Addition of an EGFP-tag to the N-terminal of influenza virus M1 protein impairs its ability to accumulate in ND10

Abstract

A previous report demonstrated that influenza virus infection induces accumulation of EGFP-tagged M1 protein (EGFP-M1) in the sub-nuclear domain ND10. Here, we show that the transfection of four viral protein (NP, PB2, PB1, PA) expression vectors and eight RNA segment expression vectors induced the formation of nuclear dots of EGFP-M1 as seen in virus infections. Omission of the segment 7 RNA expression vector, however, abolished the nuclear dots of EGFP-M1. This result suggests an essential role for authentic M1 protein and/or M2 protein, both of which are encoded in segment 7, in the formation of nuclear dots of EGFP-M1. Co-expression of M1 protein but not M2 protein with EGFP-M1 induced the formation of nuclear dots of EGFP-M1. The dots co-localized with PML protein, which is an indicator of ND10. When only M1 protein was expressed, immunostaining of M1 protein clearly revealed the nuclear dots and their colocalization with PML protein. These results demonstrate that the accumulation in ND10 is an intrinsic characteristic of M1 protein and EGFP addition abolishes this characteristic. The addition of EGFP to M1 protein induced a defect in M1 protein.

Keywords: Fluorescence protein-tag; Influenza virus; M1 protein; ND10.