Screening and identification of protein interacting with goose astrovirus

Lingling Qian¹Yuwei Liu²Xiaochun Wang²Shixing Yang²Likai Ji²XiaoPeng Sun³Jianqiang Wang⁴Tongling Shan⁵Wen Zhang^2*Quan Shen^2*

• ¹Changshu Medicine Examination Institute, Changshu, China
• ²Department of Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu Province, China
• ³Department of Orthopaedics, Jiangsu University Affiliated People's Hospital, Zhenjiang, Jiangsu Province, China
• ⁴Jintan District Hospital of Traditional Chinese Medicine, Changzhou, China
• ⁵Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, Shanghai Municipality, China

Goose Astrovirus (GoAstV), a recently identified member of the Astroviridae family in China, predominantly affects goslings, resulting in substantial economic losses to the goose farming industry due to its high infection and mortality rates. In 2 order to clarify the infection mechanism and pathogenesis of this virus, the Viral Overlay Protein Blot Assay was utilized to identify and characterize proteins on the LMH (Leghorn Male Hepatoma) cell membrane that interact with Goose Astrovirus.Proteins from various components of LMH cells were extracted. Subsequently, the astrovirus was re-inoculated into goose embryos to yield high-purity viral samples. The LMH cell membrane proteins, transferred onto a PVDF membrane, were co-incubated with the concentrated astrovirus. This successfully identified a 70kDa protein within the LMH cell membrane protein extract capable of interacting with the astrovirus protein. Following gel excision and LC-MS mass spectrometry analysis, the selected protein was identified as Glucose-regulated protein 78. The identity of the candidate protein was confirmed as HSPA5 through bioinformatics analysis and a UniProt database search. The interaction between HSPA5 and the astrovirus protein was further validated in vitro through Western blot and Co-immunoprecipitation experiments.Finally, bioinformatics tools such as SWISS-MODEL, AlphaFold, and ZDOCK were employed to construct and analyze the docking complex model between the candidate protein and GoAstV protein, including their key binding residue sites. This approach helps to explore the role of the candidate protein in the process of GoAstV infecting host cells from multiple perspectives and provides a theoretical basis for further identification of GoAstV receptors and elucidating its infection mechanism.