σNS蛋白与宿主TRAM1因子互作对鸭呼肠孤病毒复制的影响

doi:10.3969/j.issn.1004-1524.2022.07.06

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1396-1401.DOI: 10.3969/j.issn.1004-1524.2022.07.06

σNS蛋白与宿主TRAM1因子互作对鸭呼肠孤病毒复制的影响

刘志艺(), 米晓云, 黄聪, 李传峰, 陈宗艳()

中国农业科学院上海兽医研究所,上海 200241

收稿日期:2020-11-12 出版日期:2022-07-25 发布日期:2022-07-26
通讯作者: 陈宗艳
作者简介:* 陈宗艳,E-mail: zychen@shvri.ac.cn
刘志艺(1995—),女,湖南邵阳人,硕士研究生,研究方向为预防兽医学。E-mail: zhiyiliu0422@163.com
基金资助:
上海市自然科学基金(19ZR1468800);国家自然科学基金-新疆联合基金(U1703117);上海市科委科技创新行动计划(13391901602)

Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus

LIU Zhiyi(), MI Xiaoyun, HUANG Cong, LI Chuanfeng, CHEN Zongyan()

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2020-11-12 Online:2022-07-25 Published:2022-07-26
Contact: CHEN Zongyan

摘要/Abstract

摘要：

宿主因子TRAM1(translocation-associated membrane protein 1,TRAM1)是一种参与I型膜蛋白质分解的蛋白,通过升高含量来缓解内质网(ER)在应激时的压力。前期研究发现,鸭呼肠孤病毒(duck reovirus,DRV)感染原代鸭成纤维细胞后,TRAM1宿主因子变化异常。为确定宿主细胞TRAM1因子与DRV非结构蛋白σNS是否存在相互作用以及相互作用对DRV复制的影响,本实验首先表达σNS蛋白,通过GST融合蛋白沉降技术(GST pull-down)发现TRAM1蛋白与σNS蛋白在细胞外存在相互作用;通过免疫共沉淀(co-immunoprecipitation, Co-IP)试验证实二者在细胞内存在相互作用。过表达TRAM1能够从转录水平抑制DRV σNS的表达,而抑制TRAM1提高σNS的转录水平。本研究为进一步分析σNS蛋白在DRV复制过程中的功能以及为σNS蛋白与宿主因子TRAM1互作对DRV复制的影响提供试验依据。

关键词: 鸭呼肠孤病毒, σNS蛋白, 病毒-宿主相互作用, TRAM1

Abstract:

Translocation-associated membrane protein 1 (TRAM1) is a host protein which is associated membrane protein 1, found within the endoplasmic reticulum (ER). Studies have shown that TRAM1 can relieve the pressure of endoplasmic reticulum (ER) during stress by increasing its content. Previous studies found that TRAM1 had been changed in duck reovirus infected duck embryo fibroblasts cells. In this study, the interaction between DRV non-structural protein σNS and TRAM1 had been further confirmed experimentally. σNS protein was expressed firstly. Glutathione S-transferase pull-down was utilized to confirm the interaction between TRAM1 and σNS protein extracellular while co-immunoprecipitation (Co-IP) was utilized to confirm the interaction between TRAM1 and σNS protein intracellular. In addition, knockdown of TRAM1 using siRNA and overexpression of TRAM1 gene were conducted to explore its effect on virus replication. The interaction between σNS and TRAM1 has been confirmed. The results showed that TRAM1 overexpression degraded the levels of σNS transcription while TRAM1 silencing enhanced the levels of σNS transcription. In conclusion, our studies could afford help to explore the function of σNS protein on DRV replication. It also provided data to explore the role of interactions between TRAM1 and σNS during DRV infection.

Key words: duck reovirus, σNS, virus-host interaction, TRAM1

中图分类号:

S858.32

刘志艺, 米晓云, 黄聪, 李传峰, 陈宗艳. σNS蛋白与宿主TRAM1因子互作对鸭呼肠孤病毒复制的影响[J]. 浙江农业学报, 2022, 34(7): 1396-1401.

LIU Zhiyi, MI Xiaoyun, HUANG Cong, LI Chuanfeng, CHEN Zongyan. Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1396-1401.

图/表 6

表1 实时荧光定量PCR引物序列

Table 1 qRT-PCR primer sequence

基因Gene	上游引物Forward primer (5'→3')	下游引物Reverse primer(5'→3')
σNS	GTTCCTTTCGACCACGTCTATC	CATGAGACGAGAGACAGCATTAG
β-actin	CCTCTATGCCAACACAGTGC	CCTGCTTGCTGATCCACATC

图1 GST和GST-σNS蛋白的诱导表达 M,蛋白Marker;GST,GST空载对照,IPTG的终浓度为0.2 mmol·L-1;GST-σNS-1,IPTG终浓度为0.5 mmol·L-1诱导GST-σNS融合蛋白的表达;GST-σNS-2,IPTG终浓度为0.2 mmol·L-1诱导GST-σNS融合蛋白的表达。

Fig.1 Inducible expression of GST and GST-σNS M, Protein marker; GST, GST no-load control with the final concentration of IPTG was 0.2 mmol·L-1; GST-σNS-1, GST-σNS was induced at the final IPTG concentration of 0.5 mmol·L-1; GST-σNS-2, GST-σNS was induced at the final IPTG concentration of 0.2 mmol·L-1.

图2 GST-σNS融合蛋白诱导条件的优化 S,上清;I,沉淀。

Fig.2 Optimization design of inducing conditions for pGEX-4T-1-σNS expression S, Supernatant from inducible expression; I, Precipitation from inducible expression.

图3 σNS蛋白与TRAM1蛋白互在体外相互作用分析

Fig.3 Analysis of interaction between σNS protein and TRAM1 protein by Co-IP

图4 σNS蛋白与TRAM1蛋白在细胞中相互作用分析

Fig.4 Analysis of the interaction between σNS protein and TRAM1 protein by GST pull-down

图5 TRAM1蛋白对DRV-TH11复制的影响 A,siRNA抑制TRAM1的表达;B,抑制TRAM1的表达后,qRT-PCR检测DRV-TH11σNS的转录水平;C,上调TRAM1的表达后,qRT-PCR检测DRV-TH11σNS的转录水平。****,P<0.001;####,P<0.000 1。

Fig.5 Effects of TRAM1 protein on DRV-TH11 replication A, Western blot was used to detect the effect of siRNA knockdown on TRAM1; B, The mRNA level of DRV-TH11 σNS gene was detected by using qRT-PCR after knocking down TRAM1; C, The mRNA level of DRV-TH11 σNS gene was detected by using qRT-PCR after upregulating TRAM1. ****, P<0.001; ####, P<0.000 1.

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σNS蛋白与宿主TRAM1因子互作对鸭呼肠孤病毒复制的影响

Effect of interaction between translocation-associated membrane protein 1 and σNS protein on replication of duck reovirus

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