Authors:

Sam Eger

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About Paper:

Abstract: Ebola Virus' structure contains 7 proteins, including the matrix protein, VP40, which is sufficient for virus-like particle (VLP) formation. VP40 localizes at the host's plasma membrane inner leaflet in a phosphatidylserine (PS)-dependent manner via key positively charged residues in its basic patch. Further recruitment of VP40 then forms a hexamer, and eventually a filament. Mutations in the VP40 basic patch region have been discovered in a recent virus outbreak in the Democratic Republic of Congo which include G226R, S228N, S228G, and H269R, however, little is known about their effect on infectivity of the virus. The goal of this study is to characterize these mutants for their stability and binding affinity to PS containing membranes. It is hypothesized that the G226R and H269R will increase viral egress, as they mutate to more positively charged residues in the basic patch. S228N and S228G will likely decrease viral egress however, as both mutate a H- bonding serine in the basic patch. Plasmids harboring these mutants were transformed into DH5α competent cells to be midi prepped for eventual protein expression. These mutants were then transformed into BL21 competent cells, expressing VP40 via an IPTG induction mechanism, then purified. SEC profiles for S228N showed only Dimer formation while S228G showed dimer and octamer formation, similar to the Wild Type. Stability and binding affinity for PS will also eventually be tested via DSF and SPR, respectively. This will elucidate potentially dangerous mutants, which could lead to procuring specific antibodies to prevent further spread of concerning mutants.

Source:

Purdue University / 2023

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Co-authors:

Sam Eger