关键词: 纳尔逊海湾正呼肠孤病毒, 酵母双杂交实验, σNS蛋白, 蛋白互相作用, 生物信息学

Abstract:

Objective To discuss the host proteins that interact with the Nelson Bay orthoreovirus （NBV） σNS protein in the fibroblasts L929 of the mice， and to clarify the effect of host proteins on the viral replication. Methods The bait plasmid pGBKT7-S3 expressing σNS protein was constructed， and sequencing technology was used to verify the accurate expression of the bait plasmid in the Y2H yeast cells. The pGBKT7-S3 and pGADT7 plasmids were separately and jointly transformed into the Y2HGold yeast cells， plated on solid medium， and the colony growth was observed to confirm that the σNS protein was non-toxic to the yeast cells and could not self-activate the reporter gene. The bait plasmid pGBKT7-S3 was hybridized with the cDNA library in fibroblasts L929 of the mice， and the plasmids encoding the interacting proteins were extracted from the positive clones. The positive sequencing results were screened for the proteins interacting with NBV σNS through the Uniprot database. Gene Ontology（GO） functional enrichment analysis， Kyoto Encyclopedia of Genes and Genomes（KEGG） signaling pathway enrichment analysis， and STRING bioinformatics analysis were performed on the interacting proteins. Results A total of 61 positive clones were successfully screened. The DNA sequencing analysis and BLAST alignment removed 23 positive clones that did not match the database or were similar in sequence. The positive sequencing results identified 38 proteins interacting with NBV σNS through the Uniprot database. Thirty-one proteins were involved in cellular biological processes； thirty-six proteins were cellular anatomical components； thirty-one proteins had binding functions. Five proteins were part of the mitochondrial respiratory chain； seven proteins were ribosomal proteins and components of the ribosomal subunits； two proteins were involved in iron metabolism homeostasis.The GO functional enrichment analysis results showed that the interacting proteins were enriched in biological processes（BP） such as cellular processes， metabolic processes， biological regulation， localization， and response to stimuli； the cellular components were mainly cellular anatomical components and protein-containing complexes； the molecular functions were concentrated in binding， catalytic activity， structural molecule activity， and transporter activity. The KEGG signaling pathway enrichment analysis results showed that the proteins were highly enriched in translation， folding， sorting， and degradation pathways of genetic information processing and were mainly associated with the digestive system in the organism； they were linked to various viral infections and cancers. The STRING analysis results showed that the interacting proteins included ribosomal proteins， protein modification proteins， metabolic proteins， and immune proteins. Conclusion The host proteins that interact with NBV σNS protein are successfully screened， and these host proteins play important roles in viral replication.

Key words: Nelson Bay orthoreovirus, Yeast two-hybrid assay, σNS protein, Protein interaction, Bioinformatics