A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Abstract

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. 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 conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific 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 at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing. INTRODUCTIONThe mechanism of membrane fusion is currently a field of intense investigation, both for intracellular trafficking and enveloped viral infection. For viral fusion protein function, we have previously suggested that fusion protein oligomers form transient and cooperative higher-order structures or rings . These transient ring structures are proposed to facilitate conversion of kinetic energy, released during protein conformational change, into work on the target and host membranes required for membrane fusion. Indeed, biochemical evidence for baculovirus GP64 aggregates during membrane fusion has recently been reported (Markovic et al. 1998). Whether and how viral fusion proteins might function in a coordinated and cooperative manner is a major question in the field of membrane fusion. Because hydrophobic 4-3 heptad repeats are observed...

Expression and Localization of Bombyx mori V-ATPase 16 kDa Subunit c

“…It has already been known that baculovirus budded viruses (BVs) enter the cell via clathrinmediated endocytosis (Long et al, 2006). V-ATPase may play a role in regulating the pH value to promote the membrane fusion between virus and endosome (Kingsley et al, 1999). This is a necessary step for the virus release into the cytosol.…”

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“…The budded virus (BV) phenotype of group I NPVs contains a GP64-like major envelope glycoprotein. This protein is involved in viral attachment to host cells (11), triggers low-pH-dependent membrane fusion during BV entry by endocytosis (4,20,35,42), and is required for efficient budding from the cell surface (27,29).…”

Furin Is Involved in Baculovirus Envelope Fusion Protein Activation

“…Upon acidification of the endosome, the viral and endosomal membranes fuse, thereby allowing entry of the nucleocapsid into the cytoplasm (4,17). For group I NPVs, e.g., Autographa californica AcMNPV, Bombyx mori BmNPV, and Orgyia pseudotsugata OpMNPV, this fusion is mediated by envelope fusion protein GP64 (4,24,39). This protein is also required for efficient budding of newly synthesized nucleocapsids at the plasma membrane (30,33).…”