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

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.


Background
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 [1]. The virus, which was identified in 1937 [2], has been the cause of sporadic cases and outbreaks of disease in Africa, Australasia, Europe, and Middle East [3][4][5]. Since 1996, WNV has gained growing importance in the western world, causing massive outbreaks and/or small clusters of encephalitis in Europe [6][7][8]. The virus was introduced for the first time in the North America in 1999 [9,10], spreading to several countries [11], and becoming a major public health problem in the USA [12]. WNV is a small enveloped virus [13] belonging to the Flaviviriade family, genus Flavivirus [14]. Phylogenetic analysis reveals two distinct viral lineages, lineage I and lineage II [15]. Lineage I is involved in human and equine outbreaks while lineage II is not associated with clinical manifestations in humans [15][16][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) [18]. Overall, the structural proteins are involved in virus binding and penetration of host cells [19], while the non-structural proteins are involved in the replicative cycle [20] 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 [23]. Melian et al. demonstrated that the NS1' protein, a known variant of the canonical NS1 protein, results from a ribosomal frame shifting process [24]. 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 [25]. 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 order to demonstrate the in vitro production of the WARF4 protein following WNV lineage I infection, a mouse monoclonal antibody to His-WARF4 fusion protein was generated. The selected MAb 3A12 recognized the His-WARF4 by western blotting while it did not show cross-reactivity with the crude lysate from E. coli transformed with the empty vector ( Figure 1).
In silico aminoacid alignment and identification of the N-NS4B/WARF4 region detected by MAb 3A12 WARF4 is an alternative gene overlapping the COOHterminal region of the NS4B gene and a small NH2terminal portion of the NS5 gene ( Figure 2). The genomic organization of WNV implies that a −1 ribosomal frameshifting process translates WARF4, thus the novel protein has been renamed N-NS4B/WARF4. The amino acid composition of N-NS4B/WARF4 is completely different from the NS4B viral protein in the COOH-terminal region, this is shown by the amino acid alignment analysis in Figure 3A. To further support the bioinformatics evidence, a western blotting analysis of the recombinant His-WARF4 and His-NS4B proteins employing the polyclonal anti-NS4B antibody and MAb 3A12 was performed to demonstrate that His-WARF4 and NS4B proteins are dissimilar ( Figure 3B). The commercial polyclonal antibody anti-NS4B was developed to a NS4B fragment from AA 126 to AA 145, which thus overlaps the first 14 AA of the N-NS4B/WARF4 COOH-terminal region. As shown in Figure 3B (lane 4), the anti-NS4B antibody did not recognized the recombinant His-WARF4 protein. The recombinant His-NS4B fragment encompasses the COOH-terminal portion of NS4B starting from AA 120 thus including the entire ARF coding sequence. In addition, MAb 3A12 did not recognize the His-NS4B fragment (lane 9).
The N-NS4B/WARF4 region recognized by MAb 3A12 was identified by analyzing MAb 3A12 reactivity to four synthetic overlapping peptides (SP1, SP2, SP3, SP4) which cover the full N-NS4B/WARF4 COOH-terminal amino acids sequence (Figure 4). The four synthetic peptides, as well as recombinant His-WARF4, His-NS4B, Env proteins and Bovine Serum Albumin (BSA) were spotted in replicates on nitrocellulose membranes. Membranes (panels A, B, C) were then reacted with MAb 3A12, anti-NS4B antibody and anti-His antibody respectively. As shown in Figure 4, MAb 3A12 recognized only SP2, while lacking reactivity to His-NS4B, His-EnV and BSA. Conversely, anti-NS4B or anti-His antibody did not recognize any of the Figure 2 Proposed mechanism of N-NS4B/WARF4 synthesis. In the center the WNV 3 0 genomic organization is shown. WARF4 is dashed, the first and the last base of the alternative reading frame are pointed by stars. On the top, the synthesis of NS4B by canonical translation mechanism in 0 frame and maturation is shown. Below is displayed the proposed mechanism of N-NS4B/WARF4 synthesis through translation in −1 frame. The amino acids sequences of the two proteins are showed, the different COOH terminals of the two proteins are underlined.
WARF4 peptides. These results corroborate the specificity of MAb 3A12 to N-NS4B/WARF4 protein and allow to recognize the epitope of MAb 3A12 between amino acids 165-212 of N-NS4B/WARF4 sequence.

Expression of N-NS4B/WARF4 protein in WNV lineage I infected cells
Expression of N-NS4B/WARF4 following WNV lineage I infection of VERO cells in vitro was demonstrated by immunofluorescence and western blotting analyses employing MAb 3A12. Figure 5 shows the reactivity of MAb 3A12 with uninfected and infected VERO cells. MAb 3A12 showed a strong cytoplasmic immunoreactivity in infected cells (panel b), while it did not react with uninfected cells (panel c). MOPC-21 was used as negative control (panel a and c).
In order to determine the apparent molecular weight of the in vitro produced N-NS4B/WARF4 protein, western blotting analysis was carried out. As shown in Figure 6, in infected VERO cell lysate MAb 3A12 detected a protein showing an apparent molecular weight of about 28 kDa (lane 3). Recombinant His-WARF4 (lane 1) was used as positive control. No reactivity of MAb 3A12 was observed with uninfected VERO cells (lane2). The commercially available anti-NS4B antibody was used as positive control to monitor the infection of VERO cells by WNV (lane 4) and to compare the apparent molecular weight of NS4B protein to N-NS4B/WARF4 protein. Our result shows that the electrophoretic mobility of N-NS4B/WARF4 is slightly lower than that of NS4B. The recombinant His-NS4B was used as positive control for anti-NS4B antibody (lane 6).
Next, the expression of N-NS4B/WARF4 was evaluated and compared to the expression of NS4B protein through a time-course infection (24-72 hours). Figure 7 shows the results of western blotting analysis performed with MAb

N-NS4B/WARF4 induces antibodies in WNV infected individuals
In order to determine whether human sera from WNV infected individuals were able to recognize N-NS4B/ WARF4 protein, western blotting analysis was carried out. Reactivity of human sera to His-WARF4 protein was compared to that of 3 other recombinant WNV proteins, including the domain III of the envelope, a preM/M fragment and the NH 2 -terminal portion of NS5 ( Figure 9A). Eight human sera, 4 of which positive for IgGs anti-WNV, were assayed. Sera from individuals testing negative for WNV by IFA and seroneutralization test were also negative for reactivity with recombinant WNV proteins. Conversely, sera from individuals testing positive for WNV by IFA and seroneutralization test showed different patterns of reactivity with the recombinant proteins analyzed ( Figure 9B). Two sera were able to react with His-WARF4. Of these, one was able to recognize also the His-preM/M, His-NS5 and His-EnvIII proteins while the other recognized only the preM/M. In addition, one serum was able to detected EnvIII only, while another produced a weakly positive signal for the   envelope and NS5 only after a long exposure (data not shown).

Bioinformatic results
Alignment and cluster analysis of 384 WNV strains assigned 368 samples to lineage I according to previous studies [15] ( Figure 10, Table 1

Discussion
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 [25]. 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 [31].
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 [32]. The sequence encoding N-NS4B/WARF4 COOH-terminal is in −1 frame, moreover it is far from the 5 0 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][34][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 [36]. 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.

Conclusions
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. Expression of WARF4, NS4, envelope, preM/M and NS5 recombinant proteins

Cell culture and virus strain
Since the previously described recombinant His-WARF4 protein [25] does not comprise the full alternative reading frame, we cloned a novel fragment which includes the entire ARF. The 444 bps fragment spanning the WARF4 position 7311-7754 [accession number AF260967] was amplified in a single step by Superscript III One step RT-PCR, (Invitrogen, California). The fragment was cloned in pRSET vector and expressed in the BL21 Star (DE3)pLysS competent cells (Invitrogen, California). Other WNV recombinant proteins including the domain III of the Envelope (Env III), a preM/M and a NS5 fragment were produced as described above using primers and vectors reported in Table 2. The NS4B fragment was expressed by Rapid Translation System 100 E.coli HY (www.5PRIME.com). The His-tagged recombinant proteins were purified by Ni-NTA affinity chromatography kit according to manufacturer's instructions (QIAGEN, Hilden, Germany). All the oligonucleotides were synthesized by Eurofins MWG Operon (www.eurofinsdna.com).

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][38][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.

Indirect immunofluorescence assay (IFA)
The VERO E6 cell line was grown in eight wells Chamber slides ™ (Nunc, USA). 200 μl of a viral suspension (10 4 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).

Human sera
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 [50]