Subcellular Localization,tissue Expression Of Chicken ANP32 Family Proteins And Their Roles In Newcastle Disease Virus Replication

Acidic leucine nuclear phosphoprotein 32(ANP32)is a family of small molecular proteins that are highly evolutionarily conserved,which includes ANP32 A,ANP32B and ANP32 E.ANP32 family protein members have similar structural characteristics,of which the N-terminal contains a leucine-rich repeats(LRRs),which provides a framework for protein interaction;the C-terminus comprises a low complexity acidic region(LCAR),which facilitates its binding to basic proteins in the nucleus.It has been reported that chicken ANP32 B protein interacts with Newcastle disease virus(NDV)M protein and promotes the nuclear aggregation of M protein,but whether other ANP32 proteins are involved in the replication process of NDV is unclear.In addition,the subcellular localization and expression characteristics of chicken ANP32 family proteins in immune organs(spleen,thymus,bursa of Fabricius)are not yet clear.Therefore,in this study,the subcellular localization of chicken ANP32 family proteins as well as the key amino acid sites determining their nuclear localization were performed.In addition,the expression levels of ANP32 family protein genes in the chicken immune organs(spleen,thymus,bursa of Fabricius)at different months and the immune organs of chickens infected with NDV were detected by real-time quantitative PCR.Moreover,the relationship between chicken ANP32 family proteins and NDV replication was also studied.The relevant research results are as follows:1 The secondary structure,tertiary structure,amino acid homology,domain conservation and nuclear localization signal(NLS)conservation of chicken ANP32 family proteins were analyzed by bioinformatics software.At the same time,the recombinant eukaryotic expression vectors expressing chicken ANP32 family proteins NLS and its basic amino acid site mutants were constructed and transfected into cells.The key basic amino acid sites in NLS were identified by observing and analyzing their subcellular localization.The results showed that the secondary structure and tertiary structure of chicken ANP32 family proteins were mainly composed of α-helix and random coil,which had the highest amino acid homology with duck ANP32 family proteins,and their LRRs and LCAR domains were completely conserved.In addition,the nuclear localization signal(NLS)of the chicken ANP32 family proteins was highly conserved among different species,all of which were KRKR.Subcellular localization analysis showed that the tag protein EGFP was localized in the nucleus and cytoplasm,while the fusion proteins EGFP-ANP32 A,EGFP-ANP32 B and EGFP-ANP32 E were mainly localized in the nucleus,indicating that the chicken ANP32 family proteins were localized in the nucleus.The results of NLS point mutation experiments showed that the first K,first R and second K mutations in NLS changed the NLS fusion protein from nuclear localization to nuclear and cytoplasmic localization,indicating that the three sites were the key basic amino acid sites mediating the nuclear localization of chicken ANP32 family proteins.2 RT-qPCR was used to detect the expression level of ANP32 family protein genes in immune organs(spleen,thymus,bursa of Fabricius)of 1-6 months old chickens.The results showed that ANP32 family protein genes were expressed in the immune organs(spleen,thymus,bursa of Fabricius)of 1-6 months old chickens.Among them,the expression of ANP32 A and ANP32 E genes in the immune organs of 1-6 months old chickens increased first and then decreased,and the relative expression levels in immune organs of 2-month-old chickens reached the peak.The relative expression of ANP32 A gene in spleen,thymus,bursa of Fabricius of 2-month-old chickens was 1.6-10.9 times,1.6-18.8 times and 3.4-14.5 times higher than that in other months,respectively.The relative expression of ANP32 E gene in spleen,bursa of Fabricius of2-month-old chickens was 3.4-61.9 times,1.7-132.4 times higher than that in other months,respectively.The expression of ANP32 B gene showed a downward trend,and the relative expression level in immune organs of 1-month-old chickens was the highest.The relative expression levels of ANP32 B gene in spleen,thymus,bursa of Fabricius of1-month-old chickens were 1.7-16.8 times,2.2-31.3 times and 2.8-20.1 times higher than that in other months.3 RT-qPCR was used to detect the expression level of NDV in immune organs(spleen,thymus and bursa of Fabricius)on the 3 rd-5 th day after NDV infection.The results showed that the ANP32 family protein genes were expressed in the immune organs(spleen,thymus,bursa of Fabricius)of chickens infected with NDV on the 3 rd-5 th day.The ANP32 A gene showed an increasing trend,and the ANP32 B and ANP32 E genes showed a trend of increasing first and then decreasing.In addition,the expression of ANP32 A gene in bursa of Fabricius was the highest at 3 rd and 4 th day after NDV infection,which was 1.0-2.1 times and 1.1-1.4 times of other tissues,respectively.The expression of ANP32 A gene in spleen was the highest at 5 th day after NDV infection,which was 1.5-2.6 times of other tissues.The expression of ANP32 B gene in bursa of Fabricius was the highest on the 3 rd day after NDV infection,which was 3.2-4.2 times of other tissues.The expression of ANP32 B gene in spleen was the highest on the 4 th and 5 th day after NDV infection,which was 1.3-1.6 times and 2.9-4.4 times of other tissues,respectively.The expression of ANP32 E gene in bursa of Fabricius was the highest on the 3 rd day after NDV infection,which was 2.2-2.6 times of other tissues.The expression of ANP32 E gene in spleen was the highest on the 4 th and 5 th day after NDV infection,which was 3.3-3.4 times and 2.1-3.2 times of other tissues,respectively.4 The RT-qPCR method was used to detect the expression of chicken ANP32 family genes at different time points after NDV infection of chicken embryo fibroblasts(DF-1).The results showed that the expression levels of chicken ANP32 family protein genes were significantly changed at 6,12 and 24 h in DF-1 cells infected with NDV.The relative expression levels of ANP32 A,ANP32B and ANP32 E genes reached the peak at 24 h,24 h and 12 h,respectively.The peak values of ANP32 B gene and ANP32 E gene were 1.7 times higher than that of ANP32 A gene.Overexpression of chicken ANP32 A,ANP32B and ANP32 E genes obviously promoted NDV replication and cytopathogenicity.The chicken ANP32 B gene was further selected for RNA interference test.The results showed that interfering with the expression of chicken ANP32 B gene did not affect the replication and pathogenicity of NDV.The above results indicated that chicken ANP32 family proteins were mainly located in the nucleus,and were expressed in immune organs of chickens of different months and after NDV-infected chickens.At the same time,there is a certain relationship between chicken ANP32 family proteins and NDV replication and pathogenicity,which lays a foundation for the subsequent molecular mechanism of nuclear localization of chicken ANP32 family proteins and the study of candidate genes against Newcastle disease(ND).