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. Author summary Vaccinia virus is a complex large DNA virus with a large number of viral membrane proteins to facilitate cell entry. Although it is well established that vaccinia mature virus uses endocytosis to enter cells, it remains unclear how it triggers membrane fusion in the acidic environment of endosomes. Recently, we hypothesized that A26 protein in vaccinia mature virus functions as an acid-sensitive membrane fusion suppressor, which suggests a novel viral regulation not present in other enveloped viruses. We postulated that conformational changes of A26 protein at low pH result in de-repression of viral fusion complex activity to trigger viral and endosomal membrane fusion. Here, we provide structural, biochemical and biological evidence demonstrating that vaccinia A26 protein does indeed function as an acid-sensitive fusion suppressor protein to regulate vaccinia mature virus membrane fusion during endocytosis. Our data reveal an important and unique checkpoint for vaccinia mature virus endocytosis that has not been described for other viruses. Furthermore, by isolating adaptive vaccinia mutants that escaped endocytic blockage, we discovered that mutations within the A26L gene serve as an effective strategy for switching the viral infection route from endocytosis to plasma membrane fusion, expanding viral host range.