Vaccinia virus is a large double-stranded DNA virus closely related to variola virus, the causative agent of smallpox. Although a concerted campaign of vaccination successfully eradicated smallpox over 20 years ago, the re-emergence of smallpox in society by its deliberate release and/or the emergence of monkeypox virus that can also infect humans pose a serious threat to human health.
While it is known that poxviruses enter the host cell by the action of a large, multi-protein membrane-fusion machine, the details of how this machine operates are poorly understood. At least nine proteins have been identified as interacting in the context of a membrane fusion machine and are essential to successful viral entry: A28, H2, A21, L5, G3, A16, G9, J5, and F9. Biochemical techniques have shown that these proteins form a fusion complex (biotin-tags and co-immmunoprecipitation, B. Moss personal communication). These proteins all possess an N-terminal transmembrane helix, similar disulphide bonding patterns and are conserved across the poxviruses. They are expressed at the same time during the viral lifecycle and a viral encoded redox pathway ensures that correct disulphide bond formation occurs within the viral factories where the IMVs are assembled.
This project aims to determine the structure of the vaccinia host-cell fusion machine with a view to designing prophylactics that would prevent entry of vaccinia into host cells. This project has strong focus upon protein-protein interactions and developing techniques in co-transfection and co-expression in mammalian cells, active areas of current OPPF development.
STRUBI have developed a mammalian transient expression system in 293T cells that is rapid and produces protein suitable for structure determination experiments, including SeMet labelled material (Butters et al. 1999 and unpublished results). This technology is well suited to producing the viral fusion proteins. By adding a signal peptide to the sequence, these proteins can be targeted for secretion, ensuring the correct disulphide bond formation. Oxford has initial results for A28 expressed in 293T cells that shows good expression and very clean production of deglycosylated protein (Figure 1 shows the effective deglycosylation of A28 by Endo H).
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 346
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Jonathan M Grimes | Structural Biology | Oxford University | UK | jonathan@strubi.ox.ac.uk |
| Prof David Stuart FRS | Structural Biology | Oxford University | UK | stuart-pa@strubi.ox.ac.uk |
2006. Poxvirus entry and membrane fusion. Virology, 344 (1), pp. 48-54. Read abstract | Read more
The study of poxvirus entry and membrane fusion has been invigorated by new biochemical and microscopic findings that lead to the following conclusions: (1) the surface of the mature virion (MV), whether isolated from an infected cell or by disruption of the membrane wrapper of an extracellular virion, is comprised of a single lipid membrane embedded with non-glycosylated viral proteins; (2) the MV membrane fuses with the cell membrane, allowing the core to enter the cytoplasm and initiate gene expression; (3) fusion is mediated by a newly recognized group of viral protein components of the MV membrane, which are conserved in all members of the poxvirus family; (4) the latter MV entry/fusion proteins are required for cell to cell spread necessitating the disruption of the membrane wrapper of extracellular virions prior to fusion; and furthermore (5) the same group of MV entry/fusion proteins are required for virus-induced cell-cell fusion. Future research priorities include delineation of the roles of individual entry/fusion proteins and identification of cell receptors. Hide abstract
2005. Poxvirus multiprotein entry-fusion complex. Proc. Natl. Acad. Sci. U.S.A., 102 (51), pp. 18572-7. Read abstract | Read more
Poxviruses have evolved elaborate mechanisms for cell entry, assembly, and exocytosis. Recently, four vaccinia virus membrane proteins, namely A21, A28, H2 and L5, were reported to be necessary for cell entry and virus-induced cell-cell fusion but not for virion morphogenesis or attachment of virus particles to cells. Using immunoaffinity purification followed by mass spectrometry, we now show that these four proteins as well as four additional previously uncharacterized putative membrane proteins (A16, G3, G9, and J5) form a stable complex. These proteins fall into two groups: A21, A28, G3, H2, and L5 have an N-terminal transmembrane domain, 0-2 intramolecular disulfide bonds, and no sequence similarity, whereas A16, G9, and J5 have a C-terminal transmembrane domain and 4-10 predicted disulfide bonds and are homologous. Studies with conditional-lethal null mutants indicated that the viral membrane was crucial for assembly of the complex and that the absence of individual polypeptide components profoundly decreased complex formation or stability, suggesting a complicated interaction network. Analysis of purified virions, however, demonstrated that the polypeptides of the complex trafficked independently to the viral membrane even under conditions in which the complex itself could not be isolated. All eight proteins comprising the entry-fusion complex are conserved in all poxviruses, suggesting that they have nonredundant functions and that the basic entry mechanism evolved before the division between vertebrate and invertebrate poxvirus species. Hide abstract
2006. Vaccinia virus F9 virion membrane protein is required for entry but not virus assembly, in contrast to the related L1 protein. J. Virol., 80 (19), pp. 9455-64. Read abstract | Read more
All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively. Hide abstract