Gen Virol 1988, 69(Pt 1):1-21.
• [19]Allison SL, Schalich J, Stiasny K, Mandl CW, Kunz C, Heinz FX: Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J Virol 1995, 69:695-700.
• [20]Westaway EG, Mackenzie JM, Khromykh AA: Replication and gene function in Kunjin virus. Curr Top Microbiol Immunol 2002, 267:323-351.
• [21]Guo JT, Hayashi J, Seeger C: West Nile virus inhibits the signal transduction pathway of alpha interferon. J Virol 2005, 79:1343-1350.
• [22]Samuel MA, Diamond MS: Pathogenesis of West Nile Virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion. J Virol 2006, 80:9349-9360.
• [23]Evans JD, Seeger C: Differential effects of mutations in NS4B on West Nile virus replication and inhibition of interferon signaling. J Virol 2007, 81:11809-11816.
• [24]Melian EB, Hinzman E, Nagasaki T, Firth AE, Wills NM, Nouwens AS, Blitvich BJ, Leung J, Funk A, Atkins JF, Hall R, Khromykh AA: NS1’ of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness. J Virol 2010, 84:1641-1647.
• [25]Faggioni G, Ciammaruconi A, De Santis R, Pomponi A, Scicluna MT, Barbaro K, Masuelli L, Autorino G, Bei R, Lista F: Evidence of a humoral response to a novel protein WARF4 embedded in the West Nile virus NS4B gene encoded by an alternative open reading frame. Int J Mol Med 2009, 23:509-512.
• [26]Walewski JL, Keller TR, Stump DD, Branch AD: Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 2001, 7:710-721.
• [27]Bain C, Parroche P, Lavergne JP, Duverger B, Vieux C, Dubois V, Komurian-Pradel F, Trépo C, Gebuhrer L, Paranhos-Baccala G, Penin F, Inchauspé G: Memory T-cell-mediated immune responses specific to an alternative core protein in hepatitis C virus infection. J Virol 2004, 78:10460-10469.
• [28]Firth AE, Atkins JF: A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1’ may derive from ribosomal frameshifting. Virol J 2009, 6:14. BioMed Central Full Text
• [29]Diamond MS, Pierson TC, Fremont DH: The structural immunology of antibody protection against West Nile virus. Immunol Rev 2008, 225:212-225.
• [30]Nelson S, Jost CA, Xu Q, Ess J, Martin JE, Oliphant T, Whitehead SS, Durbin AP, Graham BS, Diamond MS, Pierson TC: Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog 2008, 4:e1000060.
• [31]Baril M, Dulude D, Gendron K, Lemay G, Brakier-Gingras L: Efficiency of a programmed −1 ribosomal frameshift in the different subtypes of the human immunodeficiency virus type 1 group M. RNA 2003, 9:1246-1253.
• [32]Egloff MP, Benarroch D, Selisko B, Romette JL, Canard B: An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J 2002, 21:2757-2768.
• [33]Farabaugh PJ: Programmed translational frameshifting. Microbiol Rev 1996, 60:103-134.
• [34]Giedroc DP, Theimer CA, Nixon PL: Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting. J Mol Biol 2000, 298:167-185.
• [35]Giedroc DP, Cornish PV: Frameshifting RNA pseudoknots: structure and mechanism. Virus Res 2009, 139:193-208.
• [36]Howard MT, Aggarwal G, Anderson CB, Khatri S, Flanigan KM, Atkins JF: Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons. EMBO J 2005, 24:1596-1607.
• [37]Bei R, Moretti A, Visco V, Defilippi R, Tsang K, Frati L, Muraro R: Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules. Int J Oncol 1996, 8:1127-1135.
• [38]Bei R, Masuelli L, Trono P, Orvietani PL, Losito S, Marzocchella L, Vitolo D, Albonici L, Mrozek MA, Di Gennaro E, Lista F, Faggioni G, Ionna F, Binaglia L, Manzari V, Budillon A, Modesti A: The ribosomal P0 protein induces a spontaneous immune response in patients with head and neck advanced stage carcinoma that is not dependent on its overexpression in carcinomas. Int J Oncol 2007, 31:1301-1308.
• [39]De Smaele E, Di Marcotullio L, Moretti M, Pelloni M, Occhione MA, Infante P, Cucchi D, Greco A, Pietrosanti L, Todorovic J, Coni S, Canettieri G, Ferretti E, Bei R, Maroder M, Screpanti I, Gulino A: Identification and characterization of KCASH2 and KCASH3, 2 novel Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in medulloblastoma. Neoplasia 2011, 13:374-385.
• [40]Masuelli L, Focaccetti C, Cereda V, Lista F, Vitolo D, Trono P, Gallo P, Amici A, Monaci P, Mattei M, Modesti M, Forni G, Kraus MH, Muraro R, Modesti A, Bei R: Gene-specific inhibition of breast carcinoma in BALB-neuT mice by active immunization with rat Neu or human ErbB receptors. Int J Oncol 2007, 30:381-392.
• [41]Masuelli L, Marzocchella L, Focaccetti C, Lista F, Nardi A, Scardino A, Mattei M, Turriziani M, Modesti M, Forni G, Schlom J, Modesti A, Bei R: Local delivery of recombinant vaccinia virus encoding for neu counteracts growth of mammary tumors more efficiently than systemic delivery in neu transgenic mice. Cancer Immunol Immunother 2010, 59:1247-1258.
• [42]Bei R, Budillon A, Masuelli L, Cereda V, Vitolo D, Di Gennaro E, Ripavecchia V, Palumbo C, Ionna F, Losito S, Modesti A, Kraus MH, Muraro R: Frequent overexpression of multiple ErbB receptors by head and neck squamous cell carcinoma contrasts with rare antibody immunity in patients. J Pathol 2004, 204:317-325.
• [43]Masuelli L, Budillon A, Marzocchella L, Mrozek MA, Vitolo D, Di Gennaro E, Losito S, Sale P, Longo F, Ionna F, Lista F, Muraro R, Modesti A, Bei R: Caveolin-1 overexpression is associated with simultaneous abnormal expression of the E-cadherin/α-β catenins complex and multiple ErbB receptors and with lymph nodes metastasis in head and neck squamous cell carcinomas. J Cell Physiol 2012, 227:3344-3353.
• [44]Bei R, Mentuccia D, Trono P, Masuelli L, Cereda V, Palumbo C, Marzocchella L, Mrozek MA, Pallotta P, Di Lella G, Modesti M, Cerilli M, Frajese GV, Frajese G, Zambruno G, Modesti A: Immunity to extracellular matrix antigens is associated with ultrastructural alterations of the stroma and stratified epithelium basement membrane in the skin of Hashimotos thyroiditis patients. Int J Immunopathol Pharmacol 2006, 19:661-674.
• [45]Masuelli L, Marzocchella L, Quaranta A, Palumbo C, Pompa G, Izzi V, Canini A, Modesti A, Galvano F, Bei R: Apigenin induces apoptosis and impairs head and neck carcinomas EGFR/ErbB2 signaling. Front Biosci 2011, 16:1060-1068.
• [46]Masuelli L, Bei R, Sacchetti P, Scappaticci I, Francalanci P, Albonici L, Coletti A, Palumbo C, Minieri M, Fiaccavento R, Carotenuto F, Fantini C, Carosella L, Modesti A, Di Nardo P: Beta-catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts. Cardiovasc Res 2003, 60:376-387.
• [47]Visco V, Bei R, Moriconi E, Gianni W, Kraus MH, Muraro R: ErbB2 immune response in breast cancer patients with soluble receptor ectodomain. Am J Pathol 2000, 156:1417-1424.
• [48]Bei R, Masuelli L, Moriconi E, Visco V, Moretti A, Kraus MH, Muraro R: Immune responses to all ErbB family receptors detectable in serum of cancer patients. Oncogene 1999, 18:1267-1275.
• [49]Masuelli L, Marzocchella L, Focaccetti C, Tresoldi I, Palumbo C, Izzi V, Benvenuto M, Fantini M, Lista F, Tarantino U, Modesti A, Galvano F, Bei R: Resveratrol and diallyl disulfide enhance curcumin-induced sarcoma cell apoptosis. Front Biosci 2012, 17:498-508.
• [50]Capobianchi MR, Sambri V, Castilletti C, Pierro AM, Rossini G, Gaibani P, Cavrini F, Selleri M, Meschi S, Lapa D, Di Caro A, Grossi P, De Cillia C, Venettoni S, Landini MP, Ippolito G, Nanni Costa A: Italian Transplant Network: Retrospective screening of solid organ donors in Italy, 2009, reveals unpredicted circulation of West Nile virus. Euro Surveill 2010, 15:pii: 19648.