| Rabies is a kind of viral encephalomyelitis for people or other warm-blooded animals, It can be infection and transmission in natural between domestic and wild animals, Once appear clinical symptoms after infection, the fatality rate is almost 100%. The pathogen is Rabies virus, which belong to the Lyssavirus genus of the Rhabdoviridae family. Rabies virus is compose of a negative strands of RNA and five structural protein, including the phosphorylated proteins(P), which was uncatalytic activity protein with multi-function to play an important role in the virus replication and transcription and interferon antagonism of virus. So far, although many studies have shown that the rabies virus P not only interact with its own structural protein, but also many proteins of host cells, and participate in the life cycle of the virus, but the relative data are quite limited, and pathogenesis of rabies virus is still far from understanding. This research employed the phage display technology screening direct interaction protein(ribosomal protein L9)with rabies virus P from the brain c DNA library after five rounds of biological elutriation.1.screening L9 In order to further understand the causes and pathogenesis of rabies virus, and to explore the more biological function of protein P, P gene was amplifyed from c DNA of rabies virus and cloned into expression vactor.P protein was expressed in E. coli BL21 and purified by Ni beats; Further, T7 phage c DNA library of the human brain has selected by phage display technology, after five rounds of selection, 13 candidate proteins,including L9, which could interact with P protein was identified by sequence analysis, Blast contrast, reading code calibration and ELISA assay.2.L9 and P protein interaction validationIn order to verify the 13 candidate proteinsif thy really interacted with rabies virus P, plasmids of expression GST-tagged fusion protein with all candidate proteins were constructed, and the proteins were expressed in E.coli BL21 and purified by GST beat, respetivly. DOT- pull- down assay showed that L9 has interacted with RABV- P strongly. Then the further experiment result of GST- pull- down was verified again that L9 direct interaction with RABV- P in vitro. To get rid of nonspecific in vitro environment cause the result of false positives, CO-IP assay identified that was RABV- P proteins interact with L9 in vivo. Where is interaction of P protein and L9 in the cell?In order to answer this question, the GFP-tagged P plasmid which express green fluorescent and the Ds Red-tagged L9 plasmid which express red fluorescent were constructed andco-transinfected into HEK293 T cells, and the results of confocal fluorescence microscope observation show that the P recruited L9 from nucleus to cytoplasm and located in the cytoplasm.It surggested that P and L9 may interact in the cytoplasm. In order to further confirm these results in the natural conditions can work, HEK-293 T cells were infected by RABV, and tested by indirect immunofluorescence staining and confocal fluorescence microscope observation, thd results show that the L9 with P protein is still located in the cytoplasm.3.determination the essential domain of L9 interact with PIn order to determine the crucial binding domain of P and L9 interaction, the protein pull-down assay was performed to a series of research. First of all, 8 truncated P proteins P gene P(19-297, P52-297, P82-297, P138-297, P172-297, P1-137, P1-180 and P1-218) were expressed, purificated, respectively, and then were done GST- pull- down assay with GST tagged L9 protein, The results showed that the 138-180 of P protein was essential domain interaction with L9. To further confirm the binding site, the Pâ–³ 138-180 proteinwhichdeletedthe centraldomain(residues 138-180) was constructed and analyzed by the pull-down assay. As expected, the Pâ–³ 138-180 protein could not pull down the L9 protein.It further verified the previous results. In order to determine the sites of L9 protein binding P, according to the L9 protein structure, we constructed and expressed L91-39, L91-61, L91-85, L962-192, L986-192, L997-192 truncated mutants. His- pull- down results show that the nterminal 39 aa of L9 has nothing to do with the interaction of P protein.4. Biology function of L9 to RABVOverexpression L9 in HEK293 T cel L, then detected RABV report gene expression levels that reflected influence of viral replication for L9, The results show that overexpression of L9 can significantly inhibit the replication of RABV(10 to 20 times); Western blot results show that the amount of P protein expression decreased 60-70% than control. In order to further confirm whether has specificity for L9 inhibit RABV replication, the growth curve of VSV were measured under overexpressed L9,and results showed that there was no significant difference in VSV of replication between overexpressed L9 and contrast.The above results show that the L9 expression can specifically inhibit RABV replication. So what was for RABV replication to reduce the expression of L9 or knockout L9? To answer this question, L9 specific si RNA and scramble si RNA were synthesis. The si RNA transfection into HEK- 293 t cells,then infected RABV, results show that the interference L9 can significantly enhance RABV copy(4-5 times); P protein expression quantity increased more than 50% control. The above results show that L9 has an important regulatory role to the replication of RABV.5. L9 interaction with RABV effaction the transcription of RABVTo investigate which step of the viral cycle is affected by P-L9 interaction, RNA and protein translation analysis in the presence of cycloheximide were performed by q RT-PCR and Western blotting, respectively. The q RT-PCR results show that viral genomic RNA and m RNA copy numbers in L9-overexpressing cells were both significantly reduced(31.5-76.2% and 38.6-70.2%, respectively) compared with the controls whether treated or not treated with CHX from 6 to 24 h. Furthermore, the Western-blotting results show that viral translation was totally inhibited by CHX and the expression of P was inhibited by overexpressed L9 in the control without CHX treatment. As a control, the level of VSV N m RNA was unaffected in L9-overexpressing cells treated with or without CHX from 6 to 24 h. Overall, our results show that the initial transcription and replication of RABV are affected by P-L9 interaction. |
PDF Full Text Request