OBJECIVE: To identify the interaction between Newcastle disease virus (NDV) matrix (M) protein and avian nucleophosmin B23. 1 in HEK-293T cells.
Methods: Specific primers used to amplify M gene and B23. 1 gene were designed and synthesized according to JS/5/05/Go whole gene sequence (JN631747) and avian nucleophosmin B23. 1 gene sequence (NM_205267). Viral RNA and cellular RNA were extracted from allantoic fluid of NDV JS/5/05/Go strain and DF1 cells with TRIzol reagent, respectively. The M gene and B23. 1 gene were amplified by RT-PCR and then subcloned into eukaryotic expression vectors to generate the recombinant plasmids pEGFP-M, pCMV-HA-M and pDsRed-B23. 1. To investigate the localization features of M protein and B23. 1 protein, we transfected the plasmids pEGFP-M and pDsRed-B23. 1 simultaneously into HEK-293T cells and observed the results by fluorescence microscopy. We further confirmed the interaction between the two proteins by co-immunoprecipitation (Co-IP) assays.
Results: The fusion proteins were successfully expressed in transfected HEK-293T cells by Western blot analysis. The NDV M protein and avian nucleophosmin B23. 1 showed co-localization features in the nucleolus in co-transfected HEK-293T cells. Furthermore, the binding of M with B23. 1 was demonstrated by Co-IP assays.
Conclusion: These results suggested that NDV M protein interacted with avian nucleophosmin B23. 1 and the nucleolar localization of M might be regulated via interaction with B23. 1.