In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121

Abstract

The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies

“…These include, for example, ankyrin repeat proteins, C-type lectins, immunoglobulin domain genes, and members of the N1R/p28 family (24). Furthermore, FP9 still possesses IFN-␥-binding (37) and putative interleukin 18-binding proteins, as well as chemokine-like proteins (21). The interpretation is hindered by incomplete understanding of the avian immune system, quite apart from the more complex question of the function of avian viral immunomodulators in a xenologous host.…”

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

“…The origins and propagation of FWPV FP9 (FP9), as well as the isolation and purification or rFWPV, were as described previously (4,26,28).…”

Avian Immunosuppressive Diseases and Immunoevasion

“…The avipoxviruses also encode multiple chemokinelike molecules (e.g., fpv060, fpv061, fpv116, fpv121). These appear to be secreted, although as yet no biochemical activity [194] has been attributed to any of them, partly because of the lack of identified chicken chemokine and chemokine receptor reagents [198]. It is therefore unclear whether they behave as agonists or antagonists and whether they target bona fide chemokine receptors or similar receptors for other host ligands.…”