Wild Type Rabies Virus Strategies For Evading Dendritic Cells Recognitions

Despite the fact that rabies is one of the oldest human infectious diseases, it continues to present public health threat by causing more than 55,000 human deaths every year around the globe. Its causative agent, rabies virus (RABV), belongs to the Rhabdoviridae family and its genome encodes five structual proteins. Among these, RABV glycoprotein (G) is the only viral protein that is glycosylated and exposed on the surface of the virion. RABV G is responsible for binding to cell receptors and fused with cellular membranes to release viral genome. Moreover, RABV G is the only protein capable of inducing virus neutralizing antibodies (VNA) that are protective against rabies.It has been known for a long time that most of the human rabies patients (80%) do not develop VNA at the time of death. The inability of wild-type (wt) RABV to induce VNA has also been reported in other animal species. Recent studies indicate that lab RABV activates, while wt RABV evades, the host innate immune responses. The evasion of innate immunity by wt RABV has been correlated with its low level of G protein expression. It was found that over-expression of innate immune genes stimulated higher levels of VNA production and provided better protection by activating more dendritic cells (DCs) than the parental virus. It has been reported that RABV activates DC and induces type I IFN production in an IPS-1 dependent manner. However, all these studies were performed with laboratory-attenuated RABV.To determine the roles of DC activation in RABV immunogenicity, adoptive transfer of DCs treated with RABVs was performed.50% of the mice transferred with DCs primed with lab RABV survived the challenge, whereas only 20% of the mice resisted the challenge when transferred with mock-primed DCs or DCs primed with wt RABV. In mice survived, VNA was higher than 0.75 IU/ml and no RABV was found in the brain. To further assess the immune responses, immune cells in spleen and mesenteric lymph nodes were analyzed. There were significantly more activated DCs, B cells, and plasma cells in mice transferred with DCs primed with laboratory-attenuated RABV than in those transferred with mock-primed DCs or DCs primed with wt RABV. Altogether, these results demonstrate the critical roles of DC activation in RABV immunity and protection.To investigate the mechanism(s) by which step of RABVs infection DCs are activated with laboratory-attenuated RABV, virus binding to, entry into, and fusion with DCs were investigated after infection with the RABVs. Cells surface-bound viruses and those uptaken by cells were detected by FACS and qRT-PCR. Significantly more RABVs were detected in DCs treated with laboratory-attenuated RABVs or RABV expressing the G from laboratory-attenuated RABV than wt RABV or laboratory-attenuated RABV expressing the G from wt RABV. The next crucial steps after virus entry are associated with virus fusion with cellular endosomes and virus uncoating. Results from low pH induced membrane fusion indicate that only the G from laboratory-attenuated, but not from wt RABV, has the abilities to fuse efficiently with endosomes. Western blotting was used to detect the level of G expression and electron microscopy used to evaluate the G incorporation into the virions. It was found that laboratory-attenuated RABVs or RABV expressing the G from laboratory-attenuated RABV expressed higher level of G and incorporated more G molecules in to the virions than wt RABV or laboratory-attenuated RABV expressing the G from wt RABV. Overall, these results confirm that the G from laboratory-attenuated mediates more efficient binding to, entry into, and fusion with host cells than the G from wt RABV.It has been shown that DC activation is due to recognition of viral RNA, particularly leader RNA, by RIG-1. To determine if RABV leader RNA is associated with DC activation, the level of leader RNA in DCs were determined by using qRT-PCR. Significantly higher level of leader RNA were detected in DCs infected with laboratory-attenuated RABVs or RABV expressing the G from laboratory-attenuated RABV than in DCs infected wt RABV or laboratory-attenuated RABV expressing the G from wt RABV. To further understand the role of leader RNA in DCs activation, synthesized leader RNAs from laboratory-attenuated or wt RABV were used to transfect DCs. Interestingly, both leader RNAs were capable of inducing DC activation. Analysis of the minimum free energy secondary structure indicates that both leader RNA have similar hairpin structures.In summary, our studies found that only laboratory-attenuated, but not wt, RABV is capable of activating DCs in vitro and in vivo. Consequently, mice passively transferred with DCs primes with laboratory-attenuated, but no DCs primed with wt, RABV were protected against lethal challenge. Furthermore the inability of wt RABV to activate DCs is due to the low level of its G expression and fewer G molecules incorporated into the virions. As a result, wt RABV is less capable of binding to, entering into and fusing with DCs than laboratory-attenuated RABV. Consequently, transcription of leader RNA was severely reduced in DCs infected with wt RABV. Thus it is the level of G expression ultimately determines the status of DC activation by different RABVs.