Once inside a host cell, the Ebola virus interacts with the plasma membrane (PM) to facilitate viral budding, as part of the infective process. Without this interaction, viral transcription and eventual replication within host tissues is not possible. Researchers have established that the viral protein VP40, one of seven encoded by the virus’s negative-sense RNA genome, plays a key role in this process; however, the mechanics are as yet unknown.
Adu-Gyamfi et al. (2014) sought to characterize VP40 binding domains involved in this essential step of the Ebola virus lifecycle.1 The research team created mutated sequences in a loop region near the VP40 N-terminus implicated in the process; the loop region is highly conserved among isolated virus strains associated with recent outbreaks. Through this method, the team identified the regions important for PM interaction and translocation of the viral nucleocapsid.
First the researchers created mutant constructs, which they fusion-tagged with EGFP (enhanced green fluorescent protein) before transfecting two different cell cultures, HEK293 (derived from human embryonic kidney) and CHO-K1 (from Chinese hamster ovarian cells). The scientists then examined the cell cultures for VP40 localization within the cell and for evidence of oligomerization, which is necessary for functionality.
Using fluorescence microscopy to visualize the EGFP tags, Adu-Gyamfi et al. found that, compared to the wild-type constructs, mutants affecting residues Lys127, Thr129 and Asn130 exhibited reduced association with the PM and localized predominantly within the cytoplasm. Furthermore, in association with reduced PM interaction, they also noted lower levels of VP40 co-association in the formation of oligomers. The researchers measured this using Total Internal Reflection Fluorescence (TIRF) microscopy with Numbers and Brightness counting. Because each VP40 construct contained one EGFP fusion tag, the research team could assess the degree of association between VP40 monomers as a factor of the standard brightness of a single monomer.
The study also exploited a unique feature of the Ebola virus VP40: the protein can stimulate release of non-infective virus-like particles (VLPs) from host cells in the absence of other virus constituents. To investigate this, the researchers harvested both conditioned media and cell lysates from each culture following transfection. They examined samples using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins before assessing EGFP abundance via immunoblotting. Cells transfected with specific mutant VP40 constructs released fewer VLPs into the medium and showed higher abundance of VP40 in lysates, as compared with those expressing the wild-type viral protein.
In summary, Adu-Gyamfi and co-workers identified residues within the VP40 N-terminus loop region, Lys127, Thr129 and Asn130—essential for VP40 oligomerization, VLP release and PM localization. The researchers note that, although they have not characterized direct protein–protein or protein–lipid interactions, they have shown where these events occur within the VP40 protein structure. Further studies can develop these findings and establish the mechanisms behind this important step in viral infectivity.
1. Adu-Gyamfi, E., et al. (2014, October) “A loop region in the N-terminal domain of Ebola virus VP40 is important in viral assembly, budding, and egress,” Viruses, 6 (pp. 3837–54), doi: 10.3390/v6103837.
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