Panel A shows the 4 recombinant proteins stained with the comassie-blue
Panel A shows the 4 recombinant proteins stained with the comassie-blue. 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. family, genus 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 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. Results Generation of a mouse monoclonal antibody against WARF4 protein In order to demonstrate the 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 Rabbit polyclonal to ZNF268 the His-WARF4 by western blotting while it did not show cross-reactivity with the crude lysate from transformed with the empty vector (Figure ?(Figure11). Open in a separate window Figure 1 Reactivity of MAb 3A12 with WARF4 recombinant protein. Protein extracts from 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 produced N-NS4B/WARF4 protein, western blotting analysis was carried out. As shown in Figure ?Figure6,6, 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 Ziprasidone 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). Open in a separate window 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 Ziprasidone 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 Ziprasidone of about twenty minutes (lane 6). Next, the expression of N-NS4B/WARF4 was evaluated and.