Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus

Abstract

Genes encoding fowlpox virus (FWPVAvipoxviruses, which replicate only in avian cells, have been developed as safe recombinant vectors for human vaccination (23,30). In mammalian cells, avipoxvirus replication is blocked but early gene expression occurs (45,46). Late gene expression occurs in some mammalian cell types infected by fowlpox virus (FWPV), the prototypic avipoxvirus, without productive virus replication, as morphogenesis appears to be blocked (42). FWPV has been much less intensively studied than vaccinia virus (VACV). It appears to form extracellular enveloped virus (EEV) by budding rather then wrapping (6,16 (27). The complete sequence of a pathogenic strain of FWPV (1) includes genes encoding homologs of at least 21 known VACV structural proteins, but FWPV appears to lack homologs of intracellular mature virus (IMV) proteins encoded by A27L and D8L as well as an intracellular enveloped virus protein encoded by A36R and the EEV proteins encoded by B5R, A33R, and A56R (1).Although genes encoding FWPV structural proteins have been identified by sequence homology, very little is known about the proteins they encode. We produced monoclonal antibodies (MAbs) and polyclonal antibodies against FWPV proteins as markers to study FWPV morphogenesis and identified genes encoding three FWPV immunodominant structural proteins. MATERIALS AND METHODSVirus and cells. The origins, propagation, and purification of FWPV strains (FP9, HP1, and Poxine) and canarypox virus (CNPV) have been described previously (5).MAbs. BALB/c mice were immunized twice intraperitonally 3 weeks apart with a crude preparation of Poxine in Quil A, a saponin-derived adjuvant (Superfos, Vedbaek, Denmark). For the second fusion, mice were injected twice 4 weeks apart with 100 g of sucrose gradient-purified FP9 virus (5). Hybridoma supernatants were screened by enzyme-linked immunosorbent assay or by indirect immunofluorescence assay, with Poxine-infected or noninfected chicken embryo fibroblasts (CEFs) as the antigen. The 3D9 MAb directed against the 39-kDa protein was kindly provided by C. P. Czerny.Polyclonal sera. Chicken hyperimmune sera against FP9, Poxine, or CNPV were produced as described previously (5).Radioimmunoprecipitation. Infected (multiplicity of infection, 10) or uninfected CEFs were incubated in methionine-free medium containing 2% dialyzed fetal calf serum. Two hours later, the medium was replaced with fresh medium containing 1.85 MBq of [ 35 S]methionine/ml and incubated for 2 h. The medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and lysed in 2 ml of radioimmunoprecipitation assay (RIPA) buffer (2.5 mM Tris [pH 7.2], 0.15 M NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% sodium deoxycholate). Lysates were centrifuged for 15 min (Eppendorf centrifuge), and supernatants were stored at Ϫ70°C.Ascitic fluid (10 l) or hybridoma supernatant (500 l) was incubated for 1 h at 4°C with 50 l of 10% protein A-Sepharose CL-4B (Pharmacia) in 0.1 M sodium phosphate buffer, pH 8.1. After...

Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection against Lethal Challenge in Mice

“…(A separate group possibly studied the same protein but misidentified it as the VV IHD H6R gene product [25,26]). Similarly, antibodies to the orthologs of H3L are also seen after infection with other orthopoxviruses, including orf virus, capripox virus, and fowlpox virus (8,28,33). The H3L gene is strongly conserved among the orthopoxviruses.…”

Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

“…However, the protein has been shown to be present in IMV and EV, but, unlike Fpv140, Fpv168, and Fpv198, the protein does not colocalize with viral factories in FWPV-infected cells (3). The origins, nature, and relationships of this protein and its orthologs have been considered in considerable detail elsewhere (3,30). The manner of association of Fpv191 with FWPV IMV is unclear but is probably equiv- alent to the IMV association of VACV proteins p4c and A27 (as they do not colocalize with virus factories but bind after formation of IMV [41,44,49], involving protein-protein interactions with other VACV IMV membrane proteins, such as A17 [23]).…”

“…However, the protein has been shown to be present in IMV and EV, but, unlike Fpv140, Fpv168, and Fpv198, the protein does not colocalize with viral factories in FWPV-infected cells (3). The origins, nature, and relationships of this protein and its orthologs have been considered in considerable detail elsewhere (3,30).…”

“…Fpv140 contains a C-terminal hydrophobic sequence but lacks a signal sequence, and the protein is expected to be incorporated posttranslationally into the IMV membrane via the Cterminal hydrophobic domain, as is the VACV H3 protein (12). Like VACV H3 (29), Fpv140 contains potential heparan sulfate binding sites (3), which in VACV allow IMV entry. However, no interaction between H3 and kinesin has been reported for VACV.…”

Genetic Screen of a Library of Chimeric Poxviruses Identifies an Ankyrin Repeat Protein Involved in Resistance to the Avian Type I Interferon Response

“…MAb DE9 recognizes the 63-kDa FWPV equivalent of VACV p4c, encoded by fpv191 (38), located in fragment 50 of the chimeric library. Three independent chimeras (MVA-f50a, -b, and -c) were isolated following separate transfections with fragment 50.…”