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
© Faggioni et al.; licensee BioMed Central Ltd. 2012
Received: 1 September 2011
Accepted: 12 November 2012
Published: 22 November 2012
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.
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.
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.
KeywordsWest Nile Virus WNV WARF4 N-NS4B/WARF4 Alternative open reading frame
West Nile virus (WNV) is an arthropod-borne virus maintained in a bird-mosquito transmission cycle. Birds are the natural reservoir hosts while humans and other mammals are dead-end hosts occasionally infected through mosquito bite . The virus, which was identified in 1937 , has been the cause of sporadic cases and outbreaks of disease in Africa, Australasia, Europe, and Middle East [3–5]. Since 1996, WNV has gained growing importance in the western world, causing massive outbreaks and/or small clusters of encephalitis in Europe [6–8]. The virus was introduced for the first time in the North America in 1999 [9, 10], spreading to several countries , and becoming a major public health problem in the USA .
WNV is a small enveloped virus  belonging to the Flaviviriade family, genus Flavivirus. Phylogenetic analysis reveals two distinct viral lineages, lineage I and lineage II . Lineage I is involved in human and equine outbreaks while lineage II is not associated with clinical manifestations in humans [15–17]. WNV genome is a positive single-stranded RNA of about 11 kb containing a single open reading frame flanked by two untranslated regions. RNA translation produces a long polyprotein processed by viral and cellular proteases in three structural (C, preM/M and E) and seven non structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) . Overall, the structural proteins are involved in virus binding and penetration of host cells , while the non-structural proteins are involved in the replicative cycle  and induce immunological evasion mainly through the inhibition of type I interferon signaling [21, 22]. Still, it has been suggested that other unidentified factors could play a role in the pathogenesis of WNV neuroinvasive disease . Melian et al. demonstrated that the NS1’ protein, a known variant of the canonical NS1 protein, results from a ribosomal frame shifting process . The variant protein appears to play a role in WNV neuroinvasiveness. We recently reported the existence of six alternative open reading frames (ARFs) in the WNV genome by in silico analysis. We also demonstrated a significant antibody response to one of this six novel putative proteins (WARF4) in the serum of horses testing positive for antibodies to WNV. However, there was no direct experimental proof of the in vivo existence of this novel protein . The aim of this study was to demonstrate that WARF4 protein is synthesized following WNV infection of mammalian cultured cells. To address this objective, a monoclonal antibody against WARF4 protein was produced. In addition, sera of WNV infected individuals were analyzed in order to test the capacity of WARF4 to induce an immune response in humans as well.
Generation of a mouse monoclonal antibody against WARF4 protein
In silico aminoacid alignment and identification of the N-NS4B/WARF4 region detected by MAb 3A12
Expression of N-NS4B/WARF4 protein in WNV lineage I infected cells
N-NS4B/WARF4 expression is restricted to WNV lineage I
N-NS4B/WARF4 induces antibodies in WNV infected individuals
WNV accession numbers
WARF4 Group 1
WARF4 Group 2
The use of ARFs in viruses belonging to the Flaviviriade family was first reported for the hepatitis C viruses [26, 27]. Recently, it has been demonstrated that WNV uses a short ARF, termed foo, for the synthesis of NS1’ , a known variant of the canonical NS1 [24, 28]. We earlier reported the presence of other ARFs embedded in the coding frame of the WNV genome . Our bioinformatic analysis detected six ARFs, one of which, designed WARF4, was the longest and restricted exclusively to lineage I of WNV. Since WARF4 is embedded in the NS4B gene, the novel protein has been renamed N-NS4B/WARF4. Our results suggested the production of N-NS4B/WARF4 protein in WNV infected horses because of their ability to mount a humoral immune response to N-NS4B/WARF4. However, there was no direct evidence proving the actual existence of the N-NS4B/WARF4 protein. In order to demonstrate the production of N-NS4B/WARF4 in vitro after WNV cells infection, we produced a monoclonal antibody to the N-NS4B/WARF4 COOH-terminal amino acid sequence (MAb 3A12). MAb 3A12 strongly reacted with VERO WNV infected cells by immunofluorescence and detected a ~ 28-kDa protein by western blotting. The predicted aminoacids of N-NS4B/WARF4 preclude the possibility that MAb 3A12 could react with epitopes shared with the NS4B protein (Figure 3A), however to support this prediction a western blotting analysis was performed. As shown in Figure 3B, MAb 3A12 did not recognize the recombinant COOH terminal portion of the recombinant his-tagged NS4B protein. In addition, the anti-NS4B did not recognize the recombinant His-tagged WARF4 protein. To definitely asses the specificity of MAb 3A12 against the alternative reading frame, four overlapping peptides covering the full N-NS4B/WARF4 COOH- terminal amino acid sequence were synthesized and analyzed for their reactivity to MAb 3A12. As shown in Figure 4 the results confirm the specificity of the monoclonal antibody and allow to recognize its epitope between the amino acids 165–212 of N-NS4B/WARF4 sequence. In addition we demonstrated that N-NS4B/WARF4 protein expression is restricted to WNV lineage I infection and that it is expressed at high level in the late phase of infection (Figures 7, 8). Overall, our results demonstrate that N-NS4B/WARF4 is a novel protein, different from NS4B, and that is expressed in WNV infected cells.
Furthermore, we indirectly demonstrated the “in vivo” production of N-NS4B/WARF4 by showing its immunoreactivity with human sera obtained from WNV infected patients (Figure 9). The heterogeneous reactivity to the recombinant WNV antigens displayed by sera testing positive for WNV reflects the complex humoral response elicited by WNV infection [29, 30]. In addition, it is known that the ARF proteins are expressed with both less and variable efficiency if compared to the canonical proteins .
To date, we have no experimental information on N-NS4B/WARF4 protein translation, but it appears reasonable to assume that a −1 ribosomal frame shifting mechanism produces the novel protein. Indeed, in Flaviviriade the translation process is implemented by a cap-dependent scanning process, which produces a single polyprotein . The sequence encoding N-NS4B/WARF4 COOH-terminal is in −1 frame, moreover it is far from the 5′ terminal end, lacks an AUG codon and no internal ribosomal entry site (IRES) is described for WNV. The translation by ribosomal frame shifting is the only realistic explanation for N-NS4B/WARF4 protein synthesis. Since the proposed model requires the presence of specific RNA structures such as slippery sequences associated with pseudknot [33–35], a bioinformatics analysis was performed to predict these structures. All the complete genomes of WNV available on gene bank were aligned and assigned to the two main lineages (Figure 10, Table 1). The strains belonging to the lineage 1 were first analyzed to confirm the association with WARF4 and then a further analysis was carried out looking for slippery sequences and pseudknot structure within the NS4B coding region. WARF4 was detected in 361 out of 368 genomes belonging to lineage 1. Seven genomes lacked WARF4 because of a single nucleotide substitution that interrupts the alternative frame. In the WARF4 group, two different and mutually exclusive slippery sequences with downstream frameshift-stimulating pseudknot structures were predicted. The first UUUUUUG sequence is the most representative (91%). The pseudknot structure is 80 nucleotides long and includes the initial aminoacids codified by the −1 frame. This slippery sequence is associated with the American viral strains. The second CCCUUUG/T sequence is present in 5% of the genomes, it is positioned 129 nucleotide upstream of WARF4 and has a pseudoknot structure of 40 nucleotides. The second slippery sequence is associated with circulating viral strain in Mediteranean Bacin and Est-Europe. The strain analyzed in this work belongs to this second group [Egyptian strain, an. AF260968]. It should be noticed that both the structures must promote the suppression of a termination codon (UGA) located just before the first codon of −1 frame .
The ribosomal frameshifting model also explains the discrepancy between the predicted and the observed molecular weight of the alternative N-NS4B/WARF4. The predicted alternative reading frame protein consists of 148 AA that account for a molecular weight of 16,7 kDa. However, the protein detected by MAb 3A12 in WNV infected cells migrates with an apparent molecular weight of about 28–30 kDa. Although this discrepancy could be due to a post-translation modification, the proposed model appears the most reasonable explanation for the observed molecular weight of N-NS4B/WARF4 protein. The novel protein would exist as COOH-terminal variant of the NS4B protein, indeed, the ribosomal shift in −1 frame would give rise to a NS4B variant protein where the last 123 AA should be replaced by a longer amino acid tail of 148 A. Thus, the variant protein should exhibit a molecular weight of about 30 kDa, consistent with our results (Figure 6, lane 3). The proposed ribosomal frameshifting model implies that the expression kinetic of N-NS4B/WARF4 should be like that of NS4B protein even if the amount of the novel protein should be less than that of NS4B. Figure 7 shows the expression of N-NS4B/WARF4 and NS4B in time-corse infection, the two proteins exhibit a similar kinetics. N-NS4B/WARF4 is clearly detected in the late phase of infection, such as the NS4B protein. The level of NS4B and N-NS4B/WARF4 proteins expression based on a densitometric analysis (data not shown) indicates a ratio of about 25 to 1 respectively. It should be highlighted that this ratio is estimated for the strain Eg101 [an. AF260968] that our biofinformatic analysis associates with the strain circulating in the Mediterranea area (Figure 10). The predicted pseudoknot associated with the American strain is thermodinamically more stable. It should be intersting to estimate this ratio in the American viral strain.
Overall, our results show for the first time that the novel ARF protein, N-NS4B/WARF4, is produced during the late stage of WNV lineage I infection and that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. To date, the biological function of N-NS4B/WARF4 and the role of anti- NS4B/WARF4 antibodies are unknown; however, it is suggestive that N-NS4B/WARF4 is restricted exclusively to the lineage I of WNV, which is known to be associated with the more severe clinical manifestations of WNV disease. This protein might represent a novel tool for a better understanding of WNV biology and for an improved characterization of immune response in WNV infected individuals.
Cell culture and virus strain
The VERO E6 cell line was cultured in Eagle’s minimal essential medium (MEM) with 10% Fetal Calf Serum (FCS), 100 U/mL penicillin, 200 μg/mL streptomycin in the presence of 5% CO2. WNV lineage I [strain Eg101, an. AF260968] and WNV lineage II [strain B956, an. AY532665] were propagated by infecting VERO E6 cells monolayers in MEM with 2% FCS and titrated according to the Reed and Muench formula.
Additional WNV genomic RNA [strain NY 1999, an. AF260967] was obtained from European Network for Diagnostics of Imported Viral Disease (http://www.enivd.de/ENIVD_P.HTM) during the External Quality Assurance (EQA) for molecular detection of West Nile virus. The nucleic acids were extracted by NucleoSpin RNA Virus kit according to manufacturer’s instructions (Macherey-Nagel, Düren, Germany).
Expression of WARF4, NS4, envelope, preM/M and NS5 recombinant proteins
Primers and vectors used to generate WNV recombinant proteins
5′-atggcggatcc aggcaatgcgctcagcccagcg-3′ BamHI
5′- tctttgaagctt ctagtgaactcttcttttgtc-3′ HindIII
5′- ggaaaggatcc agttgaagggaacaacc -3′ BamHI
5′-ctcctgaattc gtggttgtaaaggctttgcc-3′ EcoRI
5′-cagcgggatcc cagttaccctctctaacttcc-3′ BamHI
5′-caagggaattc aagctgtaagctggggccacc-3′ EcoRI
5′-gaggcggatcc gcggggggaatgtcacac-3′ BamHI
Production of a monoclonal antibody (MAb) recognizing the WARF4 recombinant protein
Four week-old BALB/mice were immunized twice by intraperitonal injection with 25 μg of purified His-WARF4 protein emulsified in RIBI adjuvant (RBI Immununochemical Research). Mice were then given a booster immunization intravenously with 10 μg of the immunogen, and immune splenocytes were removed 3 days later. Somatic cell hybrids were prepared with NS-1 mouse non secreting myeloma cells as previously described [37–39]. Hybridoma supernatants were screened for differential immunoreactivity to His-WARF4 and His-purified control proteins by enzyme linked immunosorbent assay [40, 41]. Positive hybridoma cell lines were cloned twice by limiting dilution. One MAb was selected and designed 3A12.
Peptide scanning analysis
Four synthetic peptides named SP1, SP2, SP3 and SP4, which cover the full N-NS4B/WARF4 COOH-terminal amino acid sequence and which have amino acids overlapping sequences were designed (Figure 4).
SP1 (H-GNALSPAADSGWNHEKRCSGWHRGHGRPRIRAHHTHHAEESWANHA), SP2 (H-THHAEESWANHADLGVSSCSSSEPVCEDSARSRNSDHGSSGDTLGEW), SP3 (H-SDHGSSGDTLGEWSKLCLECNNCHRTLPHHAWGLVVMLIHNMDTHKE) and SP4 (HVVMLIHNMDTHKEHGKTRTKKRWGKGTHLGRGLERKTQPDDKRRVH) were synthesized by PEPSCAN (http://www.pepscan.com). Five hundred ng of peptide as well as Env and BSA proteins, 50 ng of His-WARF4 and 200 ng of His-NS4B were spotted in replicates on nitrocellulose membranes. Membranes were blocked for 4 h at RT in 5% non-fat dry milk-PBS-0.1% Tween-20 and incubated 1 hour at RT with MAb 3A12, anti-NS4B antibody (Abcam plc, Cambridge, UK) and MAb anti-Histidine (Invitrogen). The membranes were washed four times and then incubated with goat anti-mouse or rabbit IgG peroxidase-conjugated antibodies (Sigma, MI, IT) and developed by a chemiluminescent kit (Sigma, MI, IT) as previously described [42, 43].
Indirect immunofluorescence assay (IFA)
The VERO E6 cell line was grown in eight wells Chamber slides™ (Nunc, USA). 200 μl of a viral suspension (104 xTCID50/ml) were used to infect VERO cells monolayers (40-50% confluent). WNV was subsequently allowed to adsorb for 1 hour at 37°C. MEM medium with 2% FCS, was then added to the infected cells monolayer. After 36 hours, the cells monolayer was washed 2 times in PBS 1X and fixed with 4% paraformaldehyde for 20 min at room temperature, followed by treatment with 0.1 M glycine for 20 min at 25°C and with 0.1% Triton X-100 for an additional 5 min at 25°C to allow permeabilization. Cells were incubated for 30 min at room temperature with MAb 3A12 or the antibody MOPC-21 used as negative control as previously described [44, 45]. Nuclei were stained with Hoechst 33342 (blue).
Western blotting analysis
The VERO cells were infected with a multiplicity of infection (MOI) of 0.1 for 24 to 72 hours or for 96 hours at 37°C in 5% CO2. Cells were scraped and harvested by centrifugation at 1,500 X g at 4°C and rinsed in ice-cold phosphate-buffered saline (PBS). Cell lysates were prepared in Staph A buffer (10 mM sodium phosphate pH 7.4, 100 mM NaCl, 5 mM EGTA, 1% Triton, 0.1% SDS, 0.5% deoxycholate) containing 1 mM PMSF [46, 47] and a protease inhibitor cocktail (complete Mini EDTA-free, Roche). Proteins from uninfected and infected VERO cells lysate (50 μg/well) or purified recombinant WARF4 (10–100 ng/well), Env III (10–100 ng/well), preM (100 ng/well), NS5 (100 ng/well), NS4B (10 ng/well) protein fragments were separated on a NuPAGE 4-12% or 12% Bis-Tris gel and transferred onto nitrocellulose membranes (Invitrogen) . Membranes were blocked for 6 h at 4°C in 5% nonfat dry milk-PBS-0.1% Tween-20 and incubated overnight at 4°C with MAb 3A12, anti-NS4b antibody, anti-M (Abcam plc, Cambridge, UK) or human sera (dilution 1:100). The membranes were washed four times and then incubated with goat anti-mouse IgG, anti-rabbit or anti-human IgG peroxidase-conjugated antibodies (Sigma, MI, IT) and developed by a chemiluminescent kit (Sigma, MI, IT) as previously described [42, 49].
Human serum samples, obtained from convalescent patients suffering from neuro-invasive WNV infection, testing positive for IgGs anti-West Nile by IFA and confirmed by Micro-Neutralization Test Assay - MNTA  were kindly provided by Dr. Vittorio Sambri (St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy). Human sera from healthy donors testing negative for IgGs anti-West Nile were used as negative controls.
384 full genomes of WNV were retrived from gene bank and aligned with BioNumerics software package (version 6.5 Applied-Maths, Belgium). Genomes were further analized by KnotInFrame software (http://bibiserv.techfak.uni-bielefeld.de/knotinframe/) to predict ribosomal −1 frameshift sites with a pseudoknot strucuture.
Human sera and experimental animals
Human sera were collected after informed consent and sent to the Regional Reference Centre for Microbiological Emergencies (CRREM, Prof. Vittorio Sambri, St. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy). The protocol was approved under the regional plan of surveillance and control for west nile disease in Emilia Romagna (5 June 2009, prot. PG/2009/128190).
Mice were sacrificed by cervical dislocation and later the spleens were removed by surgical resection. The procedures were carried out in the presence of a veterinarian who monitored proper compliance with the decree law 116/92. The protocol was approved by the ethics committee of the Station Technology for Animal (STA) (http://www.sta.uniroma2.it/) of the University of Rome “Tor Vergata” on 06/12/2010.
West Nile Alternative open Reading Frame 4
West Nile Virus
Alternative Reading Frame.
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