The Study Of SRSF10 Inhibiting The Polymerase Activity Of Avian Influenza Virus By Regulating The Alternative Splicing Of Avian ANP32A

The host range of avian influenza virus is wide,which not only brings huge economic loss to poultry breeding,but also seriously threatens human health.Wild waterfowl are the natural hosts of avian influenza virus,which must overcome many biological barriers to achieve cross species transmission successfully.Compared with human ANP32A,which limits the polymerase activity of avian influenza virus,there are 33 or 29 amino acids inserted into avian ANP32A.Both insertions restored the activity of avian polymerases in mammalian cells,but the full-length subtype was more effective than the truncated subtype.It has also been found that the species diversity of ANP32A splicing modes was related to the cross species transmission of avian influenza virus.But the mechanism of regulating alternative splicing of avian ANP32A have not been found.The mechanism of alternative splicing of avian ANP32A and the effect of this alternative splicing mechanism on the polymerase of avian influenza virus were studied in this paper.1.Identification of splicing factors regulating alternative splicing of avian ANP32AThe high-resolution polyacrylamide gel electrophoresis was used to identify and distinguish the two splicing variants,ch-ANP32A-33 and ch-ANP32A-29,which are only 12 nucleotides apart.The two transcripts produced by ANP32A alternative splicing were detected in DF-1,DEF cells,and ch-ANP32A-29 were found to account for about 22.4%and 17.4%of the total avian ANP32A,respectively.Using this method,the splicing patterns of ANP32A in chicken heart,liver,spleen,lung and kidney were detected.The results showed that the distribution of ch-ANP32A-33 and ch-ANP32A-29 in chicken heart,liver,spleen,lung and kidney were similar,without tissue specificity.There are two 3' splice sites in intron 4 of ANP32A.The splicing of the proximal splice site produces the full-length subtype(ch-ANP32A-33)and the distal splice site produces the truncated subtype(ch-ANP32A-29).The selection of splice sites are regulated by cis-elements on adjacent exons and trans acting factors combined with them.In order to find the cis-elements that affect the selection of the 3' splice sites of ANP32A intron 4,the minigene was constructed.Part of the sequence of ANP32A gene(complete sequence of exon 4,intron 4,exon 5 and the first 150 bases of intron 5)was connected to the eukaryotic expression vector.Furthermore,four minigene mutants were constructed by deletion of the 16-30,31-45,46-60,61-75 and 76-80 bases of exon 5.These vectors were transfected into DF-1 cells.By RT-PCR and polyacrylamide gel electrophoresis,it was found that the deletion of the 31-45 bases of exon 5 could reduce the amount of ch-ANP32A-29 and increase the amount of ch-ANP32A-33,and the ratio of ch-ANP32A-29 decreased from 37%to 16%.It is suggested that the cis-element affecting the alternative splicing of ANP32A is located at the 31-45 bases of exon 5.We designed and synthesized biotin labeled RNA probes containing the 31-45 bases of exon 5(cis-element).Meanwhile,RNA probes containing the 46-60 bases of exon 5 and random sequences were used as controls for RNA affinity purification experiments and mass spectrometry analysis.The results showed that 78 proteins were specifically captured by RNA probe containing cis-element,from which the trans acting factor SRSF10 protein was screened.The specific binding of SRSF10 protein to ANP32A mRNA was confirmed by RNA immunoprecipitation assay.2.SRSF10 can inhibit the polymerase activity of avian influenza virus by regulating the alternative splicing of avian ANP32AAfter overexpression of SRSF10,the amount of ch-ANP32A-29 increased,the amount of ch-ANP32A-33 decreased,and the proportion of ch-ANP32A-29 increased from 23%to 39%.This indicated that SRSF10 could change the ratio.of ch-ANP32A-33 and ch-ANP32A-29 by regulating the alternative splicing of ANP32A.The small interference RNA for the common sequence of ch-ANP32A-33 and ch-ANP32A-29 was transfected.The high-resolution polyacrylamide gel electrophoresis showed that the two subtypes decreased and the proportion remained unchanged,showing that the small interference RNA had the same interference effect on the two subtypes.On the basis of interference,we carried out the experiment of polymerase activity.The results showed that the knockdown of ANP32A inhibited the polymerase activity of H9N2 virus.In order to analyze the effect of the ratio of ch-ANP32A-33 and ch-ANP32A-29 on the activity of polymerase.ch-ANP32A-29 was supplemented separately on the basis of interference.With the increase of dose of ch-ANP32A-29 in ch-ANP32A-knockdown DF-1 cells,the polymerase activity of H9N2 virus gradually recovered but could not return to the normal level.This result showed that overexpression of ch-ANP32A-29 could only partially restore the activity of avian influenza virus polymerase in a dose-dependent manner which revealed that ch-ANP32A-29 was less efficient than ch-ANP32A-33 at promoting avian influenza virus polymerase activity.In order to further confirm that the inhibition of polymerase activity of avian influenza virus caused by SRSF10 is related to the decrease of ch-ANP32A-33.With or without co-transfection of ch-ANP32A-33,SRSF10 and plasmids of polymerase activity assay were transfected.It was found that ch-ANP32A-33 could partially recover the decrease of polymerase activity caused by SRSF10.The 627 position of PR8 PB2 gene was mutated from mammalian characteristic lysine(K)to avian characteristic glutamate(E)by inverse PCR.It was found that the overexpression of SRSF10 inhibited the polymerase activity of PR8 PB2 K627E and H9N2 virus.Furthermore,the mutant PR8 PB2 K627E virus was obtained by reverse genetics.DF-1 cells were infected with PR8 PB2 K627E and H9N2 virus after overexpression of SRSF10.The results showed that the overexpression of SRSF10 inhibited the expression of NP protein of PR8 PB2 K627E and H9N2 virus at 24 hours,and reduced the virus titers of the two viruses.However,the expression of NP and the titers of PR8 PB2 K627E and H9N2 virus could not be affected at 48 h post-infection.This indicated that the overexpression of SRSF10 inhibited the replication of avian influenza virus in the early stage.In contrast,the overexpression of SRSF10 inhibited the polymerase activity of mammalian adapted PR8 virus,but the inhibition was weaker than that of avian influenza virus.Furthermore,SRSF10 was overexpressed in 293T cells and DF-1 cells,PR8 virus was infected at 24 h post-transfection.It was found that the overexpression of SRSF10 had no effect on the expression of NP protein and virus titer of PR8 virus,indicating that the overexpression of SRSF10 had no effect on the replication of mammalian adapted influenza virus.We preliminarily explored the mechanism of avian ANP32A alternative splicing.For the first time,we found that SRSF10 could regulate the alternative splicing of the 3' splice sites of avian ANP32A intron 4 by combining with its corresponding cis-element,which is manifested in reducing the amount of ch-ANP32A-33 and increasing the amount of ch-ANP32A-29,which can inhibit the polymerase activity of avian influenza virus.