Ebola virus VP40 contains 2 overlapping late domains (7-PTAP-10 and 10-PPEY-13) that are essential for its interaction with Tsg101 and Nedd4 in the promotion of viral egress. Deletion of the late domains inhibits VP40-induced virus-like particles (VLPs). However, a truncated form of VP40, which lacks a late domain because of the deletion of amino acids 1-30, is released into supernatant as a VLP, indicating that the remaining portion of VP40 contains the structural elements required for VLP release. Thus, the purpose of this study was to identify the VP40 sequence essential for VLP budding, through the generation of deletion and alanine-scanning mutants. We found that the amino acid sequence around the proline at position 53 plays a critical role in VLP production and intracellular transport. These data also may suggest that a novel host factor(s) is involved in virus budding.