Vaccinia Virus A56/K2 Fusion Regulatory Protein Interacts with the A16 and G9 Subunits of the Entry Fusion Complex

Abstract

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cellsto form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.The simplest infectious form of vaccinia virus (VACV), called the mature virion (MV), consists of an outer membrane with at least 20 associated proteins surrounding a core containing the double-stranded DNA genome, enzymes and factors required for the transcription of early genes, and numerous structural proteins (9, 19). During virus assembly, some MVs are wrapped with additional membranes that facilitate intracellular movement, exocytosis, and cell-to-cell spread (29). Extracellular enveloped virions (EVs) are essentially MVs with an additional membrane, which is opened or removed prior to the fusion of the MV and cell membrane during virus entry (18). Thus, the viral fusion proteins are components of the MV membrane rather than the EV-specific membrane (20). Mutagenesis and affinity purification studies have shown that at least eight MV membrane polypeptides are required for entry and have no other known function (4,16,21,22,25,26,28,30,31). The components of this entry/fusion complex (EFC) are conserved in all poxviruses, indicating a common mechanism of infection. Studies with VACV indicate that fusion may occur in a pH-independent manner at the plasma membrane (1, 7, 8) or in a pH-dependent manner within endosomes (32, 33).The EFC is also required for cell-cell fusion (28, 37), which can be induced by briefly lowering the pH (10, 13) or preventing expression of either the A56 (15, 24) or K2 (17, 34, 38) polypeptide. The glycosylated A56 polypeptide, referred to as the hemagglutinin (HA), is found on the plasma membrane of i...

A Novel Mode of Poxvirus Superinfection Exclusion That Prevents Fusion of the Lipid Bilayers of Viral and Cellular Membranes

“…Deletion or mutation of either of these proteins results in the formation of syncytia at neutral pH (62)(63)(64)(65)(66). The finding that the A56/K2 heterodimer interacts specifically with two interacting proteins of the EFC provided a mechanism for preventing syncytium formation (7,8). Turner and Moyer (5) reported that cells infected with A56 or K2 mutants have increased susceptibility to superinfection at late times.…”

“…They concluded, mainly based on UV inactivation of virus particles, that early gene expression by the primary virus was responsible for resistance to superinfection and that early gene expression by the secondary virus was prevented. Subsequent studies provided evidence that SIE can be mediated by a heterodimer formed by the A56 and K2 proteins on the cell membrane (5, 6), which interact with a protein complex on the virus surface that is required for fusion and entry (7,8). Whether this mechanism, which was demonstrated at a late phase of virus replication, is related to the early SIE was not assessed.…”

Transcriptional Repression and RNA Silencing Act Synergistically To Demonstrate the Function of the Eleventh Component of the Vaccinia Virus Entry-Fusion Complex

“…Because of its small size and hydrophobicity, the I2 protein, like the O3 EFC protein (30), may have been missed by mass spectroscopy of the purified complex (33). The overall architecture of the EFC has not been determined, although a few protein-protein interactions that resist destabilization of the complex have been identified (25,42,43).…”

“…DsRed fluorescent protein under the control of the VACV p11 late promoter into the intergenic region between J4R and J5L with DsRed transcription leftward. The recombinant virus vA16iflagJ5 was derived from vA16i (28) by replacement of the original J5R gene by enhanced green fluorescent protein (GFP) regulated by the I1L intermediate promoter followed by a new J5R gene containing three tandem copies of the sequence encoding the flag epitope immediately after the N-terminal methionine as described for vA28iG93XFlag (42). Recombinant viruses were clonally purified by several rounds of fluorescentplaque isolation.…”

Poxvirus Proteomics and Virus-Host Protein Interactions

“…A serine protease inhibitor (SPI-3) encoded by K2 depends on the association with A56 for membrane localization (166). By direct interaction with the poxvirus EFC, the A56/K2 complex prevents virus entry and fusion and likely mediates the phenomenon of superinfection exclusion (167)(168)(169)(170). Additional viral PPI studies have shown that both A16 and G9, which are physically associated within the EFC, are required for binding to A56/K2 (170).…”