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

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.

Differential Innate Immune Signaling in Macrophages by Wild-Type Vaccinia Mature Virus and a Mutant Virus with a Deletion of the A26 Protein.

The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of the A26 protein from the WR strain mature virus generates a mutant, WRΔA26, that enters HeLa cells through plasma membrane fusion. Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type strain WR and the WRΔA26 mutant and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies, in which both HeLa cells infected with WR and HeLa cells infected with WRΔA26 expressed abundant viral late proteins, we found that WR expressed much less viral late protein than WRΔA26 in BMDM. Microarray analysis of the cellular transcripts in BMDM induced by virus infection revealed that WR preferentially activated type 1 interferon receptor (IFNAR)-dependent signaling but WRΔA26 did not. We consistently detected a higher level of soluble beta interferon secretion and phosphorylation of the STAT1 protein in BMDM infected with WR than in BMDM infected with WRΔA26. When IFNAR-knockout BMDM were infected with WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WR and, in turn, restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRΔA26 infection than to that from WR infection, whereas IFNAR-knockout mice were equally susceptible to WR and WRΔA26 infection, demonstrating that the ability of WRΔA26 to evade IFNAR signaling has an important influence on viral pathogenesis in vivoIMPORTANCE The vaccinia virus A26 protein was previously shown to mediate virus attachment and to regulate viral endocytosis. Here, we show that infection with strain WR induces a robust innate immune response that activates type 1 interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas infection with the WRΔA26 mutant does not. We further demonstrated that the differential activation of IFNAR-dependent cellular signaling between WR and WRΔA26 not only is important for differential host restriction in BMDM but also is important for viral virulence in vivo Our study reveals a new property of WRΔA26, which is in regulating host antiviral innate immunity in vitro and in vivo.