Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor

Abstract

Native rabies virus glycoprotein (RVGvir) is a trimeric, membrane-anchored protein that has been shown to interact with the p75 NTR neurotrophin receptor. In order to determine if the RVG trimeric oligomerization state is required for its binding with p75 NTR , different soluble recombinant molecules containing the entire RVG ectodomain (RVGect) were expressed alone or fused at its C terminus to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon'. The oligomerization status of recombinant RVG was investigated using sedimentation in sucrose gradient and p75 NTR binding assays. It was found that, in the absence of the fibritin foldon, recombinant RVGect forms unstable trimers that dissociate into monomers in a concentration-dependent manner. C-terminal fusion with the foldon induces stable RVG trimerization, which is concentration-independent. Furthermore, the fibritin foldon maintains the native antigenic structure of the carboxy part of RVGect. Cell binding experiments showed that RVG trimerization is required for efficient interaction with p75 NTR . However, the exact mode of trimerization appears unimportant, as trimeric recombinant RVGect (fused to the fibritin foldon) and RVGvir both recognize p75 NTR with similar nanomolar affinities, as shown by surface plasmon resonance experiments. Altogether, these results show that the C-terminal fusion of the RVG ectodomain with the fibritin foldon is a powerful way to obtain a recombinant trimeric native-like structure of the p75 NTR binding domain of RVG.

The Rabies Virus Glycoprotein Receptor p75 NTR Is Not Essential for Rabies Virus Infection

“…Consistent with this view, we have demonstrated that RVG trimerization is required for p75 NTR interaction (46) and that RVG interacts with the oligomeric but not with the monomeric form of p75 NTR (C. Tuffereau, unpublished results). These results suggest the need for a 3-to-3 complex for efficient binding.…”

“…First, p75 NTR might require a cofactor that is not found in these neurons. This is unlikely, because we have shown that a soluble form of p75 NTR is able and sufficient to interact with RV and RVG on its own (31,46) and its affinity constant is 2 nM (46). Second, binding of RV is prevented, and there are several molecules which are tightly associated with p75 NTR and could modify its binding affinity (14).…”

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

“…Also, the G glycoprotein from rabies virus is a relatively complex trimeric, membrane-anchored protein (Sissoëff et al 2005), and the expression of membrane proteins is not a simple process (Grisshammer 2006). In the case of transmembrane proteins, folding occurs in three different environments: the endoplasmic reticulum (ER) lumen, the ER membrane and the cytosol.…”

Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co‐transfected cell line

“…It has been shown that G can be found in three different states, a "native" state, an activated hydrophobic state and a fusion inactive conformation. Antigenic differences between these forms have been reported [10][11][12]. The immune protection mounted during rabies vaccination is considered to be very much dependent on the virus G, inducing virus neutralizing antibodies, production of T helper, cytotoxic T cells and protection against intracerebral challenge [13][14][15][16].…”