Study On The Mechanism Of Snakehead Vesiculovirus Leader RNA Promoting Virus Proliferation

The infection of the snakehead vesiculovirus?SHVV?has caused significant economic losses in Chinese aquaculture industry,and there are no effective prevention measures to control.The non-coding RNA of the virus plays an important regulatory role during viral infection.Therefore,discovering the non-coding RNA of the virus and elucidating its molecular mechanism regulating viral proliferation is an important basis for revealing the pathogenic mechanism of the virus to prevent and treat the viral disease.In current study,we established the SHVV reverse genetic system and identified that SHVV can produce a non-coding RNA called Leader RNA during infection.The discovery of Leader RNA can promote SHVV proliferation by constructing a Leader RNA deletion mutant and overexpressing the Leader RNAs.Further studies revealed that Leader RNA interacts with viral N protein to promote SHVV proliferation.The main results of this study are as follows:1.Establishment of SHVV reverse genetic system.The reverse genetic system is an important technique used to study the pathogenesis of viruses and vaccines development.To establish the SHVV reverse genetic system,firstly,a plasmid was constructed,containing the viral genomic cDNA,and helper plasmids encoding N,P and L proteins.Subsequently,these plasmids were co-transfected to 293T cells in a certain ratio.After 3 days of transfection,transferred to CCO cells,and viral protein expression and virus titer were detected by western blot and TCID₅₀,indicating that the rescue of rSHVV was successful.To compare the growth curve of wild virus strain?wtSHVV?and recombinant virus strain?rSHVV?,it was found that there was no significant difference about the proliferative capacity of wtSHVV and rSHVV.Establishment of the reverse genetic system laid a foundation for construction of Leader RNA mutant virus strain.2.Identification of SHVV Leader RNA.There is no report on Leader RNA in fish viruses.In this report,SHVV isolated in our laboratory was used as the research object,we identify whether SHVV?Fish virus?produces Leader RNA.Firstly,SHVV infected CCO cells,through small RNA sequencing,it was found that SHVV can produce three groups of Leader RNAs,named le_group1,le_group2 and le_group3.In order to exclude the influence of cells,SHVV-infected SSN-1 cells were subjected to small RNA sequencing,and the sequencing results were basically consistent with the results of CCO cells,which confirmed that SHVV can produce three groups of Leader RNAs.For the results of small RNA sequencing,we verified the transcription initiation and termination sites by RT-PCR,and the verification results were basically consistent with the above small RNA sequencing results.At the same time,we found that the expression levels of le_group1roup1 and le_group3 increased significantly with the proliferation of the virus,but the expression level of le_group2 did not change significantly.3.The effect of Leader RNA on SHVV proliferation.To construct mutant strains lacking the Leader RNAs,we mutated the transcription initiation sites of the three groups of Leader RNAs.It was found that the mutation of the transcription initiation sites of le_group1 and le_group2 could not rescued.During the mutation of the transcription initiation site of le_group3?38-42 nt?,the mutant(rSHVV-mut_38-42)was rescued successfully.We tested the generation of Leader RNA by the mutant strain and found that the mutant strain infected CCO cells could only produce le_group1,however le_group2 or le_group3 cannot be generated.Moreover,there was no significant difference in the proliferation ability of rSHVV-mut_38-42 compared with rSHVV,indicating that le_group2 and le_group3 are not essential for SHVV proliferation.Three Leader RNAs(le_1-22,le_19-48,le_41-65)and NC?Negative control?were synthesized based on the three groups of Leader RNAs.Through qRT-PCR,western blot and TCID₅₀ detection,it was found that le_1-22 can significantly enhance virus proliferation,while le_19-48 and le_41-65 have no segnificant positive effect.At the same time,le_1-30 also have the effect of induced virus proliferation,it indicates that le_group1 plays an important role in virus proliferation.4.The effect of le_group1 interaction with viral proteins on SHVV proliferation.To explore the mechanism by which le_group1 promotes viral proliferation,we investigated the effect of le_group1 on the expression of interferon and inflammatory factors.The results showed that overexpression of le_group1 had no significant effect on the expression of interferon and inflammatory factors.Further studies have found that le_group1can promote the transcription and replication of viral genomic RNA to promote viral proliferation.The transcription and replication of viral genomic RNA may be linked to viral protein interaction.Our study found that le_group1 interacts directly with the N proteins of five viral proteins.In order to study the effect of interaction between le_group1 and N protein on virus proliferation,the eukaryotic expression plasmid of viral N protein was transfected into CCO cells with le_1-22 alone/simultaneously and infected with SHVV.It was found that le_group1 interacts with N protein to promote SHVV.Further studies have found that interaction between le_group1 and N protein can promote the transcription and replication of viral genomic RNA,thereby promoting virus proliferation.In order to explore the essential domain of the interaction between le_group1 and N protein,the viral N protein was segmentally expressed and purified to determine the essential domain interaction with le_1-22,we found that the 1-45 amino acids of the viral N protein were interacted with le_1-22 by EMSA.We synthesized Synthetic segmentation deletion of le_1-22,it is determined that the 6-12 nt of Leader RNA are the essential domain for the interaction with N protein.It was also found that the region in which le_group1 interacts with the N protein differs from the region in which the viral genomic RNA binds to the N protein.