THE BACULOVIRUS GP64 ENVELOPE FUSION PROTEIN - SYNTHESIS, OLIGOMERIZATION, AND PROCESSING

The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I i ntegral membrane protein that enters the secretory pathway and is olig omerized and extensively processed during transport to the plasma memb rane. The kinetics of GP64 EFP biosynthesis, oligomerization, and proc essing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of G P64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i. , and the maximal rate of synthesis was observed in the period of 24-2 6 hr p.i., a time coincident with the onset of high level production o f budded virus in OpMNPV-infected L. dispar cells. To determine the ol igomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatograph y, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP wa s identified as a trimeric molecule, that migrates as two discrete ban ds on nonreducing SDS-PAGE. Pulse-chase studies, performed at both ear ly (12 hr p.i.) and late (36 hr p.i.) stages of the infection cycle, s howed that GP64 EFP oligomerization is complete within 15 min after sy nthesis. Efficiency of oligomerization however was relatively low, wit h less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be de graded within 30 to 45 min after synthesis. Analysis of the kinetics o f carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for both early and late phases of infe ction, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during th e late phase. Thus, carbohydrate processing appeared to become less ef ficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results i n a homotrimeric structure for GP64 EFP. While carbohydrate addition i s rapid, carbohydrate processing requires prolonged periods of time (w ith half-times of 45 to 75 min) and appears to become less efficient d uring the late phase of the infection. (C) 1995 Academic Press, Inc.