Vaccinia viral A26 protein is a fusion suppressor of mature virus and triggers membrane fusion through conformational change at low pH

Abstract

Vaccinia mature virus requires A26 envelope protein to mediate acid-dependent endocytosis into HeLa cells in which we hypothesized that A26 protein functions as an acid-sensitive membrane fusion suppressor. Here, we provide evidence showing that N-terminal domain (aa1-75) of A26 protein is an acid-sensitive region that regulates membrane fusion. Crystal structure of A26 protein revealed that His48 and His53 are in close contact with Lys47, Arg57, His314 and Arg312, suggesting that at low pH these His-cation pairs could initiate conformational changes through protonation of His48 and His53 and subsequent electrostatic repulsion. All the A26 mutant mature viruses that interrupted His-cation pair interactions of His48 and His 53 indeed have lost virion infectivity. Isolation of revertant viruses revealed that second site mutations caused frame shifts and premature termination of A26 protein such that reverent viruses regained cell entry through plasma membrane fusion. Together, we conclude that viral A26 protein functions as an acid-sensitive fusion suppressor during vaccinia mature virus endocytosis.

Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex

“…The wild-type Western Reserve (WR) strain of vaccinia virus, the A26L deletion virus (WRΔA26), WRΔA26-A4mCherry, and VTF7-3 (ATCC VR-2153) were propagated in BSC40 cells, and virus titers were determined by plaque assays as described previously (27, 28, 50). The generation of HeLa cells expressing GFP or RFP was performed as described previously (27,29). Anti-H3 (4), anti-D8 (5), anti-G3 (10), anti-A4 (51), anti-L1 (28), anti-G9 (28), anti-L5 (28), and anti-A16 (28) antibodies were all described previously.…”

“…The MV-specific A26 protein binds to individual A16 and G9 proteins of the EFC and suppresses EFC fusion activity at neutral pH (27,28). Only upon MV being internalized into endosomes can the acidic environment of these organelles trigger conformational changes in the A26 protein to induce EFC fusion activation and subsequent viral membrane fusion with endosomes, releasing viral cores into the cytoplasm (29). Unlike A26, the A56/K2 protein complex is expressed on cell surfaces in the early stage of virus infection to inhibit EV-mediated fusion to infected cells in a process termed "superinfection interference" (25,(30)(31)(32)(33)(34)(35).…”

“…Both A26-mediated and A56/K2-mediated fusion inhibition are sensitive to acid conditions, which induce the dissociation of these viral fusion suppressor proteins from the EFC and prompt "acid-induced membrane fusion." The crystal structure of the A26 protein was recently resolved (29), showing that when residues His48 and His53 of the N-terminal ␣2-helical domain become protonated at low pH, they form His-cation pairs with nearby basic amino acids that induce acid-dependent repulsion to create the conformational changes that result in fusion activation. Further in vitro mutagenesis and mutant virus characterization clarified the molecular mechanism by which MV undergoes acid-induced membrane fusion (29).…”

“…The crystal structure of the A26 protein was recently resolved (29), showing that when residues His48 and His53 of the N-terminal ␣2-helical domain become protonated at low pH, they form His-cation pairs with nearby basic amino acids that induce acid-dependent repulsion to create the conformational changes that result in fusion activation. Further in vitro mutagenesis and mutant virus characterization clarified the molecular mechanism by which MV undergoes acid-induced membrane fusion (29). In contrast, it had been unclear how the A56/K2 protein complex mediates membrane fusion inhibition and if acid conditions trigger similar conformational changes of A56/K2 to abrogate the inhibition of EV membrane fusion.…”

LINE-1 retrotransposons facilitate horizontal gene transfer into poxviruses

“…Non-essential (Blasco and Moss, 1991) HGT14 H4L Essential (Kane and Shuman, 1992) HGT15 C19L/B25R Non-essential (Perkus et al, 1991) HGT16 A26L Non-essential (Howard et al, 2008, Chang et al, 2019 HGT17 G6R Non-essential (Senkevich et al, 2008) HGT18 D6R Essential (Broyles and Fesler, 1990) HGT19 D11L Essential (Seto et al, 1987)…”