Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein

The baculovirus GP64 envelope fusion protein (GP64EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I. nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role the region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP