Live-cell diffracted X-ray blinking reveals Ebola virus VP40-driven budding dynamics and their regulation by cytoskeletal and lipid factors

Ebolavirus VP40 drives viral budding by remodeling the plasma membrane, yet the underlying real-time dynamics remain poorly understood. Here, we applied Diffracted X-ray Blinking (DXB) to investigate how E bolavirus VP40 affects membrane dynamics in living cells by monitoring millisecond-scale fluctuations in X-ray diffraction intensity. Expression of wild-type VP40 (wtVP40) from Zaire ebolavirus induced a progressive increase in membrane fluctuations, whereas the dimerization-defective mutant L117R and the oligomerization-deficient mutant I307R failed to enhance membrane motion despite comparable protein expression levels. Consistent with these findings, only wtVP40 was efficiently released into the culture medium, indicating that increased membrane fluctuations correlate with budding competence. DXB analysis further showed that wtVP40 increased the fast-moving population with higher decay constants, whereas the L117R and I307R mutants showed limited changes in the decay-constant distributions. Pharmacological perturbation studies demonstrated that VP40-induced membrane fluctuations are sensitive to both cytoskeletal integrity and phosphatidylserine availability. Expression of VP40 from Reston ebola virus similarly enhanced membrane dynamics, suggesting that VP40-mediated membrane remodeling is conserved among E bolaviruses . DXB-based membrane-motion analysis may therefore provide biophysical markers for viral assembly and a basis for evaluating budding-targeted antiviral strategies.