A coronavirus (CoV) has been shown to be the etiologic agent of the severe acute respiratory syndrome (SARS) epidemic, which affected about 30 countries in late 2002. The viral genome is almost 30 kb in length and contains at least 11 open reading frames, whereas the exact number depends on the strain and the minimal count of coded amino acid residues. Coronaviruses are positive-strand RNA viruses that code for the characteristic proteins replicase (R), spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. In addition, SARS-CoV codes for subgroup-specific accessory proteins that are thought to be dispensable for viral replication in cell culture, but may be important for virus-host interactions and thus contribute to the virus' fitness. The important role of these so-called "accessory" proteins for viral infectivity, replication efficiency and pathogenic effects is well established and investigated.
The accessory protein 7a of SRAS-CoV- structure based prediction of function
As was also found by others, SARS CoV accessory proteins does not show any significant sequence homology to other proteins in the data bases. Therefore, no obvious function could be derived from any sequence similarity to proteins with known function. Structure based predictions of functions on the basis of similarities to proteins with known functions have been successfully used in the past, and such kind of approach is a major driving force for structural genomics projects. In order to identify potential functions we use the solution structure to search for proteins with similar three-dimensional structures.
The structure of protein 7a:
The protein 7a (figure 1) is a type I membrane protein composed of 122 AA, with the amino-terminal hydrophilic domain (residues 16-98) oriented inside the lumen of the ER/Golgi or on the surface of the cell membrane or virus particle, depending on the localization of the protein.
The well structured part of 7a ectodomain is built up from seven beta-strands so that four strands form one beta sheet and three strands form a second. The sheets are closely packed or ‘‘sandwiched'' against each other. Each sheet is amphipathic with the hydrophobic side facing inward. The larger four-stranded beta-sheet consists of strands A, G, F, and C, the smaller three-stranded beta-sheet consists of strands B, E, and D. All beta-strands align in anti-parallel fashion, with the exception of strand A, which aligns parallel to strand G. Two disulfide bonds link both sheets on opposite edges therefore stabilizing the beta-sandwich structure (immunoglobulin (Ig) like fold). Residues 66-84 appear to be unstructured.
Because 7a does not reveal significant sequence homologies to proteins in the data bases, we carried out a structure based similarity search for proteins with known function. High structural similarity to D1 domains of ICAM-1 and ICAM-2, and common features in amino acid sequence between 7a and ICAM-1, suggest 7A to possess binding activity for the α L integrin I domain of LFA-1 (lymphocyte function associated antigen 1). ICAM-1 and ICAM-2 are known to specifically interact with LFA-1 (also known as CD11a/CD18, aLb2 integrin) that is expressed exclusively on leucocytes.
SARS-CoV accessory protein 7a directly interacts with human LFA-1:
To prove or disprove the structure based prediction that SARS-CoV protein 7a binds to the I domain of the αL subunit of LFA-1, we carried out a series of experiments using recombinant protein 7a and recombinant wild type and mutant LFA-1 I domain, as well as LFA-1 expressing Jurkat cells.
We demonstrated that protein 7a binds directly and specifically to human lymphocyte function-associated antigen 1 (LFA-1) on the cell surface of Jurkat cells. The binding of 7a to the cell surface is concentration-dependent and saturable and the affinity increases after cell stimulation with Phorbolester (PDBu). PDBu stimulates the cells and brings LFA-1 in an active conformation with high affinity to the ligand. A mutant K287C/K294C (Shimaoka et al. 2003) of I domain copy the activated state of LFA-1 and the wild typ I domain represents the inactive state of LFA-1. The binding are confirmed by direct in vitro binding of recombinant protein 7a to wild type and mutant K287C/K294C I domain showing that the I domain is the 7a binding site in the αL chain of LFA-1.
Wild type (open circles) I domain bound 7a with a virtually linear concentration dependence (figure 2). Up to 20 mM 7a, no saturation effect could be observed. Mutant I domain (K287C/K294C, closed circles), as well, bound 7a in a strong concentration-dependent manner, but in contrast to wild type mutant I domain binding to protein 7a reached saturation at 7a concentrations above 5 mM. This clearly shows that 7a binds significantly tighter to mutant I domain as compared to wild type I domain. This is in full accordance with the 7a binding characteristic observed with activated and non activated Jurkat cells.