[Show abstract][Hide abstract] ABSTRACT: Infectious poxvirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxvirus particles. In the case of vaccinia virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant virus particles. Furthermore, the morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.
Journal of Virology 08/2009; 83(18):9140-50. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.
The Journal of Cell Biology 10/2005; 170(6):971-81. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.
Journal of Virology 08/2005; 79(13):8046-56. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature.
[Show abstract][Hide abstract] ABSTRACT: Temperature-sensitive mutants of vaccinia virus, with genetic changes that map to the open reading frame encoding the F10 protein kinase, exhibit a defect at an early stage of viral morphogenesis. To further study the role of the enzyme, we constructed recombinant vaccinia virus vF10V5i, which expresses inducible V5 epitope-tagged F10 and is dependent on a chemical inducer for plaque formation and replication. In the absence of inducer, viral membrane formation was delayed and crescents and occasional immature forms were detected only late in infection. When the temperature was raised from 37 to 39 degrees C, the block in membrane formation persisted throughout the infection. The increased stringency may be explained by a mild temperature sensitivity of the wild-type F10 kinase, which reduced the activity of the very small amount expressed in the absence of inducer, or by the thermolability of an unphosphorylated kinase substrate or uncomplexed F10-interacting protein. Further analyses demonstrated that tyrosine and threonine phosphorylation of the A17 membrane component was inhibited in the absence of inducer. The phosphorylation defect could be overcome by transfection of plasmids that express wild-type F10, but not by plasmids that express F10 with single amino acid substitutions that abolished catalytic activity. Although the mutated forms of F10 were stable and concentrated in viral factories, only the wild-type protein complemented the assembly and replication defects of vF10V5i in the absence of inducer. These studies provide evidence for an essential catalytic role of the F10 kinase in vaccinia virus morphogenesis.
Journal of Virology 02/2004; 78(1):257-65. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An early step in vaccinia virus morphogenesis, the association of crescent membranes with electron-dense granular material, is perturbed when expression of the viral protein encoded by the A30L or G7L open reading frame is repressed. Under these conditions, we found that phosphorylation of the A17 membrane protein, which is mediated by the F10 kinase, was severely reduced. Furthermore, A30 and G7 stimulated F10-dependent phosphorylation of A17 in the absence of other viral late proteins. Evidence for physical interactions between A30, G7, and F10 was obtained by their coimmunoprecipitation with antibody against A30 or F10. In addition, phosphorylation of A30 was dependent on the F10 kinase and autophosphorylation of F10 was stimulated by A30 and G7. Nevertheless, the association of A30, G7, and F10 occurred even with mutated, catalytically inactive forms of F10. Just as A30 and G7 are mutually dependent on each other for stability, F10 was nearly undetectable in the absence of A30 and G7. The reverse is not true, however, as repression of F10 did not diminish A30 or G7. Interaction of F10 with A30 and G7 presumably occurred within the virus factory areas of the cytoplasm, where each was concentrated. F10 localized predominantly in the cortical region of immature virions, beneath the membrane where A17 is located. F10 remained associated with the particulate core fraction of mature virions after treatment with a nonionic detergent and reducing agent. The formation of protein complexes such as the one involving A30, G7, and F10 may be a mechanism for the regulated packaging and processing of virion components.
Journal of Virology 02/2004; 78(1):266-74. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vaccinia virus A30L protein is required for the association of electron-dense, granular, proteinaceous material with the concave surfaces of crescent membranes, an early step in viral morphogenesis. For the identification of additional proteins involved in this process, we used an antibody to the A30L protein, or to an epitope appended to its C terminus, to capture complexes from infected cells. A prominent 42-kDa protein was resolved and identified by mass spectrometry as the vaccinia virus G7L protein. This previously uncharacterized protein was expressed late in infection and was associated with immature virions and the cores of mature particles. In order to study the role of the G7L protein, a conditional lethal mutant was made by replacing the G7L gene with an inducible copy. Expression of G7L and formation of infectious virus was dependent on the addition of inducer. Under nonpermissive conditions, morphogenesis was blocked and viral crescent membranes and immature virions containing tubular elements were separated from the electron-dense granular viroplasm, which accumulated in large spherical masses. This phenotype was identical to that previously obtained with an inducible, conditional lethal A30L mutant. Additional in vivo and in vitro experiments provided evidence for the direct interaction of the A30L and G7L proteins and demonstrated that the stability of each one was dependent on its association with the other.
Journal of Virology 04/2003; 77(6):3418-29. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.
Journal of Virology 12/2001; 75(22):11222-6. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.
Journal of Virology 08/2001; 75(13):5752-61. · 5.08 Impact Factor