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Disulfide bond formation at the C termini of vaccinia virus A26 and A27 proteins does not require viral redox enzymes and suppresses glycosaminoglycan-mediated cell fusion.

ABSTRACT: Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

SUBMITTER: Ching YC

PROVIDER: S-EPMC2698572 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vaccinia virus envelope protein A27 has multiple functions and is conserved in the Orthopoxvirus genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21-84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel ?-helices and one unusual antiparallel ?-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner in vitro in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly in vitro. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.

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Disulfide bond formation at the C termini of vaccinia virus A26 and A27 proteins does not require viral redox enzymes and suppresses glycosaminoglycan-mediated cell fusion.

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