Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik

For evaluating EBOV GP triggering under biosafety level 2 (BSL-2) conditions, we generated vesicular stomatitis virus (VSV) pseudotypes, which provide a highly validated surrogate system for recapitulating filovirus entry (25, 26) . To facilitate viral tracking in most experiments, virions were engineered to contain a fluorescent protein, monomeric NeonGreen (mNG), fused to the viral phosphoprotein (P) (27, 28) . The heavily glycosylated mucin domain (Muc) of EBOV GP is dispensable for entry, and its deletion enhances viral attachment (29, 30) . Accordingly, we primarily used VSV pseudotypes bearing mucin domain-deleted GP (GP⌬Muc). To validate our viral preparations, 1,1=-dioctadecyl-3,3,3=,3=-tetramethylindodicarbocyanine (DiD)-labeled VSV bearing mNG-P and EBOV GP⌬Muc was bound to a coverslip and incubated with Alexa Fluor 555-labeled KZ52, a neutralizing antibody that recognizes a conformational epitope at the GP1-GP2 interface (7) . On average, over 90% of DiD-labeled particles also possessed NG-P signal, and 72% of total particles exhibited triple-labeling, indicating that the preparations used were of high quality (Fig. 1A) .