Furin Is Involved in Baculovirus Envelope Fusion Protein Activation

Abstract

. A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F 0 ) protein. BV produced in the presence of the inhibitor possesses the uncleaved F 0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F 0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F 1 and against the putative N terminus (F 2 ) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60-(F 1 ) and 21-kDa (F 2 ) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominately as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.

Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (Ac M NPV): F Proteins from Group II NPVs Are Functionally Analogous to Ac M NPV GP64

“…6A). This point mutation, an arginine-to-lysine substitution in a critical residue in the consensus furin cleavage site, was previously shown to prevent F protein cleavage (33). Using two protocols for testing gp64 complementation, we found that the F protein containing the Se8 K149 mutation did not complement the gp64-null bacmid (see Table 1).…”

“…The consensus furin cleavage site [R-X-(R/K)-R] is conserved at a similar position in F proteins from group II NPVs and GVs. Recent biochemical and mutagenic studies of the SeMNPV F protein strongly suggested that the SeMNPV F protein is cleaved by a PPC or furin protease (33). To determine whether cleavage at the furin cleavage site is essential for rescue of the gp64-null virus by the SeMNPV F protein, we generated a gp64-null bacmid expressing a SeMNPV F protein with a point mutation in the furin cleavage site (Se8 K149 ) (Fig.…”

“…F proteins from LdMNPV (ORF 130, or Ld130) and SeMNPV (ORF 8,or Se8) are present in the virion membrane and mediate pH-triggered membrane fusion (15,27). Like envelope proteins from some other virus groups, the baculovirus F proteins are cleaved internally by cellular proprotein convertases such as furin, and cleavage is necessary for fusion activity (33). These observations suggest that F proteins may represent a functional analog of GP64 in some (perhaps all) baculoviruses that do not have a gp64 gene.…”

“…An envelope protein gene that is unrelated to gp64 was recently identified in Lymantria dispar MNPV (LdMNPV) (27) and Spodoptera exigua MNPV (SeMNPV) (15), and homologs of this gene have been identified in other group II NPVs and GVs. This protein has been named the F (fusion) protein (33). F proteins from LdMNPV (ORF 130, or Ld130) and SeMNPV (ORF 8,or Se8) are present in the virion membrane and mediate pH-triggered membrane fusion (15,27).…”

GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses

“…This protein is involved in attachment of BVs to the cell, is required for low-pH-triggered membrane fusion during virus entry and is necessary later in the process of infection for efficient budding of progeny nucleocapsids (NCs) into the haemolymph or cell-culture supernatant (Blissard & Wenz, 1992;Hefferon et al, 1999;Oomens & Blissard, 1999). envelope fusion proteins, the baculovirus F protein must be cleaved post-translationally by a proprotein convertase (furin) to become fusiogenic (Lung et al, 2002;Westenberg et al, 2002). Homologues of the F gene have been identified in other group II NPVs, in beta-and deltabaculoviruses and in members of the insect retrovirus family Errantiviridae, but also exist in group I NPVs (Herniou et al, 2003;Malik et al, 2000;Rohrmann & Karplus, 2001;Terzian et al, 2001).…”