: Dengler, U.; Kresin, M.; Waschütza, G.; Otto, B.

Abstract
Poxviruses have evolved different strategies to counteract the host immune system .A soluble interferon-gamma receptor encoded in the vaccinia virus (strain WR) genome (ORF B8R) posesses a broad species specificity and neutralizes the bioactivity of human bovine, rat and rabbit interferon-gamma. The only exception so far is murine interferon-gamma which was not bound by the B8R protein. The aim of our project is to understand the different species specificity of human soluble interferon-gamma receptor (sIFNGR-1) and the Vaccinia Virus B8R protein. As a first approach we have build a model for the structure of the B8R protein based an the coordinates of the human sIFNGR-1 and the extracellular domain of human tissue factor (TF). Sequence alignments with CLUSTAL X showed that the B8R sequence only shares 20.8 per cent and 11.6 per cent identical positions with the sequences of sIFNGR-1 and TF respectively. Nevertheless secondary structure predictions for the B8R sequence suggested a high sheet content which is in accordance with the secondary structure content of the known type II cytokine receptors. The initial three-dimensional model was constructed with the software COMPOSER. Structurally conserved regions (SCRs) were defined through an alignment of B8R and sIFNGR-1 sequences using secondary structure dependent gap penalties and subsequent manual editing. In order to improve the resulting model several rounds of inspection, model rebuilding and energy minimization were applied using the programs PROCHECK BRAGI and SYBYL. As the amino acid identities observed in the alignments are rather low., now the cutrent model has to be validated by experimental data. Therefore we plan to carry out a biophysical characterization of the B8R protein. Currently we are working on an E.coli expression system of B8R. The gene was PCR-amplified from Vaccinia Virus WR infected VERO-cell cultures and cloned into an pTRCHIS B expression vector. B8R was produced as a fusion protein with a n N-terminal His-taq. Surface plasmon resonance (Biacore) experiments are planed to map the IFN-gamma binding site of B8R.