宿主SLC25A6蛋白与禽腺病毒血清4型感染的互作

Interaction between the host protein SLC25A6 and fowl adenovirus serotype 4 infection

摘要:

目的 明确宿主蛋白溶质载体家族25成员6 (solute carrier family 25 member 6, SLC25A6)在禽腺病毒血清4型(fowl adenovirus serotype-4, FAdV-4)感染中的作用及分子机制，为深入阐明FAdV-4的致病机制和制定新型防控策略提供理论依据。方法 首先确认FAdV-4在不同时间和感染复数(multiplicities of infection, MOI)下感染LMH细胞可产生明显的细胞病变。其次基于前期筛选获得的宿主蛋白SLC25A6，采用Western blotting方法检测FAdV-4对细胞内源性SLC25A6蛋白表达的影响；通过转染实验调控SLC25A6的表达(过表达或干扰内源性表达)，结合实时荧光定量PCR及Western blotting技术，从mRNA、蛋白及病毒滴度水平分析SLC25A6对FAdV-4复制的影响；采用免疫共沉淀方法验证SLC25A6与FAdV-4核心衣壳蛋白Hexon的相互作用。结果 FAdV-4可显著抑制细胞内源性SLC25A6的表达。过表达SLC25A6能显著抑制FAdV-4的复制，而干扰内源性SLC25A6则促进病毒复制。SLC25A6可与FAdV-4 Hexon蛋白的直接相互作用。结论 宿主蛋白SLC25A6通过与FAdV-4 Hexon蛋白相互作用抑制病毒复制，为深入阐明FAdV-4的致病机制及制定新型防控策略提供了理论依据。

Abstract:

Objective To clarify the role and molecular mechanism of the host protein solute carrier family 25 member 6 (SLC25A6) during fowl adenovirus serotype-4 (FAdV-4) infection, thus providing a theoretical basis for elucidating the pathogenic mechanism of FAdV-4 and developing novel prevention and control strategies.Methods First, we confirmed that infection of LMH cells with FAdV-4 at different time points and multiplicities of infection (MOI) resulted in obvious cytopathic effects (CPE). Second, on the basis of the host protein SLC25A6 identified in previous screening, Western blotting was employed to examine the effect of FAdV-4 on the expression of endogenous SLC25A6 in cells. Subsequently, transfection experiments were performed to regulate the expression of SLC25A6 (overexpression or interference with endogenous expression). RT-qPCR and Western blotting were employed to analyze the effect of SLC25A6 on FAdV-4 replication from the aspects of mRNA level, protein level, and viral titer. Finally, co-immunoprecipitation (Co-IP) was employed to verify the interaction between SLC25A6 and the core capsid protein Hexon of FAdV-4.Results FAdV-4 significantly inhibited the expression of endogenous SLC25A6 in cells. The overexpression of SLC25A6 markedly inhibited FAdV-4 replication, while interference with endogenous SLC25A6 promoted viral replication. SLC25A6 could directly interact with the Hexon protein of FAdV-4.Conclusion The host protein SLC25A6 inhibits FAdV-4 replication through its interaction with the viral Hexon protein. The results provide a theoretical basis for further elucidating the pathogenic mechanism of FAdV-4 and developing novel prevention and control strategies.