Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex

Abstract

Virus-mediated membrane fusion involves conformational changes of the viral fusion protein to fuse the opposing viral and host lipid bilayers. Unlike all other known viruses that contain a single fusion protein, poxviruses harbor a multimeric protein complex of 11 subunits, termed the entry fusion complex (EFC), to mediate fusion with host membranes. Yet, how the poxviral EFC mediates membrane fusion remains enigmatic. To establish the mechanism of EFC-triggered membrane fusion, we are deciphering the structure and function of individual EFC components. Here, we determined the crystal structure of the H2 ectodomain by X-ray diffraction, revealing a folded conformation comprising a central five-stranded β-sheet and three cladding α-helices. We reconstructed the full-length H2 by in silico prediction, revealing that the N-terminal region (amino acids 51–90) of H2 protein may fold as a long helix connecting the ectodomain and transmembrane region. Using alanine-mutagenesis screening in a transient complementation system, coimmunoprecipitation, isothermal titration calorimetry, and mature virion (MV)-triggered membrane fusion assays, we concluded that the surface of the ectodomain of H2 protein, including two loop regions, 170 LGYSG 174 and 125 RRGTGDAW 132 , constitutes a broad A28-binding region. Moreover, although not involved in A28 binding, the N-terminal helical region approximal to the transmembrane part, encompassing 64 RIK 66 , 72 W, and 83 ESDRGR 88 , is also crucial for viral EFC formation and MV infectivity. IMPORTANCE: Vaccinia virus infection requires virus-cell membrane fusion to complete entry during endocytosis; however, it contains a large viral fusion protein complex of 11 viral proteins that share no structure or sequence homology to all the known viral fusion proteins, including type I, II, and III fusion proteins. It is thus very challenging to investigate how the vaccinia fusion complex works to trigger membrane fusion with host cells. In this study, we crystallized the ectodomain of vaccinia H2 protein, one component of the viral fusion complex. Furthermore, we performed a series of mutational, biochemical, and molecular analyses and identified two surface loops containing 170 LGYSG 174 and 125 RRGTGDAW 132 as the A28-binding region. We also showed that residues in the N-terminal helical region (amino acids 51–90) are also important for H2 function.