A discrete stage of baculovirus GP64-mediated membrane fusion

Viral: fusion protein trimers can play a critical role in. limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion prot eins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order ol igomers might be stabilized by similar sequences. There is a hydrophobic;4- 3 heptad repeat contiguous to a putative oligomerization domain of Autograp ha californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if th is sequence might play a role in catalysis of membrane fusion. First, leuci ne-to-alanine mutants within and flanking the amino terminus of the hydroph obic 4-3 heptad repeat motif that oligomerize into trimers and traffic to i nsect Sf9 cell surfaces were identified. These mutants retained their wild- type conformation at neutral pH and changed conformation in acidic conditio ns, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide enc oding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synth esized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conform ational changes, as determined by the loss of-a conformationally sensitive epitope. In control experiments, a peptide of identical composition but dif ferent sequence, or a peptide encoding a portion of the Ebola GP heptad mot if,had no effect on GP64-mediated fusion. Furthermore, when the hemagglutin in (X31 strain) fusion protein of influenza was functionally expressed in S f9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesti ng that the: peptide does not exert nonspecific effects on other fusion pro teins or cell membranes. Collectively, these studies suggest that the speci fic peptide sequences of GP64 that are adjacent to and: include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion a t a stage that is downstream of the initiation of protein conformational ch anges but upstream of lipid mixing.