Characterization of filovirus protein-protein interactions in mammalian cells using bimolecular complementation.

摘要

The virion protein 40 (VP40) and nucleoprotein (NP) of Ebola (EBOV) and Marburg viruses (MARV) play key roles during virion assembly and egress. The ability to detect interactions between VP40-VP40, VP40-NP, and NP-NP and follow these complexes as they traffic through mammalian cells would enhance our understanding of the molecular events leading to filovirus assembly and budding, and provide new insights into filovirus replication and pathogenesis. Here, we successfully employed a bimolecular complementation (BiMC) approach to visualize interactions between EBOV and MARV VP40-VP40, NP-NP, and VP40-NP proteins and localize these protein complexes in mammalian cells using confocal microscopy. We demonstrate that VP40-VP40 complexes localized predominantly at the plasma membrane, whereas VP40-NP and NP-NP complexes displayed a more dispersed pattern throughout the cytoplasm. As expected based on previous findings, efficient interactions between EBOV or MARV VP40-VP40 proteins were independent of L-domains PTAPPEY and PPPY, respectively. In contrast, the formation of EBOV or MARV VP40-VP40 complexes was dependent on the previously characterized LPLGVA and LPLGIM motifs of EBOV and MARV VP40 proteins, respectively, indicating that these motifs are important for VP40 oligomerization and subsequent budding. These results highlight the feasibility and usefulness of the BiMC approach as a strategy to further characterize both filovirus protein interactions as well as filovirus-host interactions in real time in the natural environment of the cell.