The Ectodomain Of Rabies Virus Glycoprotein Determines Dendritic Cells Activation

Rabies, caused by rabies virus (RABV), is an infectious zoonosis with nervous system destroyed. Every year, around 59 000 people die of rabies all over the world, and China belongs to the countries with high incidence of rabies.RABV has a non-segmented, single-strand negative-sense RNA genome which is approximately 12kb,comprising five genes which encode the nucleoprotein (N),phosphoprotein (P), matrix protein (M), glycoprotein (G), and a large RNA-dependent RNA polymerase (L) in the order of 3,-N-P-M-G-L-5’. The transmembrane G protein is the sole protein distributed on the surface of the virion, and G protein is the crucial component in development of virus-neutralizing antibodies (VNA). It has been reported that DCs (Dendritic cells) activation is RABV G protein specific. Attenuated RABV can induce high level DCs activation and induce the production of VNA, but street RABV fails to activate DCs or induce VNA production.Previous studies show that the inability of the wild-type (wt) rabies virus (RABV) to activate DCs and escape immune system have been attributed to its glycoprotein (G),particularly to their ability to bind/ enter into dendritic cells (DCs). However, the domain responsible for G mediated immune evasion is not clear. To be further, it has been reported repeatedly that the G expression level of wt RABV is much less than that of lab-attenuated strains, but it remains unknown whether low G protein on wt RABV is necessary for this immune evasion.In the present study, attempts were made to elucidate glycoprotein domain(s)involved in differential DCs activation using lab attenuated B2c and wt-SHBRV strain. To this end, we have constructed and characterized recombinant chimeric RABV viruses expressing each structural domain such as signal peptide (sp),ectodomain (et),transmembrane (tm), cytoplasmic tail (ct) of B2c glycoprotein (G) was replaced by that of wt-SHBRV G, respectively. Characterization of recombinant RABVs show that the viruses containing SHBRV sp,tm and ct are capable growing in high titer by efficient cell to cell spreading, whereas virus containing SHBRV G gene or its ectodomain was inefficient in cell to cell spreading and grew in lower levels. Thus, the ectodomain of G protein determines the invasion ability into cells and replication level of RABV.To investigate which domain regulates G protein expression, all rRABV were co-incubated with NA or BSR cell, the analysis of level of G expression in the infected cells or on the cell surface indicate that the G quantity of rRABV with the Gct of SHBRV is significantly less than that of B2c. Therefore, the level of G expression is regulated solely by the cytoplasmic tail.Meanwhile, FACS analysis of immune activation by the recombinant RABVs show that the viruses containing SHBRV G gene or et loss the ability to activate DCs. Although the viruses with low G level do not remain the same DCs activation, it is not significantly compared with B2c. In addition, the viruses containing SHBRV sp and tm are efficient in binding to and activation of DCs. On the other hand, rRABVs with Get from SHBRV are incompetent in binding to DCs. The ectodomain of RABV G protein determines DCs binding and so regulates DCs activation, indicating the level of G expression and DCs activation are two separable functions.Together, our results demonstrate that the G mediated DC activation is regulated by the ectodomain which determines the efficiency of virus spread and binding to DCs. Thus,the ability of the RABV to activate DCs depend upon the glycoprotein ectodomain’s ability to facilitate virus binding to DCs, rather than the G protein expression level.