Study On The Function Of Rabies Virus Leader RNA And Its Host Interacting Protein

Rabies is caused by the rabies virus,people and all the warm-blooded animals are susceptible and can spread the virus.It continues to be a huge threat to human health because the disease is widespread in the world,and once the disease,the mortality rate is almost 100%.More than 59,000 people die each year from rabies,and most of them occur in developing countries in Asia and Africa.Rabies virus belongs to the Rhabdoviridae family,the virus genome encods five structural proteins,the virus in the process of transcription not only produce the five structural protein mRNA,but also produce a non-coding RNA that 5 'end without cap structure,3' without Poly A tail called leader RNA.LeRNAs in RABV infected BHK-21 cells have different lengths,usually terminating at positions 55 through 58 nt,and the number of 56 nt leader RNA is twice that of 58 nt.And the results were the same when detected at 24 hours post-infection,but the amount of leader RNA was less than 12 hours after infection.In addition,the termination positions at 43 nt and 60 nt have also been found.Both the nucleoprotein of rabies virus and the vesicular stomatitis virus can interact with the leRNA to influence the transition between viral transcription and replication,and determine whether the virus is mainly transcribed or replicated.In rabies virus infection,rabies virus nucleoprotein interacts with the leRNA preferentially than with nucleoprotein mRNA,and with other non-viral RNA.When nucleoprotein and phosphoprotein are expressed at the same time,phosphoproteins reduce the ability of nucleoprotein to bind to other RNAs,but do not affect nucleoprotein binding leader RNA.However,the role of the leRNA and the regulation mechanism of the leRNA expression is not cleared.The aim of this experiment is to study the generation and function of the leRNA and the relationship with the host.In our study,rabies virus wild-type strain DRV-AH08 was used as the research object to study the leRNA of rabies virus.A more precise RACE technique was used to determine the length of RABV leRNA in the brain of mice infected with RABV DRV-AH08 and it was found that the length of leRNA varies from 40 to 79 nt,among which the most frequent size is 64 nt.To further validate the length of leRNA we extracted the total RNA from DRV-AH08-infected SK-N-SH cells at the early stage of infection,another method was used to construct leRNA library and to measure the length of different leRNA using high-throughput sequencing.Again,64 nt long RNA was found to be one of the most prominent variants.Although the length of the leRNA was different,we predicted that the secondary structure of the leRNA of 56 nt,58 nt and 64 nt found that their secondary structures were similar.In order to further study the function of the leRNA,we use the human adenovirus expression vector to overexpress the leRNA in vitro and in vivo.Firstiy,cells were transfected with expression vectors for different length of leRNA,and then infected with different doses of rabies virus(0.01 MOI and 0.1MOI)found that 56 nt,58 nt and 64 nt length of the leRNA can inhibit rabies virus infection.And in vitro synthesized 64 nt leRNA was directly transfected into SK-N-SH cells to test the inhibitory effect of leRNA on rabies virus infection.Then we injected the human adenoviruses expressing the leRNA and the control RNA into the left and right hippocampus of the same mouse through the three-dimensional stereotactic injection systerm,then challenged with the rabies virus,and finally found that the leRNA still inhibits rabies virus infection in vivo.We also confirmed in SK-N-SH cells that the genomic RNA of rabies virus was significantly reduced when overexpression of leader RNA.Through the RNA immunoprecipitation assay we found that the overexpression of leader RNA,can inhibit the rabies virus nucleoprotein and genomic RNA binding.This suggests that the leRNA can inhibit the replication of rabies virus by inhibiting the binding of the nucleoprotein to the genomic RNA.RNA and protein interaction experiments confirmed that the host heat shock cognate 70 KD protein,Hsc70,can interact with the leRNA,this interaction is a direct interaction.Hsc70 and leRNA interactions 3D modeling results show that the Hsc70 binding region binds to the leRNA 51 nt-59 nt where is AU-enriched region.Inhibition of Hsc70 expression can promote elevated levels of leRNA.While Hsc70 expression levels decreased,will lead to reduced replication of rabies virus.In contrast,rabies virus infection also affects the expression of Hsc70,2 hours after rabies virus infection,Hsc70 levels were significantly decreased,but 36 hours post-infection,Hsc70 level was significantly increased.This different change prompted us to further study the relationship between Hsc70,leRNA and viral genomic RNA.We found that rabies virus infection will inhibit the production of Hsc70 at the early stage of infection,resulting in increased leRNA level,thereby inhibiting the virus replication,and in the late stage of infection,Hsc70 expression level was increased,leRNA level decreased,the virus rised sharply from 24 h post-infection.Similar phenomenon(autoregulation of virus replication)has also been found in other viruses.Since over-expression of leRNA specifically inhibits RABV replication in vitro and in vivo,the potential to use leRNA as a novel drug in rabies post-exposure prophylaxis was determined.Experimental result demonstrates that leRNA could inhibit DRV-AH08 replication post-infection both in vitro and in vivo,implying a potential application of leRNA in rabies post-exposure prophylaxis.