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.

Figure 1

Reactivity of MAb 3A12 with WARF4 recombinant protein. Protein extracts from E. coli BL21 transformed with His-WARF4 and with the empty vector (pRSETC) were analyzed by western blotting. MAb 3A12 reacted with the recombinant His-WARF4 while it did not show reactivity with the crude lysate of E. coli.

Figure 2

Proposed mechanism of N-NS4B/WARF4 synthesis. In the center the WNV 3^′ 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.

Figure 3

(A) Comparative aminoacid sequence analysis. Amino acids alignment of the NS4B and N-NS4B/WARF4. Identical residues are shown as dots. The arrow represents the 14 AA target of the anti-NS4B antibody. (B) Western blotting analysis of His-NS4B/His-WARF4 proteins employing MAb 3A12 and the anti-NS4B antibody. The analysis was carried out to demonstrate the different amino acids composition of N-NS4B/WARF4 protein relative to the terminal region of NS4B protein. The first three lanes show the reactivity of the anti-His antibody with the His-tagged proteins used in the same western blotting as positive controls. The anti-NS4B antibody did not react with the His-WARF4 protein (lane 4). Similarly, the MAb 3A12 did not react with His-NS4B protein (lane 9). The His-Envelope protein was used as negative control.

Figure 4

Identification of the N-NS4B/WARF4 COOH-terminal amino acids sequence detected by MAb 3A12. The amino acids sequence of the N-NS4B/WARF4 COOH-terminal region with the graphical representation of the four synthetic peptides (SP1, SP2, SP3, SP4) covering the full amino acids sequence codified by WARF4 is shown on the top. The amino acids sequence of the four synthetic peptides is showed in the center, the overlapping sequences among the contiguous peptides are underlined. Peptides (500 ng), Envelope protein and BSA (500 ng) and His-WARF4 and His-NS4B proteins (50 and 150 ng, respectively) were analyzed by dot blotting employing MAb 3A12 (panel A), anti-NS4B antibody (panel B) and MAb anti-His (panel C). The star shows the peptide (SP2) recognized by MAb 3A12.

Figure 5

Expression and intracellular localization of N-NS4B/WARF4 in VERO infected cells. Reactivity of MAb 3A12 with the cytoplasm of VERO WNV infected cells (panel b). No reactivity was observed with non infected cells (panel d). MOPC-21 was used as negative control with infected (panel a) and non infected cells (panel c).

Figure 6

Reactivity of MAb 3A12 with VERO WNV lineage I infected cells by western blotting. MAb 3A12 detects a protein with an apparent molecular weight of about 28 kDa in WNV lineage I infected VERO cells (line 3), no reactivity was observed in uninfected VERO cells (lane 2). The recombinant His-WARF4 protein was used as positive control (lane 1). The commercial anti-NS4B antibody was used to monitor the infection of VERO cells and to compare the migration of NS4B protein (lane 4) with the novel protein. The electrophoretic mobility of N-NS4B/WARF4 protein (lane 3) resulted slightly less than NS4B protein (lane 4). The recombinant His-NS4B positive control was loaded with a delay of about twenty minutes (lane 6).

Figure 7

Time-course of VERO cells infection and western blot analysis. The expression of the N-NS4B/WARF4 protein (panel A) was evaluated by western blotting analysis and compared to the expression of the NS4B protein (panel B) at 24–72 hours post-infection. Both proteins show a similar behaviour with maximum expression in the late phase of infection. His-WARF4 (20 ng) and His-NS4B (50 ng) were used as positive controls.

Figure 8

N-NS4B/WARF4 expression is restricted to WNV lineage I. Panel A shows the reactivity of WNV lineage I with MAb3A12 (lane 3), no reactivity was observed in WNV lineage II (lane 4), VERO cells were used as negative control (lane 2). To monitor the infection of VERO cells with both lineage I and II, the membrane was reprobed with a commercial antibody anti-M (panel B). The M protein and other immature forms were detected in both the WNV lineage I and II (lanes 3 and 4). His-WARF4 and His-preM/M proteins were used as positive controls.

Figure 9

Immune recognition of His-WARF4 by WNV-positive human sera. Eight human sera, 4 of which positive for IgGs anti-WNV by IFA were analyzed for the presence of anti-N-NS4B/WARF4 antibodies by western blotting. The assay was performed by testing simultaneously the reactivity to 3 other recombinant WNV proteins: the domain III of the Envelope, a prem/M protein fragment and the NH₂-terminal portion of NS5. Panel A shows the 4 recombinant proteins stained with the comassie-blue. Panel B shows the results of the western blotting analysis; 4 human sera testing negative for IgGs-anti WNV (1–4) showed no reactivity with the four antigens. The WNV-positive human sera (5–8) showed a different reactivity with the four recombinant antigens. Two sera (7, 8) reacted with His-WARF4 protein.

Figure 10

UPMGA analysis of WNV genomes. The image summarizes the results of the bioinformatics analysis. Some reference strains are shown. Sequence alignment identifies WARF4 in 98% of strain belonging to lineage I (black collapsed form). The WARF4 group (dashed) may be further separated in two groups, depending on the type of programmed −1 ribosomal frame shifting sequence detected. Group 1 carries the UUUUUUG slyppery sequence. Group 2 carries the CCCUUUG/T slippery sequence. The two pseudoknot sequences with the predicted base pairing are shown. The slippery sequences are in gray while the stop codon (AUG) and the first codon (GGC) of WARF4 are underlineed.